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Keywords:

  • metastasis;
  • disseminated tumor cells;
  • bone marrow;
  • breast cancer;
  • lung cancer;
  • prostate cancer;
  • colorectal cancer

Abstract

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

The prognosis of cancer patients is largely determined by the occurrence of distant metastases. In patients with primary tumors, this relapse is mainly due to clinically occult micrometastasis present in secondary organs at primary diagnosis but not detectable even with high resolution imaging procedures. Sensitive and specific immunocytochemical and molecular assays enable the detection and characterization of disseminated tumor cells (DTC) at the single cell level in bone marrow (BM) as the common homing site of DTC and circulating tumor cells (CTC) in peripheral blood. Because of the high variability of results in DTC and CTC detection, there is an urgent need for standardized methods. In this review, we will focus on BM and present currently available methods for the detection and characterization of DTC. Furthermore, we will discuss data on the biology of DTC and the clinical relevance of DTC detection. While the prognostic impact of DTC in BM has clearly been shown for primary breast cancer patients, less is known about the clinical relevance of DTC in patients with other carcinomas. Current findings suggest that DTC are capable to survive chemotherapy and persist in a dormant nonproliferating state over years. To what extent these DTC have stem cell properties is subject of ongoing investigations. Further characterization is required to understand the biology of DTC and to identify new targets for improved risk prevention and tailoring of therapy. Our review will focus on breast, colon, lung, and prostate cancer as the main tumor entities in Europe and the United States. © 2008 Wiley-Liss, Inc.

Solid tumors derived from epithelial tissues such as breast, prostate, lung or colorectal carcinomas are the most frequent forms of cancer in industrialized countries. Most solid tumor patients undergo a complete resection (R0) of their primary tumor but they still harbor a considerable risk to die from subsequent metastatic relapse caused by minimal residual disease (MRD) not being eliminated by primary surgery, radio- or chemotherapy.

By applying highly sensitive and specific immunocytochemical and molecular methods, it is now possible to detect disseminated tumor cells (DTC) in bone marrow and circulating tumor cells (CTC) in peripheral blood at the single cell level in the background of millions of normal cells.1 Among the distant organs, bone marrow (BM) plays the most prominent role as indicator organ for MRD, because BM is easily accessible by needle aspiration through the iliac crest. BM might be a common homing organ for DTC derived from various types of epithelial tumors. Thus far unknown environmental and internal factors can promote their recirculation from the BM niche into other distant organs such as liver or lungs where they can find better growth conditions.2 In the peripheral blood, CTC are detectable months to years after complete removal of the primary tumors, indicating that these cells might circulate between different metastatic sites.1

Our current review will focus on the relevance of DTC in BM with an emphasis on studies in patients with breast, colon, lung, and prostate cancer as the most frequent solid cancer entities. In particular, we will review the currently available methods for the detection and characterization of DTC, summarize the still limited knowledge about the specific biological properties of these cells, and discuss the clinical relevance of DTC detection with regard to an improved individualized management of cancer patients.

Detection of DTC

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Due to the extremely low concentration of DTC (1 × 10−5 to 10−6) in BM, a direct detection of these cells is usually not feasible without a prior enrichment step. The most commonly used enrichment procedure is gradient centrifugation of BM through, e.g., Ficoll-Paque. With this procedure one can separate red blood cells and granulocytes from mononuclear cells and possible DTC based on their buoyant density. The separation of granulocytes is especially important in RT-PCR analyses, as, e.g., CK20 is also expressed by granulocytes.3, 4 For the isolation of CTC from peripheral blood, size-based enrichment has also been described, e.g., by membrane filter devices such as ISET (isolation by size of epithelial tumor cells5, 6) or MEMS (micro electro-mechanical system)-based microfilter approaches.7

Another enrichment approach used either alone or after gradient centrifugation is immunomagnetic bead separation which can be performed as (i) positive selection of DTC using epithelium-specific antibodies (e.g., anti EpCAM) or (ii) negative depletion of hematopoetic cells (e.g., by antibodies against the common leukocyte antigen CD45). The advantages and disadvantages of these enrichment methods have been discussed elsewhere.8, 9

Immunological detection of DTC

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Current methods to screen BM aspirates for DTC after the enrichment can be classified into cytometric/immunological and molecular approaches.1, 10, 11

Among the cytometric methods immunocytochemistry (ICC) is the most widely used approach.2, 10 ICC has the advantage to facilitate characterization of both cell size and shape as well as the nucleus-plasma relation of each individual event. Thereby illegitimate expression of the protein of interest in BM cells can be excluded as far as possible. Because of the absence of tumor-specific target antigens, monoclonal antibodies against various epithelium-specific antigens such as cytoskeleton-associated cytokeratins, surface adhesion molecules, or growth factor receptors are applied for the detection of carcinoma cells.1, 10 These antibodies bind to the tumor cells which can then be visualized directly or indirectly by fluorescent dyes or colorimetrically.

The most commonly used antibodies, such as A45-B/B3, AE1/AE3, and KL-1 are directed against various intracellular cytokeratins (CK), which are intermediate filament proteins that are expressed in all epithelial cells but usually not in haematopoietic cells. Different CK are specific for specific epithelial cell types, e.g., CK20, frequently used for the detection of CTC/DTC in colorectal cancer (CRC) patients, is not suitable for CTC/DTC detection in lung cancer patients.12, 13

The detection of DTC in BM is still not part of the routine tumor staging in the clinical practice; however, emerging data anticipate a future role of DTC detection for risk stratification and therapeutic monitoring of breast cancer patients.14–16 Nevertheless, the detection rates of DTC in BM from nonmetastatic breast cancer and other carcinoma patients vary considerably (Tables I–IV). This might reflect the different sensitivity but also specificity of the numerous detection methods and antibodies used thus far. To overcome these problems and to provide a basis for future multicentric clinical trials, a consensus concept for the detection and enrichment of DTC in BM of thus far only breast cancer patients has been proposed.17 The use of pan-anti-cytokeratin antibodies A45-B/B3 or AE1/AE3 against a wide spectrum of cytokeratins was recommended as standard application, thereby ensuring detection of DTC also in cells that have downregulated the expression of individual cytokeratins, e.g., in the course of epithelial-mesenchymal transition.17–19 Criteria to evaluate morphology and staining results after automatic microscopic screening using sophisticated imaging approaches have also been defined to avoid false-positive and false-negative results.17, 20–22

Flow cytometry-based methods have been used for the detection of CTC/DTC. However, very few studies have been conducted on larger patient cohorts. These analyses are hampered by the lower sensitivity of flow cytometry as compared with other methods and have therefore mainly been described for analysis of BM or blood taken from advanced stage cancer patients with overt metastases.23, 24

Table I. Detection of DTC in Breast Cancer
No. of patientsStageBM aspirateEnrichment procedureDetection markers (antibody)No. of cells analyzedRange of positive cellsDetection rate cases %Detection rate controls (%)Correlation to clinical/pathol. variablesMean follow-up (months)Prognostic/predictive valueReference
  1. IC, iliac crest; ST, sternum; ICC, immunocytochemistry; Q-RT-PCR, quantitative reverse transcriptase polymerase chain reaction; MAM, mammaglobin; EMA, epithelial membrane antigen; CEA, carcinoembryonic antigen; PSE, prostate-specific Ets factor; PIP, prolactin-inducible protein; ER, estrogen receptor; PR, progesterone receptor; LN, lymph node; G, grading; TS, tumor size/stage; NA, information not available; NS, not significant; ND, not determined; DFS, disease-free survival; OS, overall survival; DM/LM, distant/lymph node metastasis; DMSF, distant metastasis-free survival; LRSF, local relapse-free survival; DDFS, Distant disease-free survival; BCSS, breast cancer-specific survival; RFS, relapse-free survival; TRD, tumor-related death.

Immunocytochemistry
 155T1–46.1 ml,IC, STFicollCK18 (CK2)NAMeanM0: 2.8/3*104;M1: 8.7/3*10418%0/75 (0%)DM(p < 0.001)NDND186
 49≥T1IC, STFicollcell-surfaceantigens (C26, T16),pan-CK (AE1)NANA37%NDNSmean 29,median 30(range 12–38)early recurrence(p < 0.04)187
 100T1–4, M04–6 ml, ICLymphoprepEMA,pan-CKNANA38%NDNSmedian 34(range 7–65)RFS (p = 0.0005),OS (p = 0.017)188
 727T1–410–12 ml,ICFicollmucin TAG12 (2E11)4–5 × 1061–9055% (LN+), 31% (LN0)0/21 (0%)TS (p < 0.001),G (p = 0.002),LN (p = 0.001)median 36DDFS (p < 0.001), OS (p < 0.001)189
 109T1–4ICFicollbreast-associatedantigens: MBr1,MBr8, MOV8,MOV16, MluClNANATotal: 31%;during surgery: 38%; 2–4 weeksafter surgery:17%NDNSmedian 36(range 15–62)NS190
 350T1–T416 ml ICLymphoprepEMANANA25%NDTS (p = 0.008), LN (p = 0.005), vascular invasion(p = 0.001)median 150(range 11–180)RFS (p < 0.001),OS (p < 0.001)191
 552T1–3ICFicollpan-CK(A45-B/B32 × 1061–1223(median 3)36%2/191 (1%)TS (p < 0.001), number of LNM.(p < 0.001),tumor type(p < 0.001),G (p = 0.017),DM (p < 0.001)median 38(range 10–70)DMSF (p < 0.001), OS (p < 0.001),192
 393≥T15–15 ml,IC, STFicollCK+EMANANA42%0/20 (0%)Number of DM(p < 0.001),bone metastases(p = 0.028),LN (p = 0.001)median 75TRD (p =0.01),DSF (p = 0.0003), OS (p = 0.022)193
 554pT1-2N0/1,M05–10 ml,ICFicollCK8, 18, 19(clone 5D3)NA1–300N0: 31%N1: 37%NDTS (p < 0.0001),G (p = 0.034),lymph vesselinvasion (p = 0.005),blood vessel invasion(p = 0.047),ER (p = 0.026),Ki-67 (p < 0.0001),uPA (p = 0.016), PAI-1 (p = 0.03)10 yearsDFS (p < 0.0001),OS (p < 0.0001)194
 920T1–T440 ml, ICLymphopreppan-CK(AE1/AE3)2 × 106NA13.4%5/40 (12.5%)TS (p = 0.013),LN (p < 0.0005), vascular invasion(p = 0.045),HER2 (p = 0.024)NDND195
 817T1–440 ml, ICFicollpan-CK(AE1/AE3)2 × 106NA13%4/98 (4%)TS (p = 0.011),LN (p < 0.001), ER and/orPR(p = 0.051)median 49(range 0.5–85)DDFS (p < 0.001), BCSS (p < 0.001)196
 114T1–43–5 ml,IC or STFicollpan-CK(A45-B/B3)3 × 1061 to >1000(mean: 70,median: 6)59%NDmenopausal status(p = 0.024),ER (p = 0.026),TS (p = 0.025)median 28OS (p = 0.0004),DSF (p = 0.012)197
 228pT1, 2,pN0–3,pM0)3–8 ml, ICFicollpan-CK(A45-B/B3)2 × 106NAPersistentDTC: 13%NDNSmean 49.8RFS (p = 0.0003), DFS (p < 0.0001), OS (p = 0.002) Persistent DTC: OS (p = 0.0008)16
 265≥T12–8 ml, ICFicollpan-CK(A45-B/B3)2 × 1061–1.500(median 2)26%NDNSmedian 60.5(range 7–255)OS (p = 0.03)198
 131T1–3, M0ICFicollICC: pan-CK(A45-B/B3)QPCR: CK193–6 × 106 eachICC: 1-12ICC: 31%Q-RT-PCR: 41%Combined: 51%NDNS1212 (48) months after surgery 35% (59%) of patients had positive ICC/Q-RT-PCR results54
 112pT1–410–20 mlBiocollpan-CK(A45-B/B3)2 × 106NA83% beforechemotherapy24% after surgeryand chemo/endocrine therapyNDDTCpersistence:G (p = 0.02)mean 12(range 5–30)ND199
 621T1–4, M0ST or ICFicollpan-CK(A45-B/B3)3 × 106NA15%NDNSmedian 56(range 1–100)OS (p = 0.02),DMSF (p = 0.006), LRFS(p = 0.0009)200
RT-PCR
 46T1–45 ml ST and ICBuffy coatMUC5B, CK19, CEANDNDMUC5B:19%,CK19: 41%,CEA: 17%NDNSmedian 34(range 12–31)ND201
 55+ 4 DCIST1–410 ml ICLymphoprepCK19NDNDDCIS: 25%,T1: 43%T2–4: 60%1/10 (10%)NSat least 4 yearsDSF (p = 0.004)202
 148T1–4,M0, M+9 ml ICFicollCK19, MAMNDNDCK19, M0: 23%,M1: 47%,MAM, M0: 16%,M1: 38%MAM: 0/13(0%)PR (CK19:p = 0.028,MAM;p = 0.026)mean 786 daysOS (CK19:p = 0.0045,MAM;p = 0.025)203
 195T1–420 mlnoneCK191 × 107ND12%1/34 (3%)NSmedian 72(range 1–99)Systemic RSF(p = 0.01),Overall RSF(p = 0.005).204
 177 (RT-PCR), 83 (ICC)T1–3, M03–6 ml ICFicollICC: pan-CK(AE1/AE3)RT-PCR: MAM,CEA, PSE, PIPNDNDICC: 6% RT-PCRcomb.: 11% MAM: 4%,PIP: 2.8%,PSE: 2.8%,CEA: 1.1%n = 49 forthresholddefinitionNDNDND205
Table II. Detection of DTC in Colorectal Cancer
No. of patientsStageBM aspirateEnrichmentprocedureDetectionmarkers (antibody)No. of cells analyzedRange of positive cellsDetection rate cases %Detection rate controls (%)Correlation to clinical/pathol. variablesMean follow-up(months)Prognostic/ predictive valueReference
  • *

    CA19-9: Lewis blood group antigenes, 17-1A: membrane antigen, CD54-0: membrane antigen.

  • IC, iliac crest; ST, sternum; IMB, immunomagnetic bead separation; GCC, guanylylcyclase; CEA, carcinoembryonic antigen; LN, lymph node; NA, information not available; NS, not significant; ND, not determined; DFS, disease-free survival; OS, overall survival; RR, recurrance rate.

Immunocytochemistry
 57I–IV (18 M1)mean 6.1 ml IC or STFicollCK18 (CK2)3 × 104mean 2.8in M0, mean 8.7 in M121%0/75 (0%)NS M0 21% vrs M1 22%, S (p > 0.05), N0 9% vrs N1 35%NDND186
 82NANANACK18 (CK2)NANA27%0/75 (0%)NANDND206
 156Duke A-Dmean 5.2 ml ICFicollCK18 (CK2)1.5 × 105ND27%0/102 (0%)LN and stagemean 26RR (p < 0.05)207
 88I–IV, all R05ml ICFicollCK18 (CK2)3 × 105ND32%0/102 (0%)NSmedian 35 (range: 12–58)RR (p < 0.0035)150
 12 M1+ 7 post operativeI–IV5 ml ICFicollCK18 (CK2)1 × 1061–6016% M1, 71% post op.NDNDmedian 27 (range:11–32)NS208
 57I–IV0.5–10 ml IC or STFicollCK18 (CK2), pan-CK (A45-B/B3)4 × 105 to 1.6 × 106NDCK2: 16%,A45-B/B3: 43.5%CK2 and A45-B/B3: 4/75 (5.3%)NDNDND148
 67I–IV8 ml ICFicollCEA (C1P83), mucin (Ra96),pan-CK (KL-1), 17-1-A, CA19-9, CD54*each: 2.5 × 105ND29%0/25 (0%)NSNDND144
 48I–IIIICFlow cytometryCK181 × 105NDpreop.: 23% postop.: 27%1/63 (1.6%)NSNDRR (p < 0.01)for postoperative BM151
 34I–IV8 ml ICFicollpan-CK (KL-1),CK18 (CK2), CEA (C1P83),17-1-A*5 × 105NDtotal 74%CEA:30%, 17-1a:26, KL1:67%, CK2: 52%NDLN and stagemedian 12.5 (range: 6–18)NS145
 80all M1 (hepatic)NASmearA33, CK18, pan-CK(CAM 5.2, AE12)NANDresected: 9% nonres.: 34%0/20 (0%)NANDNS146
 109I–IV8 ml ICFicollCEA (C1P83), mucin (Ra96), pan-CK (KL-1), CA 19-9, 17-1A*each: 2.5 × 105ND49% (Ra96 and 17-1A and CEA:<10%, CA19-9:<15%, KI-1:<25%)KI-1, 17-1A, CEA: 2/45 (4.4%), CA 19-9: 1/45 (2.2%)stageNDNS147
 18Duke C and D4–10 ml ICFicollCK20 (Ks20.8) pan-CK (Cam 5.2)1 × 105CK 20: colon: 1.6*10–4,rectum: 5*10-5CK20:36% CK8: 0%CK18: 0%NDCK20 and CEA levelsin PTNDND209
 16784 N0, 43 M110 ml ICFicollpan-CK (A45-B/B3)1 × 1061–50024%0/51 (0%)NSmean 37 (range: 12–72)OS (p = 0.006), DFS (p < 0.001)152
 51I–IVICIMBpan-CK (A45-B/B3), EpCAM (Ber-Ep4), K-ras mutation2.5 × 106ND47%ND13/17 PT tumors with K-ras mutation also mutationin BMNDND67
 41Duke A-D1–3 ml ICLymphoprep + IMBpan-CK (A45-B/B3)1 × 1075–15 (mean 10)10%ND0% simultaneously stained with uPARNDND71
 275I–IV20 ml ICIMBEpCAM (MOC31)2 × 1072–120 (median 8)17%3/206 (1.5%)NSNDND149
 11Duke A-DICFlow cytometryCK18NAND38%NDNSNDND210
 47all M1 (hepatic)10 ml ICFicollpan-CK (A45-B/B3)3–4 × 1061–14 (median 1)55%NDNDmean 43 (range:26–54)NS153
RT-PCR
 10NDNDnoneCEANDND66%0/56 (0%)NDNDND162
 15Duke A-DICnoneCK 20, CK 19NDNDCK19: 40% CK20: 0%CK19: 5/12 (42%),CK20: 0/12 (0%)NDNDND211
 57I–IV5 ml ICFicollCK 20NDND35%1/16 (6.2%)stageNDND159
 65I–IV, 40% M120 ml ICFicollCK 20NDND31%2/22 (9.1%)stageNDS in comb. with CTC in blood160
 14I–III10 ml ICFicollCK 20NDND21%NDNSNDND212
 30all M1 (hepatic)10 ml ICFicollCK 20NDND27%0/30 (0%)NDNDND213
 295I–IV, R0ICFicollCK 20NDND31%2/22 (9.1%)stageNAOS154
 109I–IV, 18% M12 ml ICnoneCK20, GCCNDNDCK20: 11%GCC: 6%CK20: 10/22 (45%), GCC:0/22 (0%)NSNDND156
 103 without +24 neoadjuv. therapyI–IV10 ml ICFicollCK 20NDND33% without and 17% with neoadjuvant treatmentNDNDmedian 49 (range:15–72)OS (p < 0.04) and DFS (p < 0.03) for neoadjuvant chemoradationpatients155
 32all M1 (hepatic)3–8 ml ICFicollCK 20NDND25%NDNSmedian 18 (range:5–40)NS157
 37all M1, R0 (hepatic)10 ml ICFicollCK 20NDND16%0/30 (0%)NDmedian 38 (range: 10–63)RR: 0.013, DFS (p = 0.04)161
 71all stage II, R010 ml ICFicollCK 20NDND28%0/30 (0%)NSmedian 58 (range:3–81)NS158
Table III. Detection of DTC in Lung Cancer
No. of patientsStageBM aspirateEnrichment procedureDetection markers (antibody)No. of cells analyzedRange of positive cellsDetectionrate cases %Detection rate controls (%)Correlation to clinicalvariablesMeanfollow-up (months)Prognostic relevanceReference
  • *

    Includes patients from ref. 168;

  • **

    Same study as ref. 163 but with longer follow-up;

  • ***

    Multicenter study with data collected from 15 hospitals.

  • IC, iliac crest; ST, sternum; IMB, immunomagnetic bead separation; GRP, prepro-gastrin-releasing peptide; NS, not significant; ND, not determined; DFS, disease-free survival; OS, overall survival; RR, recurrance rate.

Immunocytochemistry
 82I–III (N0 n = 42)0.5–5 ml ICFicollCK18 (CK2)2 × 106ND22%2/117 (1.7%)tumor size and grade13.0RR: (67% vs. 37%)168
 43I–IIIRibFicollpan-CK (CAM 5.2,AE1)1 × 106ND40%0/11 (0%)ND13.6DFS (p < 0.001),RR (p < 0.001)169
 139*I–III (N0 n = 70)2–10 ml IC, RibFicollCK18 (CK2)2 × 1061–531 (mean:2)all 60, N0:54%6/215 (2.8%)NS39.0RR: (p = 0.004) for N0 patients163
 39I–III (N0 n = 23)5 ml ICFicollCK18 (CK2)2 × 106ND38%0/5 (0%)NS4.6RR: (p = 0.008)170
 139**I–III (N0 n = 66)2–10 ml IC, RibFicollCK18 (CK2)2 × 1061–531 (mean:2)all 60%,N0:52%NDNS66.0OS (p = 0.007) for pN0 patients with ≥2CK2-positive cells164
 99I–IV (N0 n = 40, M1=3)8 ml ICFicollpan-CK (CAM 5.2,AE1)5 × 106ND22%NDNS14.3NS214
 58I–III (N0 n = 40)4 ml STFicollCK18 (CK2)1 × 106ND47%NDNS36.0OS (p = 0.044)165
 115I–II (all N0)5 ml ICFicollCK18 (CK2)1 × 106ND28%NDND35.8NS215
 80I–IV (n = n = 15, M1=27)10 ml ICFicoll+ IMBpan-CK (A45-B/B3)1–2 × 107ND23%NDND12.0OS (p = 0.030)171
 351***I–III5 ml ICFicollCK18 (CK2)1 × 1061–34 (mean:2)32%NDtumor size48.0OS (p = 0.047) in stage II–III172
 11 NSCLC, 7 SCLCI–IINDLymphopreppan-CK (CAM 5.2, MAK-6)0.5–1 × 105NDNSCLC: 0%, SCLC: 71%0/5, 0%NDNDNS216
 96I–IV (N0 n = 53, M1, n = 10)10 ml ICFicollpan-CK (AE1/AE3,MNF 116), EpCAM (Ber-Ep4)6 × 106ND22%1/32 (3.1%)NS50.5NS217
 196I–IV (M1, n = 91)10–20 ml ICLymphoprep+ IMBEpCAM (MOC31)2 × 1072–204 (mean:4)55%NDNS8NS173
RT-PCR
 33T1–4 N0M01 mlnoneMAGE-A1-12NDND33%0/30 (0%)NDNDND63
 39 NSCLC, 32 SCLCI–IV2–3 mlFicollGRPNDNDNSCLC:0% SCLC: 18%0/28 (0%)NDNDND174
 50I–III, M0, R05 ml ICFicollMAGE-A1-12NDND52%0/30 (0%)grade 1 and 2 tumors92RR (p < 0.03) for N0 patients79
Table IV. Detection of DTC in Prostate Cancer
No. of patientsStageBM aspirateEnrichment procedureDetection markers (antibody)No. of cellsanalyzedRange ofpositive cellsDetection rate cases %Detection rate controls (%)Correlationto clinical/pathol. variablesMean follow-up (months)Prognostic/predictive valueReference
  1. IC, Iliac crest; S, significant; NS, not significant; ND, not determined; DFS, disease-free survival; DM, distant metastasis; OS, overall survival; RR, recurrance rate; RSF, recurrence-free survival; PSA, prostate specific antigen; GS, Gleason score; PE, prostatectomy; RT, radiotherapy; BS, bone scan; AD, androgen deprivation.

Immunocytochemistry
 84N0M06 ml ICFicollCK18 (CK2)8 × 105NA, mean 636%0/12 (0%)NANDLocal tumor extent, DM and differentiation61
 42NA0.5–10 ml ICFicollCK18 (CK2), pan-CK (A45-B/B3)2 × 106NACK2: 31%, A45-B/B3: 45.2%CK2: 4/75 (5.3%) A45-B/B3: 4/75 (5.3%)NANDND148
 44T3–4 N0M06–10 ml ICFicollCK18 (CK2)2 × 1061–38 (mean 6, median 2)55%NDNSNDND175
 36T3–4 M0 (34 N0, 2 N1, 2)3–10 ml ICFicollCK18 (CK2), pan-CK (A45-B/B3)2 × 1061–38 prior to therapy; 1–26 after therapy58% prior totherapy, 19 % after therapyNDNSNDND183
 48pT2–pT35 ml ICFicollpan-CK (CAM 5.2 + AE1/AE3 +NCL5D3)2.5 × 106NA10%0/20 (0%)NANDND218
 53T1–46–10 ml ICFicollCK18 (CK2)1.7 × 106NA38%NDNANDND62
 45T1–3 pN0M06–8 ml ICFicollCK18 (CK2)NANA20%NDNSMedian 25NS177
 154pT1–2 pN06–8 ml ICFicollCK18 (CK2)1–2 × 1061–5125.3%1/9 (11%)NAMedian 32NS141
 82N0–N16–8 ml ICFicollCK18 (CK2),pan-CK (A45-B/B3)1 × 106CK2: 1–21, A45: 1–128CK2: 20/82CK2 (24%), A45-B/B3 (40%)NDNSMedian 48Earlier biochemical relapse, p ≤0.004 for A45/B-B3180
 30T1–T3 N0M0ICFicollpan-CK (A45-B/B3)1 × 106NA30%NDNANDND63
 20pT2 pN0M0R0ICFicollpan-CK (A45-B/B3)NANA40%NDNANDND219
 220NA10 ml ICFicollEpCAM (BerEP4)NANA90% before PE, 69% 4 months after PE, 79% 5 years after PE1/48 (2%)NSNDND64
 55NA5–10 ml ICFicollpan-CK (A45-B/B3)2 × 106NA22%NDNANDND41
 266cT1–T4 pN0M020–30 ml ICLymphopreppan-CK (AE1/AE3)2 × 1061–1118–20%NDGS (p = 0.04), Gleason pattern (p = 0.04)Median 84Shorter RR (p = 0.03)in patients treated with RT, but no AD181
 69NAICAccu-Prep, MACSEpCAM (BerEP4)NANA71%NDNANDNS77
 292NA10 ml ICMACSEpCAM (BerEP4)NANA35–92%3/27 (11%)NS12NS65
 193cT1–3 pN0M05–8 ml ICFicollpan-CK (A45-B/B3)5 × 1061–50(median 2)45%NDNSMedian 16Shorter PSA-RSF(p = 0.003), predictive for PSA relapse (p = 0.014)179
RT-PCR
 35T1–35 ml ICLymphoprepPSANDND27% (7/26 BS-), 56% (5/9 BS+)NDMetastatic bone disease (incl. micrometastasis) to serum PSA, BS- to serum PSA (p < 0.01)NDND220
 71pT1–3 M05 ml ICFicollPSANDND62%0/30 (0%)NSNDND218
 86cT1–23–5 ml ICFicollPSA1 × 107ND45%NDSerum PSA (p = 0.001), tumor stage (p = 0.003)Range: 1–43DSF (p = 0.004)176
 53ND6–10 ml ICFicollPSA3.2 × 106 28%0/53 (0%)NSNDND62
 116T1–28 ml ICFicollPSANDND44–66%NDNSNDRR182
 244cT1–25–10 ml ICFicollPSANDND32%NDNANDND90
 30T1–3 N0M01 mlnoneMAGE-A1-12, PSANDND60% (at least one MAGE-A-mRNA) 27% (PSA)0/30 (0%) (MAGE-A)High risk ofmetastatic relapse (p = 0.02)NDND63
 161pT1–41 ml ICnoneMAGE-A1-12NDND16%0/13 (0)%NSNDND78
 292ND10 ml ICMACSPSANDND43–83%3.7%NSNDNS65

Discrimination between viable and apoptotic DTC is facilitated by a new technique, designated EPISPOT (epithelial immunospot). This method offers the advantage to detect viable DTC by their ability to secrete individual proteins after short term culture (48 hr). In a recently published study, Muc-1 and/or CK19 secreting DTC could be demonstrated in BM samples of breast cancer patients with (90%) and without (50%) overt metastases but not from healthy controls.25 This technique has been also applied to CTC detection and its clinical validation is ongoing.

A series of new techniques has been developed and applied for CTC detection in the peripheral blood but not for BM analysis yet. Further progress toward a standardized method for CTC detection was reached through the introduction of the CellSearch system (Veridex, Warren, NJ), an automated enrichment and immunostaining device that has been cleared by the U.S. Food and Drug Administration for the detection of CTC in the peripheral blood of patients with metastatic breast, colon and prostate cancer.26–33 Recently, Nagrath et al.34 presented a microfluid platform called the “CTC-chip,” which consists of an array of anti-EpCAM antibody-coated microposts capable to capture CTC from unfractionated blood under precisely controlled laminar flow conditions.34 With this technique in more than 95% of cancer patients CTC were detected. This high incidence of CTC and the high number of CTC especially in patients without overt metastases requires further investigations on the specificity of this assay. A similar micropost-based method has been used commercially with rather limited success over several years by another group for the detection of fetal erythroblasts in the peripheral blood of pregnant women, another rare event application [www.bioconcept.com]. Furthermore, ultraspeed automated digital microscopy fiber-optic array scanning technology (FAST) and laser printing techniques have been developed to scan 300,000 cells per second, thereby detecting cells decorated by fluorescence dye-conjugated antibodies directly on a slide. However, additional time has to be assessed for artifact rejection and visual verification of the results.35–37 CTC counts 2–3 log units higher than other groups reported were detected by Pachmann et al.38, 39 in nearly 100% of breast cancer patients using the MAINTRAC assay. These discrepancies to the results obtained with other assays have raised discussions about the specificity of this approach using only anti-EpCAM antibodies for positive selection of tumor cells.38

Molecular detection of DTC

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Molecular detection of DTC is based on PCR amplification of either DNA or cDNA (mRNA). A drawback of PCR-based methods is that the tumor cells of interest cannot be morphologically identified and isolated for further analyses.

DNA-based methods rely on the detection of known mutations, amplifications or methylation patterns in the tumor cells. Although numerous genetic alterations have been described, because of the tremendous heterogeneity of genetic alterations in tumor cells, currently no universally applicable DNA marker exists for DTC screening.1, 10, 11 Another possible confounder is the fact that DNA molecules are relatively stable, and therefore the detection of tumor specific genomic aberrations may not necessarily indicate the presence of intact DTC but also DNA fragments derived from necrotic or apoptotic tumor cells.40–42

Therefore, measurements of epithelium-specific or more organ-specific mRNA species such as CK19 and 20 or mammaglobin or PSA mRNA by RT (reverse transcriptase)-PCR have been proven as more promising to detect DTC in BM samples.43–50 The main advantage of very sensitive RT-based techniques for the detection of DTC at the single-cell level is the nearly “unlimited” availability of primers for almost every gene of interest. However, one of the major problems of RT-PCR approaches is low RNA stability. Therefore, storage and sample preparation have to be performed under conditions avoiding RNA degradation.50

RT-PCR based methods have been shown to be technically even more challenging than immunocytochemical assays with respect to specificity. The main drawback of using surrogate tissue-specific markers are false-positive results due to illegitimate low-level of epithelial- or tissue-related transcription in normal cells.51–53 Moreover, heterogeneity in the expression levels of a particular target transcript cannot be predicted. To avoid false-negative findings due to downregulation of the expression of a single gene, current RT-PCR analyses are therefore frequently performed as multimarker assays. Furthermore, quantitative real-time RT-PCR is helpful to decrease false positive findings by exclusion of very low DNA amounts deriving from illegitimate expression of noncancerous cells.45, 54–56 Marker genes currently used in RT-PCR approaches to detect DTC in BM from cancer patients are listed in several recent reviews.1, 8, 10

Specific biological characteristics of DTC

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Biological characterization of cells categorized as DTC is directed to (1) confirm their malignant origin, (2) identify diagnostically and therapeutically relevant properties, and (3) increase the current knowledge about the biology of tumor cell dissemination in cancer patients. Since there is no gold standard thus far, different methods have been applied to assess the malignant potential of DTC. In addition to the technical challenges of single cell research, these studies are all hampered by the fact that these cells can exhibit features distinct from their corresponding primary tumors.

Previous reports indicated that DTC might be genomically heterogeneous.57 DTC can disseminate in a less-progressed genomic state and might aquire genomic alterations typical for fully metastatic cells later.58 Although surprisingly characterized by normal karyotypes, the malignant origin of a DTC population isolated from BM of breast cancer patients was demonstrated by other genetic alterations, such as allelic losses, and gene amplifications.59 While DTC can loose their ability to produce PSA, a significant percentage of DTC in prostate cancer patients still expressed PSA.60–65 LOH (loss of heterozygosity) pattern of PSA-positive CTC isolated from blood of prostate cancer patients indicated that CTC derive from distinct foci within the primary tumors.66 Moreover, K-ras mutations although not necessarily identical to those found in the corresponding primary tumors were reported in DTC derived from colon cancer.67, 68

DTC are heterogeneous with regard to the expression of growth factor receptors, adhesion molecules, proteases and their inducer and receptors, major histocompatibility complex antigens, signaling kinases, melanoma associated antigens (MAGE) or telomerase activity.1, 2, 63, 69–79 Of particular importance is the epidermal growth factor receptor HER2, the expression of which in primary tumors is essential for Trastuzumab treatment decisions of breast cancer patients.80, 81 HER2 overexpression on DTC in BM was predictive for a poor clinical outcome of stage I–III breast cancer patients.82 In the study of Vincent-Salomon et al.,83 both negative and positive HER2 status remained, in the majority of the cases, stable between DTC and the corresponding primary tumors. However, there is also evidence for discrepancies between the HER2 status in primary tumors and DTC in BM82, 84, 85 and CTC in blood.86, 87 Although HER2 expression was heterogeneous in DTC from individual patients, HER2-positive DTC might identify additional patients who can benefit from Trastuzumab therapy.85 Further studies are ongoing to verify whether HER2-positive DTC have predictive value for an improved response of the patients to Trastuzumab treatment.

Negativity for Ki-67 staining pointed to the hypothesis that the vast majority of DTC persists in a nonproliferating dormant state,84, 88, 89 which might also be responsible for their partial resistance to adjuvant chemotherapy in high risk breast cancer patients.14 In contrast using MIB immunostaining, Bianco et al. found in 36% of DTC-positive prostate cancer patients proliferating DTC the presence of which revealed to be an independent predictor of disease recurrence.90 Disturbance of dormancy and entry into the dynamic phase of metastasis formation might be caused by both genetic and epigenetic changes in DTC as well as in the surrounding microenvironment or premetastatic niche.91–93 However, conditions and timing of outgrowth of DTC are largely unknown thus far.1, 2, 11

There is first evidence for a molecular signature of primary tumors spreading early into BM94, 95; however, information about global gene expression of DTC is still limited. For CTC isolated from blood of patients with metastatic colorectal, prostate and breast cancer, global gene expression profiles could be defined and a list of CTC-specific genes was obtained, which might be useful to distinguish normal donors from cancer patients.96 Furthermore, Smirnov et al.96 concluded that gene expression profiles for CTC may be used to differentiate among the 3 different metastatic cancers. Interestingly, TWIST1, a transcription factor that plays a pivotal role in metastasis by promoting epithelial-mesenchymal transition,97–100 was part of the gene expression signature identified in EpCAM-enriched cells from BM of breast cancer patients after chemotherapy.101 Not observed in EpCAM-enriched cells of BM from healthy volunteers, TWIST1 expression was associated with occurrence of distant metastasis and local progression, even in pretreatment BM samples.101

Whether DTC possess stem or progenitor cell features enabling them to both self-renew and differentiate is currently under debate.102–105 Among the various breast cancer stem cell markers, CD44 positivity and absent/weak expression of CD24 or expression of ALDH-1 seem to be characteristic for breast cancer “founder” cells with a high capacity to form tumors in immunosuppressed mice.106–111 Recently, by gene expression analysis of CD44+/CD24−/low cells separated from breast cancer tissue, a 186 gene “invasiveness gene signature” was published, which was significantly associated with a reduced overall and metastasis-free survival of the breast cancer patients analyzed.112 Furthermore, different developmental pathways such as Notch, Wnt and hedgehog have been described to play pivotal roles in regulating cancer stem cell features.113–116 Thus, several sets of genes and proteins have been identified which might be helpful for the further characterization of DTC regarding their potential stem cell-like properties. Hints for stem cell features of DTC in BM were provided by Balic et al. and Alix-Panabieres et al. who demonstrated a significant number of DTC from BM of breast cancer patients with either CD44+/CD24−/low or CK19+/Muc-1 stem cell-like phenotypes.25, 117 The nonproliferating state of DTC that renders them resistant to systemic chemotherapy and the long term persistence of these cells in BM of cancer patients is also consistent with their putative stem cell phenotype.14, 16, 118

Clinical Relevance of DTC

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Several studies have shown significant correlations between the presence of DTC in BM and metastatic relapse in various tumor types, suggesting that the founder cells of overt metastases might be among those DTC. Here we will briefly review the current state of knowledge, focusing on the main types of solid tumors (i.e., breast, colorectal, nonsmall lung, and prostate carcinomas). Additional studies have been performed in patients with other epithelial tumor entities, such as gastric cancer,119 esophageal cancer,120, 121 pancreatic cancer,122, 123 ovarian cancer and head and neck carcinomas.124–131

In colorectal cancer, approximately 50% of patients undergoing a curative resection (R0) die from metastatic disease within 5 years. Even among lymph node negative (N0) patients, the relapse rate is 30%.132, 133 In lung cancer, the prognosis is even worse, with 60% of R0 and 40% of N0 patients dying of the disease.134 Whereas in breast and prostate cancer the overall survival is today relatively high (80–90%, 5 year; 70–80%, 10 year), a considerable fraction of nodal negative patients still relapse (25–30% and 15–50%) and this can often take place many years (>10 years) after the removal of the primary tumor.135–137

Breast cancer

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Numerous studies have been published investigating the presence and clinical relevance of DTC in BM of breast cancer patients.1, 15, 138, 139 The size of the patient cohorts analyzed and the applied detection methods vary considerably between these studies (Table I). The most commonly used method for DTC detection in breast cancer is immunocytochemistry-based analysis using antibodies against either cytokeratins or mucins (e.g., MUC-1). In most studies detecting these antigens, the prevalence of DTC in early-stage disease ranges between 13 and 50% (Table I), whereas in patients with metastatic breast cancer the rate increases up to 70%.138 RT-PCR-based methods have mostly used CK19, CEA or mammaglobin as markers. The detection rates between these studies varied between 12 and 50% (Table I) and raising up to 80% in metastatic breast cancer patients.138, 139

Depending on the method used and the number of patients analyzed, the different studies have come to somewhat different conclusions regarding the clinical relevance of DTC (Table I). However, Braun et al.15 published a meta-analysis consisting of 4,703 breast cancer patients. In this pooled analysis, the presence of DTC in BM was not only predictive of the development of skeletal metastases but was also predictive for the development of metastases in other organs.15

Besides their presence at primary diagnosis and surgery, DTC have been described to survive chemotherapy and hormonal therapy,14, 88, 140 and they can persist in BM over many years post-surgery. This persistence is also linked to an increased risk of late metastatic relapse.16, 54, 118, 141, 142 For example, in high-risk breast cancer patients (>3 involved axillary lymph nodes or extensive invasion of cutaneous lymph vessels), the presence of tumor cells after therapy was associated with an extremely poor prognosis.14 A European pooled analysis involving 696 breast cancer patients confirmed these findings. Here, 16% of breast cancer patients had tumor cell persistence in BM, and their presence was an independent prognostic factor for subsequent reduced breast cancer survival.143

Colorectal cancer

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Most studies describing immunocytochemistry for the detection of DTC in colorectal cancer (CRC) patients have either used the monoclonal antibody CK2 against CK18 or the pan-cytokeratin antibody A45-B/B3 (Table II), and a few studies have used a cocktail of several (up to 5 different) antibodies against different epithelial antigens including cytokeratins.144–147 The DTC detection rate in studies using CK2 was between 16 and 32%, whereas the detection rate was clearly higher in studies using the A45-B/B3 antibody (24–55%). This difference might be due to a potential downregulation or loss of CK18 expression on DTC.18, 148 Both antibodies rarely detect CK-positive cells in BM of noncancer control patients (0–5.5%). In studies using a cocktail of different antibodies, the DTC detection rate in cancer patients has varied between 29 and 74%.

The largest study so far conducted has been performed by Flatmark et al.149 including 275 patients and 206 noncancer control patients. In this study, an immunomagnetic bead enrichment system capturing DTC by the antibody MOC31 directed against EpCAM was applied. More BM cells could be analyzed (2 × 107) than in other studies and 17% of the patients showed DTC, whereas only 1.5% of controls were positive. The BM status did not correlate significantly with disease stage or other clinical parameters. Unfortunately, no clinical follow-up was available, and the clinical significance of the findings in cancer patients remains therefore unclear.

So far 5 publications have reported a positive association between DTC in BM and an increased recurrence rate150, 151 or a reduced overall survival.145, 152 In contrast, 3 other reports, mostly on smaller patient cohorts, could not detect any association between prognostic factors and the presence of DTC.146, 147, 153

To date, CK20 is the most commonly used marker in RT-PCR analyses of BM from CRC patients (Table II), with more than 10 studies published so far. However, only in 3 studies the number of investigated patients was larger than 100 with DTC detection rates of 11–35%.154–156 The detection rate of CK20 in healthy controls ranged between 0 and 10% except for 1 study with 45% positivity.156 Only very few studies have investigated the correlation between CK20 transcripts in BM and prognosis. Two groups reported no association to survival,157, 158 whereas 4 groups found an association between the presence of CK20 transcripts and worse overall survival.154, 159–161 However, both negative reports were performed on metastatic patients only.

CEA (carcinoembryonic antigen) transcripts were also searched for in 1 small study involving only 10 patients with 66% of whom had detectable CEA transcripts, whereas none of the 56 healthy controls had CEA-positive cells in their BM.162 No follow-up information was available for these patients. In 1 other study, both CK20 and GCC (guanylylcyclase C) were investigated simultaneously.156 While CK20 transcripts could be detected in 11% of CRC cases, GCC mRNA was only detected in 6% of the patients. No GCC expression was found in healthy controls. Interestingly, only 1/109 patients showed simultaneously GCC and CK20 transcripts. None of these transcripts correlated with clinical parameters such as stage or grade and again no survival data were reported.

In conclusion, several studies have been conducted to elucidate the clinical relevance of DTC in CRC. These studies, however, present a very heterogeneous picture, with different patient groups, sample sizes, follow-up times, staining methods, and target antigens, all of which probably have contributed to the observed variation in DTC detection rates and association to clinical parameters (Table II).

Lung cancer

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Table III shows the 13 ICC studies investigating the prognostic relevance of DTC in BM of patients with nonsmall cell lung carcinomas (NSCLC). In 7 studies, the monoclonal antibody CK2 against CK18 was used, whereas 5 studies have exploited different pan-cytokeratin antibodies. The rate of CK-positive cells in the different studies ranged between 22 and 60%. While the frequency of positive patients does not seem to correlate with the antibody used, the sampling number and also the anatomic site of BM aspirations might influence the number of positive cases. A higher frequency of DTC was not only found in BM aspirations from the rib or sternum in lung cancer patients163–165 but also among breast and oesophageal cancer patients when compared with BM samples taken from the iliac crest.166, 167

Irrespective of the localization of the BM puncture, 8 studies have shown a correlation between DTC in BM and worse clinical outcome.163–165, 168–172 However, the largest study conducted so far on 196 patients could not find an association, which can, however, be explained by a very short follow up time (median, 8 months).173

Interestingly, very few studies have been performed using RT-PCR to detect DTC in BM. Sienel et al. detected MAGE-A transcripts in 50 NSCLC cases free of overt distant metastases.79 Twenty-two percent of the patients harbored MAGE-A transcripts and such transcripts were undetectable in healthy controls. Among lymph node-negative patients, the presence of MAGE-A was associated with poor prognosis and revealed to be an independent risk factor in a multivariate analysis. In another smaller study, blood and BM of both SCLC (small cell lung cancer) and NSCLC cases were investigated for the presence of gastrin-releasing peptide (GRP). While no transcripts could be detected among NSCLC cases, in 50% of the blood and 18% of BM samples from SCLC patients GRP mRNA was detected.174

In conclusion, around half of the published studies on the prognostic role of DTC in BM in nonsmall cell lung cancer have found an association between the presence of DTC and worse overall survival or recurrence rate. However, most studies conducted so far have only investigated a small number of patients, and thus larger standardized studies are needed to confirm the prognostic value in lung cancer.

Prostate cancer

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

Currently, prostate cancer biology is only incompletely reflected by the commonly available diagnostic/prognostic parameters. Consequently, additional diagnostic parameters are urgently needed. BM is the most prominent metastatic site in prostate cancer and several research groups have focused on the detection of DTC in this organ over the past 10 years (Table IV). However, most of the studies included a relatively small number of patients and/or follow up information, and data about the influence of hormonal treatment on prognosis are sparse or lacking.

Immunocytochemistry using anti-cytokeratin and anti-PSA antibodies was the most frequently conducted approach (Table IV).175–179 DTC detection rates ranged between 10 and 90% (Table IV) and a comparison between the results obtained with the CK2 (anti-CK18) and the pan-CK antibody A45-B/B3 revealed a downregulation of CK18 of DTC in a significant number of cases.148, 180

There is evidence for a correlation of DTC to clinically established risk factors. Spread of DTC seems to be linked to histological differentiation of the primary tumor.61, 181 Moreover, while in some studies DTC detection was not associated with initial PSA concentration in the serum of prostate cancer patients,177, 179 a correlation of DTC measurement to early PSA recurrence was shown by 2 studies.179, 180 Weckermann et al.180 analyzed 82 patients and observed that the mean time to PSA increase was shorter in patients with DTC-positive compared with DTC-negative BM samples. As shown by Bianco et al.,90 the presence of MIB-1-positive proliferating DTC in bone marrow was associated with increased postoperative Gleason score.

Although a DTC-positive BM status was associated with grading and increased risk of metastasis, the study by Berg et al. (2007)181 on 266 patients did not find a correlation of DTC detection and survival. Most recently, Köllermann et al179 reported on the prognostic relevance of DTC in BM found in 86/193 (44.6%) patients with clinically localized prostate cancer submitted to neoadjuvant hormonal therapy followed by radical prostatectomy and a median follow-up of 44 months. This study is the first one on a larger series of prostate cancer patients with sufficiently long follow-up, which clearly demonstrates an independent adverse prognostic effect of the presence of DTC at the time of initial diagnosis. The presence of DTC in BM of prostate cancer patients found by A45-B/B3 ICC before neoadjuvant hormonal therapy and subsequent surgery represented even an independent prognostic parameter, suggesting that DTC may contribute to the failure of current neoadjuvant hormonal therapy regimens. Addition of information about DTC detection in BM to the conventional diagnostic parameters might lead to more precise reflection of the biological potential of an individual prostate carcinoma.179

There are also studies that used RT-PCR for DTC detection, mostly amplifying PSA- or MAGE-specific cDNAs as markers with detection rates ranging between 16 and 83% (Table IV). The BM status in these studies frequently correlated to PSA serum levels; however, there are only sparse data about the prognostic relevance or potential predictive values of these findings. Results from BM analysis presented by Wood and Banerjee176 demonstrated a correlation between RT-PCR-PSA positivity and a significantly shorter disease-free survival. Furthermore, RT-PCR status correlated significantly with serum PSA and pathological stage.176 In the study by Gao et al.,182 no association between BM RT-PCR PSA positivity and grading or serum markers was observed, but PSA mRNA detection correlated significantly with early disease recurrence.

In conclusion, there is some evidence that the detection of DTC in the BM of prostate cancer patients might represent a prognostic parameter. DTC appear to survive hormonal therapy183, 184 and this might be one explanation for the well-known failure of neoadjuvant hormonal therapy. However, improved characterization of DTC and larger multicenter studies followed by nomogram testing against the established risk parameters are required to introduce DTC detection into the future clinical management of prostate cancer patients.

Conclusion and future directions

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References

The development of rare cell detection techniques allows now the reliable detection of DTC in BM of cancer patients years before the occurrence of distant overt metastases signals incurability. This information may be used to assess the individual prognosis of cancer patients and stratify the patients at risk to systemic adjuvant cancer therapies aimed to prevent metastatic relapse. However, for future implementation of DTC detection into clinical practice, it is essential to establish standardized methods with a high degree of reproducibility. An important step into this direction has been thus far made for the immunocytochemical detection of DTC using anticytokeratin antibodies.17, 20–22

Currently, most data on the prognostic value of DTC are available for breast cancer. In other tumor entities, the database is not as solid and the results are more controversial as in breast cancer, but several single institution studies including patients with colon, lung and prostate cancer have documented a link between the presence of DTC at primary surgery and subsequent metastatic relapse. Multicenter analyses are now required to confirm the prognostic value in these tumor entities.

DTC in BM have been detected in all solid tumor types suggesting that the BM might be a preferred reservoir for blood-borne DTC. The BM environment may allow these cells to persist over many years and to disseminate into other organs. Whether DTC use for this purpose the same niches as haematopoetic stem cells185 is subject of current investigations. However, it cannot be excluded that BM is simply a convenient indicator organ and that early DTC are also present in other organs such as lung or liver, which are less easily accessible than BM. Rapid autopsy programs have the potential to address this important question.

Several studies found DTC in BM even many years after surgery and adjuvant therapy, and the presence of these DTC was associated with an increased risk for metastatic relapse.16, 54, 118, 120, 141, 142 These results demonstrate that DTC can reside in a prolonged latent state referred to as dormancy before they grow out into overt skeleton metastases. To understand this peculiar stage of dormancy together with the identification of the founder cells of overt metastases (“metastatic stem cells”) are some of the most important and challenging areas of basic research on the biology of DTC. In this context, the development of appropriate mouse models, which mimic the stage of minimal residual cancer, are of utmost importance to perform functional studies for validation of the descriptive findings observed in cancer patients.

Sequential peripheral blood drawings in particular for real-time monitoring of minimal residual disease in cancer patients undergoing systemic therapies (e.g., chemotherapy, hormonal therapy or antibody therapy) should be more acceptable than repeated BM aspirations. To date, it is not clear if CTC measurements could replace the examination of BM. To answer this question, many research groups are currently assessing CTC in clinical studies with encouraging results (for review, see Ref. 9). Implementation of CTC/DTC measurements into clinical trials has the potential to obtain an important future biomarker for real-time monitoring of the efficacy of systemic adjuvant therapy in individual patients.

References

  1. Top of page
  2. Abstract
  3. Detection of DTC
  4. Immunological detection of DTC
  5. Molecular detection of DTC
  6. Specific biological characteristics of DTC
  7. Clinical Relevance of DTC
  8. Breast cancer
  9. Colorectal cancer
  10. Lung cancer
  11. Prostate cancer
  12. Conclusion and future directions
  13. References
  • 1
    Pantel K,Brakenhoff RH,Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat Rev Cancer 2008; 8: 32940.
  • 2
    Pantel K,Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004; 4: 44856.
  • 3
    Jung R,Petersen K,Kruger W,Wolf M,Wagener C,Zander A,Neumaier M. Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes. Br J Cancer 1999; 81: 8703.
  • 4
    Kruger WH,Jung R,Detlefsen B,Mumme S,Badbaran A,Brandner J,Renges H,Kroger N,Zander AR. Interference of cytokeratin-20 and mammaglobin-reverse-transcriptase polymerase chain assays designed for the detection of disseminated cancer cells. Med Oncol 2001; 18: 338.
  • 5
    Vona G,Sabile A,Louha M,Sitruk V,Romana S,Schutze K,Capron F,Franco D,Pazzagli M,Vekemans M,Lacour B,Brechot C, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulatingtumor cells. Am J Pathol 2000; 156: 5763.
  • 6
    Pinzani P,Salvadori B,Simi L,Bianchi S,Distante V,Cataliotti L,Pazzagli M,Orlando C. Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum Pathol 2006; 37: 7118.
  • 7
    Zheng S,Lin H,Liu JQ,Balic M,Datar R,Cote RJ,Tai YC. Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. J Chromatogr A 2007; 1162: 15461.
  • 8
    Zach O,Lutz D. Tumor cell detection in peripheral blood and bone marrow. Curr Opin Oncol 2006; 18: 4856.
  • 9
    Alix-Panabieres C,Riethdorf S,Pantel K. Circulating tumor cells in bone marrow micrometastasis. Clin Cancer Res, in press.
  • 10
    Lacroix M. Significance, detection and markers of disseminated breast cancer cells. Endocr Relat Cancer 2006; 13: 103367.
  • 11
    Alix-Panabieres C,Muller V,Pantel K. Current status in human breast cancer micrometastasis. Curr Opin Oncol 2007; 19: 55863.
  • 12
    Ciampa A,Fanger G,Khan A,Rock KL,Xu B. Mammaglobin and CRxA-01 in pleural effusion cytology: potential utility of distinguishing metastatic breast carcinomas from other cytokeratin 7-positive/cytokeratin 20-negative carcinomas. Cancer 2004; 102: 36872.
  • 13
    Ikeda S,Fujimori M,Shibata S,Okajima M,Ishizaki Y,Kurihara T,Miyata Y,Iseki M,Shimizu Y,Tokumoto N,Ozaki S,Asahara T. Combined immunohistochemistry of beta-catenin, cytokeratin 7, and cytokeratin 20 is useful in discriminating primary lung adenocarcinomas from metastatic colorectal cancer. BMC Cancer 2006; 6: 31.
  • 14
    Braun S,Kentenich C,Janni W,Hepp F,de Waal J,Willgeroth F,Sommer H,Pantel K. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000; 18: 806.
  • 15
    Braun S,Vogl FD,Naume B,Janni W,Osborne MP,Coombes RC,Schlimok G,Diel IJ,Gerber B,Gebauer G,Pierga JY,Marth C, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005; 353: 793802.
  • 16
    Janni W,Rack B,Schindlbeck C,Strobl B,Rjosk D,Braun S,Sommer H,Pantel K,Gerber B,Friese K. The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 2005; 103: 88491.
  • 17
    Fehm T,Braun S,Muller V,Janni W,Gebauer G,Marth C,Schindlbeck C,Wallwiener D,Borgen E,Naume B,Pantel K,Solomayer E. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 2006; 107: 88592.
  • 18
    Woelfle U,Sauter G,Santjer S,Brakenhoff R,Pantel K. Down-regulated expression of cytokeratin 18 promotes progression of human breast cancer. Clin Cancer Res 2004; 10: 26704.
  • 19
    Willipinski-Stapelfeldt B,Riethdorf S,Assmann V,Woelfle U,Rau T,Sauter G,Heukeshoven J,Pantel K. Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in human micrometastatic and primary breast carcinoma cells. Clin Cancer Res 2005; 11: 800614.
  • 20
    Borgen E,Beiske K,Trachsel S,Nesland JM,Kvalheim G,Herstad TK,Schlichting E,Qvist H,Naume B. Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998; 185: 42734.
  • 21
    Borgen E,Naume B,Nesland JM,Nowels KW,Pavlak N,Ravkin I,Goldbard S. Use of automated microscopy for the detection of disseminated tumor cells in bone marrow samples. Cytometry 2001; 46: 21521.
  • 22
    Borgen E,Pantel K,Schlimok G,Muller P,Otte M,Renolen A,Ehnle S,Coith C,Nesland JM,Naume B. A European interlaboratory testing of three well-known procedures for immunocytochemical detection of epithelial cells in bone marrow. Results from analysis of normal bone marrow. Cytometry B Clin Cytom 2006; 70: 4009.
  • 23
    Cohen SJ,Alpaugh RK,Gross S,O'Hara SM,Smirnov DA,Terstappen LW,Allard WJ,Bilbee M,Cheng JD,Hoffman JP,Lewis NL,Pellegrino A, et al. Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer. Clin Colorectal Cancer 2006; 6: 12532.
  • 24
    Garcia JA,Rosenberg JE,Weinberg V,Scott J,Frohlich M,Park JW,Small EJ. Evaluation and significance of circulating epithelial cells in patients with hormone-refractory prostate cancer. BJ U Int 2007; 99: 51924.
  • 25
    Alix-Panabieres C,Vendrell JP,Pelle O,Rebillard X,Riethdorf S,Muller V,Fabbro M,Pantel K. Detection and characterization of putative metastatic precursor cells in cancer patients. Clin Chem 2007; 53: 5379.
  • 26
    Cristofanilli M,Budd GT,Ellis MJ,Stopeck A,Matera J,Miller MC,Reuben JM,Doyle GV,Allard WJ,Terstappen LW,Hayes DF. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004; 351: 78191.
  • 27
    Allard WJ,Matera J,Miller MC,Repollet M,Connelly MC,Rao C,Tibbe AG,Uhr JW,Terstappen LW. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 2004; 10: 6897904.
  • 28
    Cristofanilli M,Hayes DF,Budd GT,Ellis MJ,Stopeck A,Reuben JM,Doyle GV,Matera J,Allard WJ,Miller MC,Fritsche HA,Hortobagyi GN, et al. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005; 23: 142030.
  • 29
    Hayes DF,Cristofanilli M,Budd GT,Ellis MJ,Stopeck A,Miller MC,Matera J,Allard WJ,Doyle GV,Terstappen LW. Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res. 2006; 12: 421824.
  • 30
    Danila DC,Heller G,Gignac GA,Gonzalez-Espinoza R,Anand A,Tanaka E,Lilja H,Schwartz L,Larson S,Fleisher M,Scher HI. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin Cancer Res 2007; 13: 70538.
  • 31
    Riethdorf S,Fritsche H,Muller V,Rau T,Schindlbeck C,Rack B,Janni W,Coith C,Beck K,Janicke F,Jackson S,Gornet T, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cellsearch system. Clin Cancer Res 2007; 13: 9208.
  • 32
    Shaffer DR,Leversha MA,Danila DC,Lin O,Gonzalez-Espinoza R,Gu B,Anand A,Smith K,Maslak P,Doyle GV,Terstappen LW,Lilja H, et al. Circulating tumor cell analysis in patients with progressive castration-resistant prostate cancer. Clin Cancer Res 2007; 13: 20239.
  • 33
    Sastre J,Maestro ML,Puente J,Veganzones S,Alfonso R,Rafael S,Garcia-Saenz JA,Vidaurreta M,Martin M,Arroyo M,Sanz-Casla MT,Diaz-Rubio E. Circulating tumor cells in colorectal cancer: correlation with clinical and pathological variables. Ann Oncol 2008; 19: 9358.
  • 34
    Nagrath S,Sequist LV,Maheswaran S,Bell DW,Irimia D,Ulkus L,Smith MR,Kwak EL,Digumarthy S,Muzikansky A,Ryan P,Balis UJ, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007; 450: 12359.
  • 35
    Krivacic RT,Ladanyi A,Curry DN,Hsieh HB,Kuhn P,Bergsrud DE,Kepros JF,Barbera T,Ho MY,Chen LB,Lerner RA,Bruce RH. A rare-cell detector for cancer. Proc Natl Acad Sci USA 2004; 101: 105014.
  • 36
    Curry DN,Krivacic RT,Hsieh HB,Ladanyi A,Bergsrud DE,Ho MY,Chen LB,Kuhn P,Bruce RH. High-speed detection of occult tumor cells in peripheral blood. Conf Proc IEEE Eng Med Biol Soc 2004; 2: 126770.
  • 37
    Hsieh HB,Marrinucci D,Bethel K,Curry DN,Humphrey M,Krivacic RT,Kroener J,Kroener L,Ladanyi A,Lazarus N,Kuhn P,Bruce RH, et al. High speed detection of circulating tumor cells. Biosens Bioelectron 2006; 21: 18939.
  • 38
    Pachmann K,Clement JH,Schneider CP,Willen B,Camara O,Pachmann U,Hoffken K. Standardized quantification of circulating peripheral tumor cells from lung and breast cancer. Clin Chem Lab Med 2005; 43: 61727.
  • 39
    Pachmann K,Dengler R,Lobodasch K,Frohlich F,Kroll T,Rengsberger M,Schubert R,Pachmann U. An increase in cell number at completion of therapy may develop as an indicator of early relapse: quantification of circulating epithelial tumor cells (CETC) for monitoring of adjuvant therapy in breast cancer. J Cancer Res Clin Oncol 2008; 134: 5965.
  • 40
    Schwarzenbach H,Muller V,Beeger C,Gottberg M,Stahmann N,Pantel K. A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer. Breast Cancer Res 2007; 9: R66.
  • 41
    Schwarzenbach H,Chun FK,Lange I,Carpenter S,Gottberg M,Erbersdobler A,Friedrich MG,Huland H,Pantel K. Detection of tumor-specific DNA in blood and bone marrow plasma from patients with prostate cancer. Int J Cancer 2007; 120: 146571.
  • 42
    Schwarzenbach H,Chun FK,Muller I,Seidel C,Urban K,Erbersdobler A,Huland H,Pantel K,Friedrich MG. Microsatellite analysis of allelic imbalance in tumour and blood from patients with prostate cancer. BJU Int 2008; 102: 2538.
  • 43
    Schoenfeld A,Kruger KH,Gomm J,Sinnett HD,Gazet JC,Sacks N,Bender HG,Luqmani Y,Coombes RC. The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 1997; 33: 85461.
  • 44
    Smith BM,Slade MJ,English J,Graham H,Luchtenborg M,Sinnett HD,Cross NC,Coombes RC. Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol 2000; 18: 14329.
  • 45
    Berois N,Varangot M,Aizen B,Estrugo R,Zarantonelli L,Fernandez P,Krygier G,Simonet F,Barrios E,Muse I,Osinaga E. Molecular detection of cancer cells in bone marrow and peripheral blood of patients with operable breast cancer. Comparison of CK19, MUC1 and CEA using RT-PCR. Eur J Cancer 2000; 36: 71723.
  • 46
    Zhong XY,Kaul S,Lin YS,Eichler A,Bastert G. Sensitive detection of micrometastases in bone marrow from patients with breast cancer using immunomagnetic isolation of tumor cells in combination with reverse transcriptase/polymerase chain reaction for cytokeratin-19. J Cancer Res Clin Oncol 2000; 126: 2128.
  • 47
    Zippelius A,Lutterbuse R,Riethmuller G,Pantel K. Analytical variables of reverse transcription-polymerase chain reaction-based detection of disseminated prostate cancer cells. Clin Cancer Res 2000; 6: 274150.
  • 48
    Bossolasco P,Ricci C,Farina G,Soligo D,Pedretti D,Scanni A,Deliliers GL. Detection of micrometastatic cells in breast cancer by RT-pCR for the mammaglobin gene. Cancer Detect Prev 2002; 26: 603.
  • 49
    Halabi S,Small EJ,Hayes DF,Vogelzang NJ,Kantoff PW. Prognostic significance of reverse transcriptase polymerase chain reaction for prostate-specific antigen in metastatic prostate cancer: a nested study within CALGB 9583. J Clin Oncol 2003; 21: 4905.
  • 50
    Becker S,Becker-Pergola G,Fehm T,Wallwiener D,Solomayer EF. Time is an important factor when processing samples for the detection of disseminated tumor cells in blood/bone marrow by reverse transcription-PCR. Clin Chem 2004; 50: 7856.
  • 51
    Ring A,Smith IE,Dowsett M. Circulating tumour cells in breast cancer. Lancet Oncol 2004; 5: 7988.
  • 52
    Balducci E,Azzarello G,Valori L,Toffolatti L,Bolgan L,Valenti MT,Bari M,Pappagallo GL,Ausoni S,Vinante O. A new nested primer pair improves the specificity of CK-19 mRNA detection by RT-PCR in occult breast cancer cells. Int J Biol Markers 2005; 20: 2833.
  • 53
    Ballestrero A,Garuti A,Bertolotto M,Rocco I,Boy D,Nencioni A,Ottonello L,Patrone F. Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer. Br J Cancer 2005; 92: 194852.
  • 54
    Slade MJ,Singh A,Smith BM,Tripuraneni G,Hall E,Peckitt C,Fox S,Graham H,Luchtenborg M,Sinnett HD,Cross NC,Coombes RC. Persistence of bone marrow micrometastases in patients receiving adjuvant therapy for breast cancer: results at 4 years. Int J Cancer 2005; 114: 94100.
  • 55
    Ring AE,Zabaglo L,Ormerod MG,Smith IE,Dowsett M. Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques. Br J Cancer 2005; 92: 90612.
  • 56
    Benoy IH,Elst H,Philips M,Wuyts H,Van Dam P,Scharpe S,Van Marck E,Vermeulen PB,Dirix LY. Prognostic significance of disseminated tumor cells as detected by quantitative real-time reverse-transcriptase polymerase chain reaction in patients with breast cancer. Clin Breast Cancer 2006; 7: 14652.
  • 57
    Klein CA,Blankenstein TJ,Schmidt-Kittler O,Petronio M,Polzer B,Stoecklein NH,Riethmuller G. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002; 360: 6839.
  • 58
    Schmidt-Kittler O,Ragg T,Daskalakis A,Granzow M,Ahr A,Blankenstein TJ,Kaufmann M,Diebold J,Arnholdt H,Muller P,Bischoff J,Harich D, et al. From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci USA. 2003; 100: 773742.
  • 59
    Schardt JA,Meyer M,Hartmann CH,Schubert F,Schmidt-Kittler O,Fuhrmann C,Polzer B,Petronio M,Eils R,Klein CA. Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer. Cancer Cell 2005; 8: 22739.
  • 60
    Riesenberg R,Oberneder R,Kriegmair M,Epp M,Bitzer U,Hofstetter A,Braun S,Riethmuller G,Pantel K. Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells. Histochemistry 1993; 99: 616.
  • 61
    Oberneder R,Riesenberg R,Kriegmair M,Bitzer U,Klammert R,Schneede P,Hofstetter A,Riethmuller G,Pantel K. Immunocytochemical detection and phenotypic characterization of micrometastatic tumour cells in bone marrow of patients with prostate cancer. Urol Res 1994; 22: 38.
  • 62
    Zippelius A,Kufer P,Honold G,Kollermann MW,Oberneder R,Schlimok G,Riethmuller G,Pantel K. Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997; 15: 27018.
  • 63
    Kufer P,Zippelius A,Lutterbuse R,Mecklenburg I,Enzmann T,Montag A,Weckermann D,Passlick B,Prang N,Reichardt P,Dugas M,Kollermann MW, et al. Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res 2002; 62: 25161.
  • 64
    Ellis WJ,Pfitzenmaier J,Colli J,Arfman E,Lange PH,Vessella RL. Detection and isolation of prostate cancer cells from peripheral blood and bone marrow. Urology 2003; 61: 27781.
  • 65
    Pfitzenmaier J,Ellis WJ,Hawley S,Arfman EW,Klein JR,Lange PH,Vessella RL. The detection and isolation of viable prostate-specific antigen positive epithelial cells by enrichment: a comparison to standard prostate-specific antigen reverse transcriptase polymerase chain reaction and its clinical relevance in prostate cancer. Urol Oncol 2007; 25: 21420.
  • 66
    Schmidt H,DeAngelis G,Eltze E,Gockel I,Semjonow A,Brandt B. Asynchronous growth of prostate cancer is reflected by circulating tumor cells delivered from distinct, even small foci, harboring loss of heterozygosity of the PTEN gene. Cancer Res 2006; 66: 895965.
  • 67
    Tortola S,Steinert R,Hantschick M,Peinado MA,Gastinger I,Stosiek P,Lippert H,Schlegel W,Reymond MA. Discordance between K-ras mutations in bone marrow micrometastases and the primary tumor in colorectal cancer. J Clin Oncol 2001; 19: 283743.
  • 68
    Conzelmann M,Linnemann U,Berger MR. Molecular detection of clinical colorectal cancer metastasis: how should multiple markers be put to use? Int J Colorectal Dis 2005; 20: 13746.
  • 69
    Pantel K,Schlimok G,Kutter D,Schaller G,Genz T,Wiebecke B,Backmann R,Funke I,Riethmuller G. Frequent down-regulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 1991; 51: 47125.
  • 70
    Klein CA,Seidl S,Petat-Dutter K,Offner S,Geigl JB,Schmidt-Kittler O,Wendler N,Passlick B,Huber RM,Schlimok G,Baeuerle PA,Riethmuller G. Combined transcriptome and genome analysis of single micrometastatic cells. Nat Biotechnol 2002; 20: 38792.
  • 71
    Werther K,Normark M,Brunner N,Nielsen HJ. Cytokeratin-positive cells in preoperative peripheral blood and bone marrow aspirates of patients with colorectal cancer. Scand J Clin Lab Invest 2002; 62: 4957.
  • 72
    Hemsen A,Riethdorf L,Brunner N,Berger J,Ebel S,Thomssen C,Janicke F,Pantel K. Comparative evaluation of urokinase-type plasminogen activator receptor expression in primary breast carcinomas and on metastatic tumor cells. Int J Cancer 2003; 107: 9039.
  • 73
    Thurm H,Ebel S,Kentenich C,Hemsen A,Riethdorf S,Coith C,Wallwiener D,Braun S,Oberhoff C,Janicke F,Pantel K. Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. Clin Cancer Res 2003; 9: 2598604.
  • 74
    Reimers N,Zafrakas K,Assmann V,Egen C,Riethdorf L,Riethdorf S,Berger J,Ebel S,Janicke F,Sauter G,Pantel K. Expression of extracellular matrix metalloproteases inducer on micrometastatic and primary mammary carcinoma cells. Clin Cancer Res 2004; 10: 34228.
  • 75
    Pierga JY,Bonneton C,Magdelenat H,Vincent-Salomon A,Nos C,Boudou E,Pouillart P,Thiery JP,de Cremoux P. Real-time quantitative PCR determination of urokinase-type plasminogen activator receptor (uPAR) expression of isolated micrometastatic cells from bone marrow of breast cancer patients. Int J Cancer 2005; 114: 2918.
  • 76
    Meng S,Tripathy D,Shete S,Ashfaq R,Saboorian H,Haley B,Frenkel E,Euhus D,Leitch M,Osborne C,Clifford E,Perkins S, et al. uPAR and HER-2 gene status in individual breast cancer cells from blood and tissues. Proc Natl Acad Sci USA. 2006; 103: 173615.
  • 77
    Pfitzenmaier J,Ellis WJ,Arfman EW,Hawley S,McLaughlin PO,Lange PH,Vessella RL. Telomerase activity in disseminated prostate cancer cells. BJ U Int 2006; 97: 130913.
  • 78
    Mecklenburg I,Weckermann D,Zippelius A,Schoberth A,Petersen S,Prang N,Riethmuller G,Kufer P. A multimarker real-time RT-PCR for MAGE-A gene expression allows sensitive detection and quantification of the minimal systemic tumor load in patients with localized cancer. J Immunol Methods 2007; 323: 18093.
  • 79
    Sienel W,Mecklenburg I,Dango S,Ehrhardt P,Kirschbaum A,Passlick B,Pantel K. Detection of MAGE-A transcripts in bone marrow is an independent prognostic factor in operable non-small-cell lung cancer. Clin Cancer Res 2007; 13: 38407.
  • 80
    Piccart-Gebhart MJ,Procter M,Leyland-Jones B,Goldhirsch A,Untch M,Smith I,Gianni L,Baselga J,Bell R,Jackisch C,Cameron D,Dowsett M, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353: 165972.
  • 81
    Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006; 12: 895904.
  • 82
    Braun S,Schlimok G,Heumos I,Schaller G,Riethdorf L,Riethmuller G,Pantel K. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. Cancer Res 2001; 61: 18905.
  • 83
    Vincent-Salomon A,Pierga JY,Couturier J,d'Enghien CD,Nos C,Sigal-Zafrani B,Lae M,Freneaux P,Dieras V,Thiery JP,Sastre-Garau X. HER2 status of bone marrow micrometastasis and their corresponding primary tumours in a pilot study of 27 cases: a possible tool for anti-HER2 therapy management? Br J Cancer 2007; 96: 6549.
  • 84
    Pantel K,Schlimok G,Braun S,Kutter D,Lindemann F,Schaller G,Funke I,Izbicki JR,Riethmuller G. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells J Natl Cancer Inst 1993; 85: 141924.
  • 85
    Solomayer EF,Becker S,Pergola-Becker G,Bachmann R,Kramer B,Vogel U,Neubauer H,Wallwiener D,Huober J,Fehm TN. Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients. Breast Cancer Res Treat 2006; 98: 17984.
  • 86
    Meng S,Tripathy D,Shete S,Ashfaq R,Haley B,Perkins S,Beitsch P,Khan A,Euhus D,Osborne C,Frenkel E,Hoover S, et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA. 2004; 101: 93938.
  • 87
    Wulfing P,Borchard J,Buerger H,Heidl S,Zanker KS,Kiesel L,Brandt B. HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clin Cancer Res 2006; 12: 171520.
  • 88
    Muller V,Stahmann N,Riethdorf S,Rau T,Zabel T,Goetz A,Janicke F,Pantel K. Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity. Clin Cancer Res 2005; 11: 367885.
  • 89
    Wikman H,Vessella RL,Pantel K. Cancer micrometastasis and tumor dormancy. APMIS, in press.
  • 90
    Bianco FJ,Jr,Wood DP,Jr,Gomes de Oliveira J,Nemeth JA,Beaman AA,Cher ML. Proliferation of prostate cancer cells in the bone marrow predicts recurrence in patients with localized prostate cancer. Prostate 2001; 49: 23542.
  • 91
    Vessella RL,Pantel K,Mohla S. Tumor cell dormancy: an NCI workshop report. Cancer Biol Ther 2007; 6: 1496504.
  • 92
    Naumov GN,Bender E,Zurakowski D,Kang SY,Sampson D,Flynn E,Watnick RS,Straume O,Akslen LA,Folkman J,Almog N. A model of human tumor dormancy: an angiogenic switch from the nonangiogenic phenotype. J Natl Cancer Inst 2006; 98: 31625.
  • 93
    Marches R,Scheuermann R,Uhr J. Cancer dormancy: from mice to man. Cell Cycle 2006; 5: 17728.
  • 94
    Woelfle U,Cloos J,Sauter G,Riethdorf L,Janicke F,van Diest P,Brakenhoff R,Pantel K. Molecular signature associated with bone marrow micrometastasis in human breast cancer. Cancer Res 2003; 63: 567984.
  • 95
    Naume B,Zhao X,Synnestvedt M,Borgen E,Giercksky Russnes H,Lingjorde OC,Stromberg M,Wiedswang G,Kvalheim G,Karesen R,Nesland JM,Borresen-Dale A-L, et al. Presence of bone marrow micrometastasis is associated with different recurrence risk within molecular subtypes of breast cancer. Mol Oncol 2007; 1: 16071.
  • 96
    Smirnov DA,Zweitzig DR,Foulk BW,Miller MC,Doyle GV,Pienta KJ,Meropol NJ,Weiner LM,Cohen SJ,Moreno JG,Connelly MC,Terstappen LW, et al. Global gene expression profiling of circulating tumor cells. Cancer Res 2005; 65: 49937.
  • 97
    Rosivatz E,Becker I,Specht K,Fricke E,Luber B,Busch R,Hofler H,Becker KF. Differential expression of the epithelial-mesenchymal transition regulators snail. SIP1, and twist in gastric cancer. Am J Pathol 2002; 161: 188191.
  • 98
    Kang Y,Massague J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004; 118: 2779.
  • 99
    Cheng GZ,Chan J,Wang Q,Zhang W,Sun CD,Wang LH. Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res 2007; 67: 197987.
  • 100
    Lo HW,Hsu SC,Xia W,Cao X,Shih JY,Wei Y,Abbruzzese JL,Hortobagyi GN,Hung MC. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 2007; 67: 906676.
  • 101
    Watson MA,Ylagan LR,Trinkaus KM,Gillanders WE,Naughton MJ,Weilbaecher KN,Fleming TP,Aft RL. Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients. Clin Cancer Res 2007; 13: 50019.
  • 102
    Ponti D,Costa A,Zaffaroni N,Pratesi G,Petrangolini G,Coradini D,Pilotti S,Pierotti MA,Daidone MG. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005; 65: 550611.
  • 103
    Ponti D,Zaffaroni N,Capelli C,Daidone MG. Breast cancer stem cells: an overview. Eur J Cancer 2006; 42: 121924.
  • 104
    Wicha MS,Liu S,Dontu G. Cancer stem cells: an old idea–a paradigm shift. Cancer Res 2006; 66: 188390; Discussion 95–6.
  • 105
    Stingl J,Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 2007; 7: 7919.
  • 106
    Sheridan C,Kishimoto H,Fuchs RK,Mehrotra S,Bhat-Nakshatri P,Turner CH,Goulet R,Jr,Badve S,Nakshatri H CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 2006; 8: R59.
  • 107
    Phillips TM,McBride WH,Pajonk F. The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 2006; 98: 177785.
  • 108
    Fillmore C,Kuperwasser C. Human breast cancer stem cell markers CD44 and CD24: enriching for cells with functional properties in mice or in man? Breast Cancer Res 2007; 9: 303.
  • 109
    Storci G,Sansone P,Trere D,Tavolari S,Taffurelli M,Ceccarelli C,Guarnieri T,Paterini P,Pariali M,Montanaro L,Santini D,Chieco P, et al. The basal-like breast carcinoma phenotype is regulated by SLUG gene expression. J Pathol 2008; 214: 2537.
  • 110
    Zucchi I,Sanzone S,Astigiano S,Pelucchi P,Scotti M,Valsecchi V,Barbieri O,Bertoli G,Albertini A,Reinbold RA,Dulbecco R. The properties of a mammary gland cancer stem cell. Proc Natl Acad Sci USA 2007; 104: 1047681.
  • 111
    Ginestier C,Hur MH,Charafe-Jauffret E,Monville F,Dutcher J,Brown M,Jacquernier J,Viens P,Kleer C,Liu S,Schott A,Hayes D, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007; 1: 55567.
  • 112
    Liu R,Wang X,Chen GY,Dalerba P,Gurney A,Hoey T,Sherlock G,Lewicki J,Shedden K,Clarke MF. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med 2007; 356: 21726.
  • 113
    Liu S,Dontu G,Mantle ID,Patel S,Ahn NS,Jackson KW,Suri P,Wicha MS. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 2006; 66: 606371.
  • 114
    Farnie G,Clarke RB. Mammary stem cells and breast cancer-role of notch signalling. Stem Cell Rev 2007; 3: 16975.
  • 115
    Farnie G,Clarke RB,Spence K,Pinnock N,Brennan K,Anderson NG,Bundred NJ. Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst 2007; 99: 61627.
  • 116
    Lindvall C,Bu W,Williams BO,Li Y. Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Rev 2007; 3: 15768.
  • 117
    Balic M,Lin H,Young L,Hawes D,Giuliano A,McNamara G,Datar RH,Cote RJ. Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 2006; 12: 561521.
  • 118
    Wiedswang G,Borgen E,Karesen R,Qvist H,Janbu J,Kvalheim G,Nesland JM,Naume B. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 2004; 10: 53428.
  • 119
    Heiss MM,Simon EH,Beyer BC,Gruetzner KU,Tarabichi A,Babic R,Schildberg FW,Allgayer H. Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow. J Clin Oncol 2002; 20: 200516.
  • 120
    Thorban S,Roder JD,Nekarda H,Funk A,Siewert JR,Pantel K. Immunocytochemical detection of disseminated tumor cells in the bone marrow of patients with esophageal carcinoma. J Natl Cancer Inst 1996; 88: 12227.
  • 121
    Kaifi JT,Yekebas EF,Schurr P,Obonyo D,Wachowiak R,Busch P,Heinecke A,Pantel K,Izbicki JR. Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer. J Natl Cancer Inst 2005; 97: 18407.
  • 122
    Thorban S,Roder JD,Pantel K,Siewert JR. Immunocytochemical detection of isolated epithelial tumor cells in bone marrow of patients with pancreatic carcinoma. Am J Surg 1996; 172: 2978.
  • 123
    Izbicki JR,Pantel K,Hosch SB. Micrometastasis in solid epithelial tumors: impact on surgical oncology. Surgery 2002; 131: 15.
  • 124
    Fehm T,Becker S,Bachmann C,Beck V,Gebauer G,Banys M,Wallwiener D,Solomayer EF. Detection of disseminated tumor cells in patients with gynecological cancers. Gynecol Oncol 2006; 103: 9427.
  • 125
    Wimberger P,Heubner M,Otterbach F,Fehm T,Kimmig R,Kasimir-Bauer S. Influence of platinum-based chemotherapy on disseminated tumor cells in blood and bone marrow of patients with ovarian cancer. Gynecol Oncol 2007; 107: 3318.
  • 126
    Gath HJ,Heissler E,Hell B,Bier J,Riethmuller G,Pantel K. Immunocytologic detection of isolated tumor cells in bone marrow of patients with squamous cell carcinomas of the head and neck region. Int J Oral Maxillofac Surg 1995; 24: 3515.
  • 127
    Pantel K,Gath H,Heissler E. Staging of head and neck cancer. N Engl J Med 1995; 332: 1788; author reply 1789–90.
  • 128
    Partridge M,Li SR,Pateromichelakis S,Francis R,Phillips E,Huang XH,Tesfa-Selase F,Langdon JD. Detection of minimal residual cancer to investigate why oral tumors recur despite seemingly adequate treatment. Clin Cancer Res 2000; 6: 271825.
  • 129
    de Nooij-van Dalen AG,van Dongen GA,Smeets SJ,Nieuwenhuis EJ,Stigter-van Walsum M,Snow GB,Brakenhoff RH. Characterization of the human Ly-6 antigens, the newly annotated member Ly-6K included, as molecular markers for head-and-neck squamous cell carcinoma. Int J Cancer 2003; 103: 76874.
  • 130
    Partridge M,Brakenhoff R,Phillips E,Ali K,Francis R,Hooper R,Lavery K,Brown A,Langdon J. Detection of rare disseminated tumor cells identifies head and neck cancer patients at risk of treatment failure. Clin Cancer Res 2003; 9: 528794.
  • 131
    Colnot DR,Nieuwenhuis EJ,Kuik DJ,Leemans CR,Dijkstra J,Snow GB,van Dongen GA,Brakenhoff RH. Clinical significance of micrometastatic cells detected by E48 (Ly-6D) reverse transcription-polymerase chain reaction in bone marrow of head and neck cancer patients. Clin Cancer Res 2004; 10: 782733.
  • 132
    Iddings D,Bilchik A. The biologic significance of micrometastatic disease and sentinel lymph node technology on colorectal cancer. J Surg Oncol 2007; 96: 6717.
  • 133
    Bilchik AJ,Hoon DS,Saha S,Turner RR,Wiese D,DiNome M,Koyanagi K,McCarter M,Shen P,Iddings D,Chen SL,Gonzalez M, et al. Prognostic impact of micrometastases in colon cancer: interim results of a prospective multicenter trial. Ann Surg 2007; 246: 56875; discussion 75–7.
  • 134
    Tonato M. Consensus conference on medical treatment of non-small cell lung cancer: adjuvant treatment. Lung Cancer 2002; 38( Suppl 3): S3742.
  • 135
    Swindle PW,Kattan MW,Scardino PT. Markers and meaning of primary treatment failure. Urol Clin North Am 2003; 30: 377401.
  • 136
    Gronau E,Goppelt M,Harzmann R,Weckermann D. Prostate cancer relapse after therapy with curative intention: a diagnostic and therapeutic dilemma. Onkologie 2005; 28: 3616.
  • 137
    Lang JE,Hall CS,Singh B,Lucci A. Significance of micrometastasis in bone marrow and blood of operable breast cancer patients: research tool or clinical application? Expert Rev Anticancer Ther 2007; 7: 146372.
  • 138
    Slade MJ,Coombes RC. The clinical significance of disseminated tumor cells in breast cancer. Nat Clin Pract Oncol 2007; 4: 3041.
  • 139
    Vincent-Salomon A,Bidard FC,Pierga JY. Bone marrow micrometastasis in breast cancer: review of detection methods, prognostic impact and biological issues. J Clin Pathol 2008; 61: 5706.
  • 140
    Kollermann MW,Pantel K,Enzmann T,Feek U,Kollermann J,Kossiwakis M,Kaulfuss U,Martell W,Spitz J. Supersensitive PSA-monitored neoadjuvant hormone treatment of clinically localized prostate cancer: effects on positive margins, tumor detection and epithelial cells in bone marrow. Eur Urol 1998; 34: 31824.
  • 141
    Weckermann D,Wawroschek F,Krawczak G,Haude KH,Harzmann R. Does the immunocytochemical detection of epithelial cells in bone marrow (micrometastasis) influence the time to biochemical relapse after radical prostatectomy? Urol Res 1999; 27: 28590.
  • 142
    Naume B,Wiedswang G,Borgen E,Kvalheim G,Karesen R,Qvist H,Janbu J,Harbitz T,Nesland JM. The prognostic value of isolated tumor cells in bone marrow in breast cancer patients: evaluation of morphological categories and the number of clinically significant cells. Clin Cancer Res 2004; 10: 30917.
  • 143
    Janni W,Wiedswang G,Fehm T,Jueckstock J,Borgen E,Rack B,Braun S,Sommer H,Solomayer E,Pantel K,Nesland JM,Genss E, et al. Persistence of disseminated tumor cells (DTC) in bone marrow (BM) during follow-up predicts increased risk for relapse up-date of the pooled European data. Breast Cancer Res Treat 2006; 100( suppl 1) abstract 18.
  • 144
    Juhl H,Stritzel M,Wroblewski A,Henne-Bruns D,Kremer B,Schmiegel W,Neumaier M,Wagener C,Schreiber HW,Kalthoff H. Immunocytological detection of micrometastatic cells: comparative evaluation of findings in the peritoneal cavity and the bone marrow of gastric, colorectal and pancreatic cancer patients. Int J Cancer 1994; 57: 3305.
  • 145
    Broll R,Lembcke K,Stock C,Zingler M,Duchrow M,Schimmelpenning H,Strik M,Muller G,Kujath P,Bruch HP. [Tumor cell dissemination in bone marrow and peritoneal cavity. An immunocytochemical study of patients with stomach or colorectal carcinoma.] Langenbecks Arch Chir 1996; 381: 518.
  • 146
    Cohen AM,Garin-Chesa P,Hanson M,Weyhrauch K,Kemeny N,Fong Y,Paty P,Welt S,Old L. In vitro detection of occult bone marrow metastases in patients with colorectal cancer hepatic metastases. Dis Colon Rectum 1998; 41: 11125.
  • 147
    Schott A,Vogel I,Krueger U,Kalthoff H,Schreiber HW,Schmiegel W,Henne-Bruns D,Kremer B,Juhl H. Isolated tumor cells are frequently detectable in the peritoneal cavity of gastric and colorectal cancer patients and serve as a new prognostic marker. Ann Surg 1998; 227: 3729.
  • 148
    Pantel K,Schlimok G,Angstwurm M,Weckermann D,Schmaus W,Gath H,Passlick B,Izbicki JR,Riethmuller G. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994; 3: 16573.
  • 149
    Flatmark K,Bjornland K,Johannessen HO,Hegstad E,Rosales R,Harklau L,Solhaug JH,Faye RS,Soreide O,Fodstad O. Immunomagnetic detection of micrometastatic cells in bone marrow of colorectal cancer patients. Clin Cancer Res 2002; 8: 4449.
  • 150
    Lindemann F,Schlimok G,Dirschedl P,Witte J,Riethmuller G. Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 1992; 340: 6859.
  • 151
    O'sullivan GC,Collins JK,Kelly J,Morgan J,Madden M,Shanahan F. Micrometastases: marker of metastatic potential or evidence of residual disease? Gut 1997; 40: 5125.
  • 152
    Leinung S,Wurl P,Schonfelder A,Weiss CL,Roder I,Schonfelder M. Detection of cytokeratin-positive cells in bone marrow in breast cancer and colorectal carcinoma in comparison with other factors of prognosis. J Hematother Stem Cell Res 2000; 9: 90511.
  • 153
    Schoppmeyer K,Fruhauf N,Oldhafer K,Seeber S,Kasimir-Bauer S. Tumor cell dissemination in colon cancer does not predict extrahepatic recurrence in patients undergoing surgery for hepatic metastases. Oncol Rep 2006; 15: 44954.
  • 154
    Vogel I,Soeth E,Röder C,Kremer B,Henne-Bruns D,Kalthoff H. Multivariate analysis reveals RT-PCR-detected tumour cells in the blood and/or bone marrow of patients with colorectal carcinoma as an independent prognostic factor. Ann Oncol 2000; 11( Suppl 4): 43.
  • 155
    Kienle P,Koch M,Autschbach F,Benner A,Treiber M,Wannenmacher M,von Knebel Doeberitz M,Buchler M,Herfarth C,Weitz J. Decreased detection rate of disseminated tumor cells of rectal cancer patients after preoperative chemoradiation: a first step towards a molecular surrogate marker for neoadjuvant treatment in colorectal cancer. Ann Surg 2003; 238: 32430; discussion 30–1.
  • 156
    Conzelmann M,Dieterle CP,Linnemann U,Berger MR. Cytokeratin 20 and guanylyl cyclase C mRNA is largely present in lymph node and liver specimens of colorectal cancer patients. Int J Cancer 2003; 107: 61728.
  • 157
    Vlems FA,Diepstra JH,Punt CJ,Ligtenberg MJ,Cornelissen IM,van Krieken JH,Wobbes T,van Muijen GN,Ruers TJ. Detection of disseminated tumour cells in blood and bone marrow samples of patients undergoing hepatic resection for metastasis of colorectal cancer. Br J Surg 2003; 90: 98995.
  • 158
    Koch M,Kienle P,Kastrati D,Antolovic D,Schmidt J,Herfarth C,von Knebel Doeberitz M,Weitz J. Prognostic impact of hematogenous tumor cell dissemination in patients with stage II colorectal cancer. Int J Cancer 2006; 118: 30727.
  • 159
    Soeth E,Roder C,Juhl H,Kruger U,Kremer B,Kalthoff H. The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease. Int J Cancer 1996; 69: 27882.
  • 160
    Soeth E,Vogel I,Roder C,Juhl H,Marxsen J,Kruger U,Henne-Bruns D,Kremer B,Kalthoff H. Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR. Cancer Res 1997; 57: 310610.
  • 161
    Koch M,Kienle P,Hinz U,Antolovic D,Schmidt J,Herfarth C,von Knebel Doeberitz M,Weitz J. Detection of hematogenous tumor cell dissemination predicts tumor relapse in patients undergoing surgical resection of colorectal liver metastases. Ann Surg 2005; 241: 199205.
  • 162
    Gerhard M,Juhl H,Kalthoff H,Schreiber HW,Wagener C,Neumaier M. Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 1994; 12: 7259.
  • 163
    Pantel K,Izbicki J,Passlick B,Angstwurm M,Haussinger K,Thetter O,Riethmuller G. Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases. Lancet 1996; 347: 64953.
  • 164
    Passlick B,Kubuschok B,Izbicki JR,Thetter O,Pantel K. Isolated tumor cells in bone marrow predict reduced survival in node-negative non-small cell lung cancer. Ann Thorac Surg 1999; 68: 20538.
  • 165
    Sugio K,Kase S,Sakada T,Yamazaki K,Yamaguchi M,Ondo K,Yano T. Micrometastasis in the bone marrow of patients with lung cancer associated with a reduced expression of E-cadherin and beta-catenin: risk assessment by immunohistochemistry. Surgery 2002; 131: S22631.
  • 166
    Relihan N,McGreal G,Kelly J,Ryan D,O'sullivan GC,Redmond HP. Combined sentinel lymph-node mapping and bone-marrow micrometastatic analysis for improved staging in breast cancer. Lancet 1999; 354: 12930.
  • 167
    Mattioli S,D'Ovidio F,Tazzari P,Pilotti V,Daddi N,Bandini G,Piccioli M,Pileri S. Iliac crest biopsy versus rib segment resection for the detection of bone marrow isolated tumor cells from lung and esophageal cancer. Eur J Cardiothorac Surg 2001; 19: 5769.
  • 168
    Pantel K,Izbicki JR,Angstwurm M,Braun S,Passlick B,Karg O,Thetter O,Riethmuller G. Immunocytological detection of bone marrow micrometastasis in operable non-small cell lung cancer. Cancer Res 1993; 53: 102731.
  • 169
    Cote RJ,Beattie EJ,Chaiwun B,Shi SR,Harvey J,Chen SC,Sherrod AE,Groshen S,Taylor CR. Detection of occult bone marrow micrometastases in patients with operable lung carcinoma. Ann Surg 1995; 222: 41523; discussion 23–5.
  • 170
    Ohgami A,Mitsudomi T,Sugio K,Tsuda T,Oyama T,Nishida K,Osaki T,Yasumoto K. Micrometastatic tumor cells in the bone marrow of patients with non-small cell lung cancer. Ann Thorac Surg 1997; 64: 3637.
  • 171
    Kasimir-Bauer S,Schleucher N,Weber R,Neumann R,Seeber S. Evaluation of different markers in non-small cell lung cancer: prognostic value of clinical staging, tumour cell detection and tumour marker analysis for tumour progression and overall survival. Oncol Rep 2003; 10: 47582.
  • 172
    Yasumoto K,Osaki T,Watanabe Y,Kato H,Yoshimura T. Prognostic value of cytokeratin-positive cells in the bone marrow and lymph nodes of patients with resected nonsmall cell lung cancer: a multicenter prospective study. Ann Thorac Surg 2003; 76: 194201, discussion 2.
  • 173
    Brunsvig PF,Flatmark K,Aamdal S,Hoifodt H,Le H,Jakobsen E,Sandstad B,Fodstad O. Bone marrow micrometastases in advanced stage non-small cell lung carcinoma patients. Lung Cancer 2008; 61: 1706.
  • 174
    Saito T,Kobayashi M,Harada R,Uemura Y,Taguchi H. Sensitive detection of small cell lung carcinoma cells by reverse transcriptase-polymerase chain reaction for prepro-gastrin-releasing peptide mRNA. Cancer 2003; 97: 250411.
  • 175
    Pantel K,Aignherr C,Kollermann J,Caprano J,Riethmuller G,Kollermann MW. Immunocytochemical detection of isolated tumour cells in bone marrow of patients with untreated stage C prostatic cancer. Eur J Cancer 1995; 31: 162732.
  • 176
    Wood DP,Jr,Banerjee M. Presence of circulating prostate cells in the bone marrow of patients undergoing radical prostatectomy is predictive of disease-free survival. J Clin Oncol 1997; 15: 34517.
  • 177
    Kollermann J,Heseding B,Helpap B,Kollermann MW,Pantel K. Comparative immunocytochemical assessment of isolated carcinoma cells in lymph nodes and bone marrow of patients with clinically localized prostate cancer. Int J Cancer 1999; 84: 1459.
  • 178
    Weckermann D,Wawroschek F,Krawczak G,Haude KH,Harzmann R. Does the immunocytochemical detection of epithelial cells in bone marrow (micrometastasis) influence the time to biochemical relapse after radical prostatectomy? Urol Res 1999; 27: 28590.
  • 179
    Köllermann J,Weikert S,Schostak M,Kempkensteffen C,Kleinschmidt K,Rau T,Pantel K. Prognostic significance of disseminated tumor cells in the bone marrow of prostate cancer patients submitted to neoadjuvant hormonal therapy. J Clin Oncol 2008, in press.
  • 180
    Weckermann D,Muller P,Wawroschek F,Harzmann R,Riethmuller G,Schlimok G. Disseminated cytokeratin positive tumor cells in the bone marrow of patients with prostate cancer: detection and prognostic value. J Urol 2001; 166: 699703.
  • 181
    Berg A,Berner A,Lilleby W,Bruland OS,Fossa SD,Nesland JM,Kvalheim G. Impact of disseminated tumor cells in bone marrow at diagnosis in patients with nonmetastatic prostate cancer treated by definitive radiotherapy. Int J Cancer 2007; 120: 16039.
  • 182
    Gao CL,Dean RC,Pinto A,Mooneyhan R,Connelly RR,McLeod DG,Srivastava S,Moul JW. Detection of circulating prostate specific antigen expressing prostatic cells in the bone marrow of radical prostatectomy patients by sensitive reverse transcriptase polymerase chain reaction. J Urol 1999; 161: 10706.
  • 183
    Pantel K,Enzmann T,Kollermann J,Caprano J,Riethmuller G,Kollermann MW. Immunocytochemical monitoring of micrometastatic disease: reduction of prostate cancer cells in bone marrow by androgen deprivation. Int J Cancer 1997; 71: 5215.
  • 184
    Wood DP,Jr,Beaman A,Banerjee M,Powell I,Pontes E,Cher ML. Effect of neoadjuvant androgen deprivation on circulating prostate cells in the bone marrow of men undergoing radical prostatectomy. Clin Cancer Res 1998; 4: 211923.
  • 185
    Wilson A,Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006; 6: 93106.
  • 186
    Schlimok G,Funke I,Holzmann B,Gottlinger G,Schmidt G,Hauser H,Swierkot S,Warnecke HH,Schneider B,Koprowski H, et al. Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17–1A monoclonal antibodies. Proc Natl Acad Sci USA 1987; 84: 86726.
  • 187
    Cote RJ,Rosen PP,Lesser ML,Old LJ,Osborne MP. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991; 9: 174956.
  • 188
    Harbeck N,Untch M,Pache L,Eiermann W. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3-year median follow-up. Br J Cancer 1994; 69: 56671.
  • 189
    Diel IJ,Kaufmann M,Costa SD,Holle R,von Minckwitz G,Solomayer EF,Kaul S,Bastert G. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 16528.
  • 190
    Molino A,Pelosi G,Turazza M,Sperotto L,Bonetti A,Nortilli R,Fattovich G,Alaimo C,Piubello Q,Pavanel F,Micciolo R,Cetto GL. Bone marrow micrometastases in 109 breast cancer patients: correlations with clinical and pathological features and prognosis. Breast Cancer Res Treat 1997; 42: 2330.
  • 191
    Mansi JL,Gogas H,Bliss JM,Gazet JC,Berger U,Coombes RC. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999; 354: 197202.
  • 192
    Braun S,Pantel K,Muller P,Janni W,Hepp F,Kentenich CR,Gastroph S,Wischnik A,Dimpfl T,Kindermann G,Riethmuller G,Schlimok G. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I. II, or. III breast cancer. N Engl J Med 2000; 342: 52533.
  • 193
    Gebauer G,Fehm T,Merkle E,Beck EP,Lang N,Jager W. Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001; 19: 366974.
  • 194
    Gerber B,Krause A,Muller H,Richter D,Reimer T,Makovitzky J,Herrnring C,Jeschke U,Kundt G,Friese K. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001; 19: 96071.
  • 195
    Naume B,Borgen E,Kvalheim G,Karesen R,Qvist H,Sauer T,Kumar T,Nesland JM. Detection of isolated tumor cells in bone marrow in early-stage breast carcinoma patients: comparison with preoperative clinical parameters and primary tumor characteristics. Clin Cancer Res 2001; 7: 41229.
  • 196
    Wiedswang G,Borgen E,Karesen R,Kvalheim G,Nesland JM,Qvist H,Schlichting E,Sauer T,Janbu J,Harbitz T,Naume B. Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003; 21: 346978.
  • 197
    Pierga JY,Bonneton C,Vincent-Salomon A,de Cremoux P,Nos C,Blin N,Pouillart P,Thiery JP,Magdelenat H. Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 2004; 10: 1392400.
  • 198
    Schindlbeck C,Kampik T,Janni W,Rack B,Jeschke U,Krajewski S,Sommer H,Friese K. Prognostic relevance of disseminated tumor cells in the bone marrow and biological factors of 265 primary breast carcinomas. Breast Cancer Res 2005; 7: R117485.
  • 199
    Becker S,Becker-Pergola G,Wallwiener D,Solomayer EF,Fehm T. Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy. Breast Cancer Res Treat 2006; 97: 916.
  • 200
    Bidard FC,Vincent-Salomon A,Gomme S,Nos C,De Rycke Y,Thiery JP,Sigal-Zafrani B,Mignot L,Sastre-Garau X,Pierga JY. Disseminated tumor cells of breast cancer patients: a strong prognostic factor for distant and local relapse. Clin Cancer Res 2008; 14: 330611.
  • 201
    Berois N,Varangot M,Sonora C,Zarantonelli L,Pressa C,Lavina R,Rodriguez JL,Delgado F,Porchet N,Aubert JP,Osinaga E. Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA. Int J Cancer 2003; 103: 5505.
  • 202
    Jung YS,Lee KJ,Kim HJ,Yim HE,Park JS,Soh EY,Kim MW,Park HB. Clinical significance of bone marrow micrometastasis detected by nested rt-PCR for keratin-19 in breast cancer patients. Jpn J Clin Oncol 2003; 33: 16772.
  • 203
    Benoy IH,Elst H,Philips M,Wuyts H,Van Dam P,Scharpe S,Van Marck E,Vermeulen PB,Dirix LY. Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer. Br J Cancer 2006; 94: 67280.
  • 204
    Farmen RK,Nordgard O,Gilje B,Shammas FV,Kvaloy JT,Oltedal S,Heikkila R. Bone marrow cytokeratin 19 mRNA level is an independent predictor of relapse-free survival in operable breast cancer patients. Breast Cancer Res Treat 2008; 108: 2518.
  • 205
    Mikhitarian K,Martin RH,Ruppel MB,Gillanders WE,Hoda R,Schutte del H,Callahan K,Mitas M,Cole DJ. Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: interim results of a prospective cohort study. BMC Cancer 2008; 8: 55.
  • 206
    Schneider BM,Schlimok G,Riethmuller G,Witte J. [Bone marrow micrometastases in colorectal cancers.] Fortschr Med 1989; 107: 5963.
  • 207
    Schlimok G,Funke I,Bock B,Schweiberer B,Witte J,Riethmuller G. Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 1990; 8: 8317.
  • 208
    Silly H,Samonigg H,Stoger H,Brezinschek HP,Wilders-Truschnig M. Micrometastatic tumour cells in bone marrow in colorectal cancer. Lancet 1992; 340: 1288.
  • 209
    Litle VR,Warren RS,Moore D,2nd,Pallavicini MG. Molecular cytogenetic analysis of cytokeratin 20-labeled cells in primary tumors and bone marrow aspirates from colorectal carcinoma patients. Cancer 1997; 79: 166470.
  • 210
    Sheehan KM,Cahill RA,McGreal G,Steele C,Byrne MF,Kirwan WO,Kay EW,Fitzgerald DJ,Redmond HP,Murray FE. Cyclooxygenase-2 expression in primary human colorectal cancers and bone marrow micrometastases. Dig Liver Dis 2004; 36: 3927.
  • 211
    Gunn J,McCall JL,Yun K,Wright PA. Detection of micrometastases in colorectal cancer patients by K19 and K20 reverse-transcription polymerase chain reaction. Lab Invest 1996; 75: 6116.
  • 212
    Weitz J,Kienle P,Magener A,Koch M,Schrodel A,Willeke F,Autschbach F,Lacroix J,Lehnert T,Herfarth C,von Knebel Doeberitz M. Detection of disseminated colorectal cancer cells in lymph nodes, blood and bone marrow. Clin Cancer Res 1999; 5: 18306.
  • 213
    Weitz J,Koch M,Kienle P,Schrodel A,Willeke F,Benner A,Lehnert T,Herfarth C,von Knebel Doeberitz M. Detection of hematogenic tumor cell dissemination in patients undergoing resection of liver metastases of colorectal cancer. Ann Surg 2000; 232: 6672.
  • 214
    Poncelet AJ,Weynand B,Ferdin F,Robert AR,Noirhomme PH. Bone marrow micrometastasis might not be a short-term predictor of survival in early stages non-small cell lung carcinoma. Eur J Cardiothorac Surg 2001; 20: 4818.
  • 215
    Osaki T,Oyama T,Gu CD,Yamashita T,So T,Takenoyama M,Sugio K,Yasumoto K. Prognostic impact of micrometastatic tumor cells in the lymph nodes and bone marrow of patients with completely resected stage I non-small-cell lung cancer. J Clin Oncol 2002; 20: 29306.
  • 216
    Thompson AB,Kessinger A,Sharp JG. A pilot evaluation of micrometastases for the prediction of outcome in lung cancer. Chest 2004; 125: 156S7S.
  • 217
    Hsu CP,Shai SE,Hsia JY,Chen CY. Clinical significance of bone marrow microinvolvement in nonsmall cell lung carcinoma. Cancer 2004; 100: 794800.
  • 218
    Melchior SW,Corey E,Ellis WJ,Ross AA,Layton TJ,Oswin MM,Lange PH,Vessella RL. Early tumor cell dissemination in patients with clinically localized carcinoma of the prostate. Clin Cancer Res 1997; 3: 24956.
  • 219
    Kollermann J,Muller M,Goessl C,Krause H,Helpap B,Pantel K,Miller K. Methylation-specific PCR for DNA-based detection of occult tumor cells in lymph nodes of prostate cancer patients. Eur Urol 2003; 44: 5338.
  • 220
    Deguchi T,Yang M,Ehara H,Ito S,Nishino Y,Takahashi Y,Ito Y,Shimokawa K,Tanaka T,Imaeda T,Doi T,Kawada Y. Detection of micrometastatic prostate cancer cells in the bone marrow of patients with prostate cancer. Br J Cancer 1997; 75: 6348.