The prognosis and treatment of patients with multiple tumors may depend on the correlation between tumors: multiple primary tumors, or recurrent tumors, and metastatic disease. The authors investigated whether the detection of molecular aberrations in multiple gynecologic tumors in individual patients provided clinically useful information on the correlation between the tumors.
Between 1999 and 2001, molecular analyses were performed on tissue from 15 gynecologic patients, all with multiple tumors. The molecular analyses included loss of heterozygosity determinations at eight DNA loci and mutation analyses of p53 exons 5–8 using the single-strand conformation polymorphism method. Previously, it was not possible to use routine diagnostic histopathology to determine accurately the correlation between multiple lesions in patients with gynecologic malignancies, information that may have an impact on clinical decision-making and prognosis.
Molecular results were obtained from all tumors from each of the 15 patients. The DNA alterations detected provided evidence that two patients had second primary tumors, nine patients had a single tumor with metastases, and four patients had two independent primary tumors as well as metastatic disease. The results provided additional diagnostic information and contributed to clinical decision-making.
From the last decades of molecular research, it has become clear that almost all malignancies exhibit genomic instability and, as a consequence, harbor DNA aberrations.1 Large numbers of nonrandom chromosomal aberration patterns have been described in a wide range of tumors, and it generally has been accepted that the mutational activation of protooncogenes and the mutational inactivation of tumor suppressor genes are related causally to the tumorigenic process.2–6 In addition, tumors contain numerous genomic alterations that, to date, have to be regarded as disease-related epiphenomena, because their relation with tumorigenesis is not clear.1 The finding of genomic aberrations in tumors not only increased our understanding of tumorigenesis but also led to numerous clinical investigations into their correlation with clinical parameters. The vast majority of these studies were performed on groups of patients with the same type of tumor, and the results were of limited value for any individual patient. In the current article, we report on the additional value that molecular determinations may have for individual patient who suffer from multiple tumors.
Patients with multiple, histologically comparable, synchronous or metachronous tumors represent a difficult clinical problem. The treatment and prognosis of patients with two or more primary tumors is different from the treatment and prognosis of patients with recurrent or metastatic disease. Thus, it is important to determine the correlation between multiple tumors within an individual patient. Accurate discrimination between multiple primary tumors, recurrent tumors, or metastatic disease is not always possible using routine pathologic examination with hematoxylin and eosin (H&E) staining and immunohistochemistry. Therefore, additional investigations that can determine the correlation between multiple tumors within an individual patient have potential value.
Most tumors are characterized by the presence of numerous genomic aberrations.1 These aberrations comprise small (point) mutations and large chromosomal deletions and amplifications. The frequency of deletion of chromosomal segments exceeds the frequency of gains in most tumors.4 Approximately 50% of all carcinomas harbor p53 gene mutations;7–9 and loss of DNA at chromosomal regions 3p, 5q, 8p, 9p, 10q, 13q, 14q, and 17p is frequent in numerous carcinoma types.4 For example, in patients with ovarian carcinoma, > 50% of tumors have p53 gene mutations,4, 10–16 and almost all tumors have a deletion of at least one of the regions 3p, 5q, 8p, 9p, 10q, 13q, 14q, or 17p.15, 17–23 These molecular aberration profiles can be regarded as tumor specific clonal markers and, as such, provide information on the possible correlation between multiple tumors within an individual patient.18, 24–28 Comparable molecular aberration profiles in different tumors indicate a clonal correlation in recurrent tumors or metastatic disease, whereas different profiles suggest independent primary tumors.
In this report, we present 15 gynecologic patients, all with multiple tumors, in whom the prognoses and/or clinical management would be influenced by identifying the nature of their lesions: primary tumor with recurrent or metastatic lesion or multiple primary tumors. Accurate discrimination has not proved possible using conventional diagnostic histopathology or immunohistochemistry; therefore, molecular determinations were carried out. Loss of heterozygosity (LOH) analyses for 3p, 5q, 8p, 9p, 10q, 13q, 14q, and 17p and p53 aberration analyses were performed on routinely formalin fixed, paraffin embedded tissues. Routinely processed biopsies and, in some instances, cytology smears were sufficient. In all biopsies and smears, the molecular results contributed substantial information toward the diagnosis.
MATERIALS AND METHODS
We investigated a series of 15 gynecologic patients with multiple synchronous or metachronous carcinomas using the following selection criteria: no accurate discrimination possible between multiple tumors within an individual patient by routine diagnostic histopathology and knowledge of the nature of the tumors would have an impact on prognosis or clinical decision-making. The requests for molecular determinations all were received between 1999 and 2001. Patient characteristics are shown in Table 1. Four patients had synchronous tumors, and 11 patients had metachronous lesions. All 15 patients had at least 1 gynecologic tumor: There were 10 ovarian tumors, 4 endometrial tumors, 4 cervical tumors and 1 ovarian teratoma. The tumors that were located outside the uterus and ovaries in these patients included four breast tumors; two sigmoid tumors; one tumor each located in the rectum, thorax lining, omentum, appendix, kidney, lung, bladder, liver, and brain; one teratoma; and two lymph node metastases. Twelve women had adenocarcinomas, 2 women (Patients 10 and 12) had squamous cell carcinomas, and 1 woman (Patient 7) had adenosquamous carcinoma. One woman (Patient 9), in addition to two ovarian adenocarcinomas, had an ovarian teratoma. For genomic analysis, routine pathology specimens were used, including resection specimens, biopsy specimens, and cytology preparations.
Table 1. Patient Characteristics and Molecular Results
S: synchronous; M: metachronous; LOH: loss of heterozygosity; Id: identical; Diff: different; Mets: metastases; 2nd prim: second primary; Adeno: adenocarcinoma; squamous: squamous cell carcinoma.
Numbers not in parenthese indicate the months after primary diagnosis. For patients with more than two tumors, the numbers of lesions are indicated in parentheses. For example, Patient 2 had three tumors: in the ovary (1), ovary (2), and thorax wall (3). The ovary (1) and ovary (2) tumors were synchronous, and the thorax wall (3) tumor was metachronous and was diagnosed 36 months later than the ovary tumors. The two ovarian tumors (1, 2) had identical LOH patterns, and the thorax wall tumor (3) had a different LOH pattern. The ovarian tumors (1, 2) were regarded as primary tumor and metastasis; whereas the thorax wall tumor (3) was a second primary tumor.
From routine formalin fixed, paraffin embedded tissues, H&E-stained sections were used to select parts of the tissues comprised of > 70% tumor cells. From the same paraffin block, normal tissue also was selected. The selected tissue parts were punched gently out of the tissue blocks. After this procedure, a new H&E-stained section was made to verify the isolated tissue parts (Fig. 1, T2; Fig. 2, T1 and T3). For sections in which manual microdissection of tissue parts comprised of 70% tumor cells was not possible, laser-capture microdissection (LCM) was used on H&E-stained sections29 (Fig. 1, T1; Fig. 2, T2). Cytology smears that contained high percentages of tumor cells were scraped manually from the glass slide. The punched tissue fragments, without deparaffinization, and the LCM and scraped tissue fragments were digested in 100–200 μL 50 mM Tris/HCl, pH 8.0, and 20 μL Proteinase K (20 mg/mL). After overnight incubation at 56 °C, the lysates were boiled for 10 minutes, and, after centrifugation, the supernatant was used for molecular analysis.
DNA Aberration Analyses
Because the DNA retrieved from routine processed tissue specimens is cross-linked, we used small amplicon (< 200 base pair) polymerase chain reaction (PCR) analysis to investigate DNA aberrations. For LOH analysis, highly polymorphic dinucleotide repeat markers were amplified from chromosomal regions that frequently are deleted in carcinomas. Two markers for each locus were used. The markers were D3S1284 and D3S1238 on 3p, D8S133 and D8S136 on 8p, D9S156 and D9S161 on 9p, D13S153 and D13S155 on 13q, and D17S786 and TP53CA on 17p. The use of these markers has been described previously.27 In addition, we used LOH markers on 5q, 10q, and 14q (Table 2) obtained from The Genome Database (www.GDB.org). In all specimens, the markers on 3p, 8p, 9p, 13q, and 17p were used. When no loss or only loss of one locus was found, the second set of six markers (three loci) was used. From all specimens, exons 5–8 of the p53 gene were investigated by PCR/single-strand conformation polymorphism (SSCP) analysis. Each exon was amplified in two overlapping fragments, as described previously.27 For both LOH analysis and SSCP analysis, tumor DNA always was compared with normal DNA from the same patient. PCR analysis was performed with 1–3 μL of isolated DNA in a final reaction volume of 15 μL containing: 1.5 mM MgCl2; 0.02 mM dATP; 0.2 mM each of dGTP, dTTP, and dCTP; 0.8 μCi α-32PdATP (Amersham, Buckinghamshire, United Kingdom); 20 pmol of each primer; and 0.2 unit Taq polymerase (Promega, Madison, WI). PCR was performed for 35 cycles (denaturing at 95 °C for 30 seconds, annealing at 55 °C for 45 seconds, and extension at 72 °C for 1 minute) in a Biometra thermocycler (Biometra, Göttingen, Germany). A final extension was carried out at 72 °C for 10 minutes. PCR products were diluted with loading buffer (95% formamide; 10 mM ethylenediamine tetraacetic acid, pH 8.0; 0.025% bromophenol blue; and 0.025% xylene cyanol), denatured at 95 °C for 4 minutes, and snap-cooled on ice. For LOH analysis, PCR products were separated on a denaturing 6% polyacrylamide gel; and, for SSCP analysis, the samples were run overnight at 7 Watts on a nondenaturing 6% polyacrylamide gel containing 10% glycerol in 1 × tris-borate-EDTA running buffer. After electrophoresis, gels were fixed in 10% acetic acid, dried on blotting paper on a vacuum gel dryer, and exposed to X-ray film overnight at − 70 °C, using intensifying screens. Films were evaluated by visual inspection.
Table 2. Loss of Heterozygosity Marker Information
Product size (bp)
bp: Base pairs; F: forward primer; R: reverse primer; GDB: The Genome Database (Available from URL: www.GDB.org).
F: GCA TAT CCT CAA TGG GAT CC; R: CAT ATT TTA TCT AGG CGG CC
F: TCC AGA GTC TTC ATT CAT ATT C; R: TCT CTG GGG AGG AGT AAG G
The results and the conclusions are summarized in Table 1. From all routinely processed tissue specimens, we were able to obtain amplifiable DNA, even from tissues that were stored for > 10 years. In all specimens, loss of at least 1 of 8 loci was found with the 16 markers (2 per locus). DNA loss was regarded as identical when, in different tumors from an individual patient, the same markers showed loss of the same alleles (Fig. 3, T1 and T2 with markers D8S133 and TP53CA; Fig. 4, markers D10S215 and D14S67, both with T2 and T3). LOH was scored as different when, in different tumors from an individual patient, different markers showed loss (Fig. 4, T1 and T2/T3 with D17S786 and D10S215) or when the same markers had loss of different alleles (Fig. 4, T1 and T2/T3 with marker D14S67).
In 9 of 15 patients, tumor specific p53 aberrations were found by SSCP analysis (Table 1). We regard the same aberrant SSCP pattern in different tumors from the same patient as a strong indication of the presence of the same mutation (Fig. 3, T1 and T2 with p53 exon 5), as is accepted generally. For the SSCP and LOH analyses, tumor DNA and normal DNA were isolated from the same paraffin blocks, and all samples from each patient were investigated in the same experiment to obtain maximal comparability. With this procedure, tumor specific mutations can be discriminated clearly from polymorphisms, and identical aberrant SSCP patterns within one experiment are a very strong indication for identical DNA aberrations. The results for each individual patient were evaluated separately. No discrepancies were found when the LOH marker results from any individual patient were compared with the results from another patients or when LOH and p53 analyses results were compared. The background and the molecular results from two patients are described as examples.
A woman age 53 years had an ovarian mass identified when she was admitted to the hospital with a nonovarian mass-related complaint. She underwent an abdominal total hysterectomy and bilateral salpingo-oophorectomy, and a well-differentiated endometrioid carcinoma was identified in the uterine corpus (Fig. 1, T1). In addition, a histologically similar carcinoma was identified in the left ovary (Fig. 1, T2). The histologic features suggested that the uterine lesion was a primary tumor that infiltrated to nearly half the thickness of the myometrium; however, whether the ovarian lesion was a second primary tumor or a metastasis was uncertain. LOH and p53 aberration analyses revealed identical alterations in both tumors (Fig. 3, T1 and T2). Therefore, the patient was diagnosed with endometrial carcinoma metastatic to the ovary, a higher stage lesion of the uterine carcinoma that required a larger field of adjuvant radiotherapy rather than chemotherapy.
A woman who was identified as BRCA1 positive had undergone left-sided mastectomy in 1998 (at the age of 49 years) for infiltrating ductal carcinoma (Fig. 2, T1). She also had positive progesterone and estrogen receptor status. Two years later, she presented at the outpatient department with a left-sided, enlarged lymph node located in the groin. This lymph node was removed and showed histologic metastatic carcinoma compatible with breast carcinoma (Fig. 2, T2). However, shortly thereafter, it was discovered that the patient had a pelvic lesion, and she underwent hysterectomy and bilateral salpingo-oophorectomy. Poorly differentiated adenocarcinoma was present in the ovary (Fig. 2, T3), morphologically more suggestive of ovarian carcinoma than metastatic breast carcinoma. LOH and p53 aberration analyses showed identical patterns in the ovarian tumor and the lymph node and clearly different patterns in the breast tumor (Fig. 4, T1–T3). Therefore, the adenocarcinoma in the lymph node was regarded as a metastasis from the ovarian tumor and not from the breast tumor. Because of these findings, it was concluded that the patient had a primary ovarian carcinoma (International Federation of Gynecology and Obstetrics Stage IIIc) and no recurrence of breast carcinoma. The hormone therapy was stopped, and the patient was treated adequately with chemotherapy, which consisted of paclitaxel and carboplatin.
Fifteen gynecologic patients, all with multiple tumors, were selected for molecular investigations. In all patients, information concerning the clonal correlations between the tumors would have an impact on prognosis or clinical decision-making. The histology and immunohistochemistry of the multiple tumors were similar, making discrimination between multiple primary tumors and recurrent or metastatic disease difficult. Tumors harbor DNA aberrations that can be regarded as tumor specific clonal markers.1 Identification of DNA aberration profiles from multiple tumor localizations within an individual patient can allow the discrimination between multiple primary tumors and recurrent or metastatic disease.18, 27, 28, 31 Therefore, we performed LOH and p53 gene aberration analyses on the multiple tumors in these 15 patients. For LOH analyses, DNA has to be isolated from parts of the tumor with few normal cells. Such parts were obtained either manually or by LCM.29 The molecular assays were based on the ability to amplify specific DNA fragments by PCR. The DNA retrieved from routine formalin fixed and paraffin embedded tissues is highly degraded; therefore, we used small-amplicon PCR (< 200 base pairs) for our analyses. With this procedure, we were able to obtain molecular information from all tumors, even from paraffin tissues that had been stored for > 10 years. In all patients, at least 1 locus showed loss of DNA, and tumor specific p53 alterations were found in 9 of 15 patients. Differing or identical LOH and p53 aberration patterns in multiple tumors point to the presence of multiple primary tumors or to recurrent or metastatic disease, respectively. The molecular information provided evidence that two patients had second primary tumors, nine patients had a single tumor with metastases, and four patients had both independent multiple primary tumors and metastatic disease. In two women (Patients 3 and 11), carcinoma tissue present in lymph nodes was investigated. In both patients, the molecular aberration profiles of the lymph node metastases were identical to one of the other tumors present in the patient. These results demonstrate that definite metastases were identified incontrovertibly as such by our molecular analyses. We recently reported identical results for patients with head and neck carcinoma and lung carcinomas.27 In two women with bilateral ovarian carcinomas (Patients 2 and 9), we found monoclonality. This has also been described by other investigators.15, 18, 24, 25 Despite these results, there remain pitfalls. Intratumor heterogeneity with regard to molecular aberrations can be present.18, 32–37 In addition, genomic instability in tumors proceeds over time, resulting in the possibility of detecting molecular aberrations present in the most recent lesion that are not present in the first lesion of clonally related tumors.38 These findings may be interpreted as suggesting independent lesions, although the tumors, in fact, are related clonally. In metachronous lesions, we regard the presence of specific molecular aberrations in the first tumor and their absence in the second tumor as a strong indication of independent entities. Furthermore, because we investigated only eight chromosomal loci and exons 5–8 of the p53 gene, the possibility that independent tumors by chance harbor the same genomic aberrations cannot be excluded completely. Although mutational hotspots are present in exons 5–8 of the p53 gene,7, 8 we regard the finding of identical aberrant p53 SSCP patterns as a strong indication of related tumors. In trying to ascertain whether these theoretical possibilities influenced the interpretation of our data, we examined every individual genomic aberration as an independent indicator for correlations. No discrepancies were found between individual LOH results in any individual patient. Furthermore, in all patients, the LOH results were consistent with the p53 results. This indicates that the loss of the investigated loci and the p53 gene alterations are present in the majority of tumor cells.
The results of the current study demonstrated that detection of molecular aberrations in multiple tumors within an individual patient can provide clinically relevant information on the correlations between tumors. Molecular analyses can be performed on most routine tissue specimens, and, using the routine molecular biology laboratory infrastructure, results can be obtained within 2–3 weeks. Therefore, it is possible to obtain molecular information from almost all patients.
The authors thank Mustaffa Abbou and Frank van der Panne for help with preparing the tables and figures.