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Keywords:

  • fine-needle aspiration biopsy;
  • in-situ hybridization;
  • polymerase chain reaction;
  • genomics

Abstract

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

The effectiveness of fine-needle aspiration biopsy (FNAB) for rendering a specific diagnosis can be improved by applying several ancillary modalities. This review details several applications of molecular techniques using FNAB specimens with an emphasis on those used for patient care. A detailed search of the literature was conducted to collect all reports that used FNAB for different types of molecular tests. Several types of molecular tests, including in-situ hybridization, polymerase chain reaction, Southern blotting, and gene microarrays using FNAB specimens have been reported. These tests have been used with different organ systems and different objectives, including the detection of cancer cells, diagnosis, distinction of benign and malignant disease, prediction of response to chemotherapy, risk assessment, and selection of patients for targeted therapy. Except for a few tests such as assessment of HER2/neu for gene amplification in breast cancer, detection of clonality in hematopoietic neoplasms, and specific chromosomal translocations in the former and in the diagnosis of soft tissue sarcoma, most of the molecular tests using FNAB specimens are currently investigational. The reported literature indicates the excellent potential of using material procured from FNAB for almost any type of molecular test. Whereas few of these tests alone are used for patient care, some of them have the potential for clinical use in the near future. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.

Fine-needle aspiration biopsy (FNAB) is often the first test for the investigation of lesions, whether palpable or nonpalpable and deep-seated in the body. With advancements in interventional radiology, FNAB has vastly improved our ability to detect and sample small lesions, and the need for open biopsy has sharply declined.

Tissue can be obtained by FNAB or by core needle biopsy (CNB) from almost anywhere in the body that can be used for diagnosis and in cases of malignant lesions for ascertaining prognostic and predictive markers. Although the use of CNB has to some extent decreased the use of FNAB, the techniques are complementary in rendering a specific diagnosis, and they are performed routinely in most institutions. FNAB is superior to CNB in that it permits the acquisition of tissue from very small lesions or, in the case of larger lesions, from multiple sites. An emerging advantage of FNAB in comparison to CNB is its ability to obtain tumor cells with much lower contamination of stroma than CNB for molecular analysis of tissues.

The effectiveness of FNAB for rendering a specific diagnosis can be improved tremendously by the application of several ancillary modalities. Although in many cases cytomorphologic features alone might be sufficient for making a diagnosis, the use of ancillary tests is often necessary not only for rendering a specific diagnosis but, where malignant lesions are involved, for determining prognostic and predictive factors from the procured aspirate. Most currently available ancillary techniques can be used on FNAB specimens. Immunocytochemistry performed on direct smears, monolayered preparation, and cell block sections of FNAB is the most commonly utilized ancillary technique. Immunocytochemistry is widely and routinely used on FNAB specimens for determining the organ of origin of a metastatic tumor, for classification and typing of tumors, and for determining prognostic and predictive markers. There are, however, no immunostains that can help in the distinction of benign from malignant lesions on FNAB. In comparison to immunocytochemistry, molecular tests can aid in the distinction of benign from malignant lesions, in determining the genetic abnormalities and genetic makeup of tumors that can be useful not only for making a more specific diagnosis but also for determining prognosis, response to therapy, and determining the presence or absence of specific molecular targets for selection of patients for targeted therapy.

Because our understanding of the molecular genetics of tumors is growing rapidly, as is the sophistication of molecular techniques, the application of molecular tests on FNAB specimens is also accelerating. Most molecular techniques, including in-situ hybridization, polymerase chain reaction (PCR), and transcriptional profiling, can be performed with FNAB material. Interphase cytogenetic studies by in-situ hybridization techniques, unlike metaphase karyotyping, allow analysis of cytogenetic alterations of individual cells independent of their ability to proliferate. In-situ hybridization with chromogenic or fluorescent signals has distinct advantages over other molecular techniques because it allows comparison of cellular morphology with chromosomal alterations in cells. FNAB specimens, unlike cell-block sections or tissue sections, are particularly suitable for in-situ hybridization. The availability of intact cells makes it possible to count hybridized signals in the nuclei without nuclear transection and the associated inaccuracy in signal counting that can occur with cell-block or tissue sections. With the availability of several commercial DNA probes, fluorescence in-situ hybridization (FISH) has several clinical applications pertaining to FNAB specimens. PCR amplification, which allows automated enzymatic in vitro synthesis of a target DNA sequence in millions of copies for subsequent sequence analysis, is 1 of the most commonly used molecular technique to demonstrate molecular alterations in FNAB specimens. Reverse transcription-polymerase chain reaction (RT-PCR) analysis allows for amplification of very limited quantities of transcripts. This technique is suitable for molecular analysis of limited amounts of material, such as that procured by FNAB. Multiplex PCR allows for amplification of several target sequences simultaneously and is increasingly used for molecular analysis. PCR can be performed using aspirated material collected solely for molecular analysis, from cells scraped from cellular smears, or from 10-μm tissue sections of cell blocks prepared from FNAB samples. The success of the test depends on the amount of viable material available for analysis. FNAB is also being used for microarray analysis. The concept of DNA chip or microarray technology relies on the accurate binding or hybridization of strands of DNA with their precise complementary copies where the known sequences are bound onto a solid-state substrate. These are hybridized with probes of fluorescent cDNAs or genomic sequences from test material. By analyzing the intensity of fluorescence on the chip the expression of several thousands of genes can be determined simultaneously. The data generated in microarray experiments is analyzed using bioinformatics statistical programs. There are few reports indicating the utility of FNAB specimens from sites such as breast, lymph node, and lung, for transcriptional profiling using any of the currently available gene chips for transcriptional profiling.

Although some molecular tests are used for patient care, most are currently investigational only. The reported literature indicates the excellent potential of material procured from FNAB for applications in molecular analysis. This review details several applications of molecular techniques using FNAB specimens, with particular emphasis on those used for patient care. Several other molecular tests that are currently investigational, but with potential for clinical practice, are also discussed. The applications of molecular techniques to FNABs will be outlined under sections pertaining to neoplasms in different organs and a separate section is devoted to the role in the field of infectious diseases.

BREAST NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

Evaluation of human epidermal growth factor receptor 2 (HER2/neu) gene amplification in breast cancer is an important application of FISH using cytology material. HER2/neu, a proto-oncogene located on the long arm of chromosome 17, is an important prognostic and predictive factor in patients with invasive breast cancer.1, 2 Its overexpression is closely related to gene amplification and is seen in 25% to 30% of patients with invasive breast cancer.

The assessment of HER2/neu status is valuable for selecting patients with breast carcinoma for treatment in an adjuvant or neoadjuvant setting with trastuzumab, a humanized monoclonal antibody that targets the HER2/neu receptor.3 HER2/neu is evaluated on all newly diagnosed cases of breast carcinoma and in many cases of metastatic or recurrent breast carcinoma.4 The FISH assay for HER2/neu in a primary breast tumor is usually performed on tissue sections of the tumor and not on an FNAB sample because of the inability of FNAB specimens to distinguishing in-situ from invasive breast cancer. However, because HER2/neu status is usually concordant between primary tumor and locoregional and distant metastasis, FNAB smears or cell block sections of metastatic tumors can be used for evaluating HER2/neu status.

Air-dried cytology smears—either stained with Diff-Quik and then destained before analysis, or unstained smears with evenly spread cells—are most suitable for FISH analysis. The FISH assay for HER2/neu is performed using either of 2 kits approved by the US Food and Drug Administration. Those kits use either an HER2/neu probe alone (Oncor, Gaithersburg, MD) or an HER2/neu probe and a centromere 17 probe (Path Vysion, Vysis, Downers Grove, IL). Using a CEP17 probe and determining the ratio of HER2/neu:CEP17 to express the results of hybridization prevents misdiagnosing cases with polysomy for chromosome 17 as HER2/neu-amplified. A ratio of HER2 to CEP17 of more than 2.0 is considered amplified. Figure 1 is an illustration of a case of FNAB of metastatic breast carcinoma in axillary lymph node showing HER2/neu gene amplification by FISH, which was performed using a direct smear.

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Figure 1. Fine-needle aspiration biopsy of a case of metastatic breast carcinoma in axillary lymph node showing marked HER2/neu gene amplification by fluorescence in-situ hybridization (FISH) using Path Vysion HER2/neu probe (Vysis). Note the markedly increased copy numbers of HER2/neu (orange signal) in contrast to 3–4 copy numbers of CEP17 (green signal) (FISH, original magnification ×1000).

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Several investigators have reported on their work with direct smears obtained from FNAB samples for HER2/neu evaluation by FISH. Bofin et al.5 reported results of HER2/neu gene amplification by FISH on FNAB smears that were comparable to results obtained with tissue sections. They concluded that, by providing whole nuclei, FNAB smears are ideal specimens for evaluation of HER2/neu gene amplification by FISH. Nizzoli et al.6 compared immunocytochemical (ICC) evaluation of HER2/neu protein overexpression with FISH on 66 cytospin smears obtained from FNAB. They reported a concordance of 78% between the 2 tests. They also indicated that, for FNA specimens, FISH was better for evaluation of HER2/neu status than was ICC because ICC offers lower reproducibility and reliability due to specimen and methodologic variability and differences in antibody sensitivity. Mezzelani et al.7 concurred; they reported that FNAB is a reliable technique for evaluation of HER2/neu gene amplification in breast cancer. The use of FNAB specimens for ascertaining HER2/neu gene amplification in patients with metastatic or recurrent breast cancer is gaining popularity for targeting therapy with trastuzumab.

Several molecular tests have been reported to be useful in the distinction of benign from malignant breast lesions if used in conjunction with conventional cytology. These tests include FISH for chromosomal aneusomy, PCR for allelotyping, determination of methylation status, and clonality assays. These tests, however, are largely investigational and are not used in clinical practice. Changes in the copy numbers of chromosomes 1, 8, 11, and 17 have been found to be early events in the development of breast cancer. Interphase FISH for detecting chromosomal aneusomy using FNAB smears of breast lesions has been reported as a reproducible technique with potential for use in conjunction with cytomorphology for diagnosis, risk assessment, and for determining any possible influence on clinical progression and therapy for malignant cases.8–10

In a pilot study of 25 cases, Magda et al.11 used a human androgen receptor clonality assay to assess clonality as a potential adjunct to conventional cytology for the distinction of benign and malignant breast lesions on FNAB specimens. This test allows determination of clonality based on X chromosome inactivation as detected by PCR of genomic DNA after methylase-sensitive restriction digestion. Loss of heterozygosity (LOH) analysis has been used to establish clonality. Certain gene products or chromosomal sites are generally expressed in a heterozygous way in somatic cells. If such a heterozygosity is shown to be lacking, then it is implied that clonality has occurred. The polymorphic sites are amplified by PCR and products are analyzed in the usual way. Euhus et al.12 performed LOH analysis with cells microdissected from Papanicolaou-stained smears of breast cancer samples and concluded that successful allelotyping can be performed with FNAB smears with accurate and reproducible results.

Analysis of the methylation status of selected genes by methylation-specific PCR (MSP) has been investigated for the distinction of benign from malignant lesions. Pu et al.13 examined the methylation status of 3 genes: RAR2, RASSF1A, and cyclin D2 by MSP on archival FNAB samples of breast tissue and found cyclin D2 methylation to be a specific marker of malignancy. In another study, Geronimo et al.14 used a panel of 4 genes, including CDH1, GSTP1, BRCA1, and RAR β, for MSP analysis but did not find them to be discriminatory for benign and malignant breast lesions.

FNAB of breast tumors has also been used for transcription profiling. Assersohn et al.15 tested the feasibility of using FNAB from primary breast tumors for cDNA microarray analysis. Those authors compared the transcriptional profiles obtained from FNAB with that of excised tissue and found them to be comparable. However, because only 15% of FNAB specimens were suitable for microarray analysis, they concluded that validated amplification techniques have to be applied for majority of the FNAB specimens for meaningful microarray analysis. Symmans et al.16 reported that both FNAB and CNB can provide equivalent amounts of RNA for genomic studies. They indicated that transcriptional profiles from FNAB may be more informative about cancer cells alone, whereas profiles generated from CNB are more likely to contain additional information about stroma. Pusztai et al.17 used single-pass FNAB to obtain comprehensive transcriptional profiles of 38 patients with stage I-III breast cancer using cDNA microarrays. There was a strong correlation between estrogen receptor (ER) and HER2/neu status as determined both by transcriptional profiling and by conventional immunohistochemistry on CNB samples. Ayers et al.18 used single-pass FNAB from 42 patients for transcriptional profiling using cDNA microarrays. They described a multigene model comprised of 74 markers that could predict pathologic complete response to chemotherapy with an overall sensitivity and specificity of 43% and 100%, respectively. Sotiriou et al.19 used serial FNAB of primary breast tumors before and after neoadjuvant chemotherapy for transcriptional profiling and identified candidate genes that could distinguish tumors with a probability of complete response from those that do not respond to chemotherapy. The overall findings of reported studies using FNAB for genomic analysis clearly indicates not only the feasibility but also the potential of such studies for clinical and research use in generating specific transcriptional profiles of patients with breast cancer.

SOFT-TISSUE NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

Translocations that lead to recombination of coding sequences of different genes and that result in the formation of pathologic fusion genes and expression of pathologic gene fusion products are common and specific in soft-tissue sarcomas.20–22 Recognition of these specific cytogenetic alterations is valuable for making accurate diagnosis of soft-tissue tumors by molecular analysis. The chromosomal translocation (11;22) (q24;q12) is specific for Ewing sarcoma, peripheral neuroepithelioma, and Askin tumor. This cytogenetic alteration rearranges and fuses the EWS gene on chromosome 11 and FLI1 genes of the ETS transcription family on chromosome 22 and is a sensitive and specific molecular genetic test for diagnosis of these tumors. EWS gene fused with genes belonging to some of the other members of the ETS transcription factor family is specific for other soft-tissue tumors. When fused with the ATF-1 gene resulting from t(12;22)(q13;q12) it is specific for clear-cell sarcoma; when fused with the WT-1 gene in t(11;22)(p13;q12) it is specific for desmoplastic small round cell tumor; when fused with the CHN gene in t(9;22)(q22;q12) it is specific for myxoid chondrosarcoma.

Alveolar rhabdomyosarcoma is characterized by 2 tumor-specific chromosomal translocations, t(2;13)(q35;q14) and t(1;13)(p36;q14), resulting in fusions of the PAX3 and PAX7 genes, which are members of the PAX transcription factor gene family mapped to 2q35 and 1p36 with the FKRH gene mapped to 13q14. Synovial sarcoma is characterized by specific t(X;18)(p11;q11) translocation involving the SYT gene on chromosome 18 and 1 of the SSX genes on chromosome X, leading to functional fusion (SYT-SSX). These translocations are specific for synovial sarcoma and are particularly valuable for diagnosis of the small-cell and monophasic variants of synovial sarcoma.

The specific cytogenetic alterations in soft-tissue tumors can be detected using karyotypic analysis, PCR, or in-situ hybridization. There are rare reports indicating the utility of FNAB specimens for karyotypic analysis of sarcomas. Saboorian et al.23 could make a definitive diagnosis of 2 cases of synovial sarcoma based on the demonstration of translocation t(x;18) (p11.2; q11.2) by karyotypic analysis of G-banded chromosome preparations. Touch preparations of CNB and cytospin or monolayer preparations of FNAB samples of primary or recurrent sarcomas are excellent specimens for FISH testing because of the availability of single cells for analysis. Figures 2 and 3 are illustrations of a case of Ewing sarcoma and synovial sarcoma showing the specific chromosomal translocation of t(11;22) (q24;q12) and t(x;18) (p11.2; q11.2) as demonstrated by FISH that was performed using touch preparations of the core biopsy. Hummel et al.24 reported the utility of FISH and conventional karyotyping to demonstrate the balanced translocation between chromosomes X and 18 in 2 cases of primary and metastatic synovial sarcoma in lung. Sapi et al,25 in reporting on their experience with diagnosis of soft tissue tumors by FNAB, emphasized ancillary techniques, such as FISH used in conjunction with immunostaining for definitive preoperative diagnosis. They performed ancillary FISH on 30 of the 94 tumors from the National Soft Tissues Consortium of Hungary and found this test to be useful for making a diagnosis of synovial sarcoma in 6 cases, Ewing sarcoma/PNET in 5 cases, myxoid liposarcoma in 5 cases, clear-cell sarcoma in 2 cases, and desmoplastic small round cell tumor in 1 case. Molecular analysis performed on a single case with an FNAB sample to demonstrate EWS/WT1 chimeric transcript leading to a definite diagnosis of desmoplastic soft tissue sarcoma has been reported by Ferlicot et al.26

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Figure 2. Fine-needle aspiration biopsy of a case of Ewing sarcoma showing small round cells in Diff-Quik-stained (A) direct smear and demonstrating (B) yellow fusion signals by fluorescence in-situ hybridization (FISH) indicating t(11:22) (q24; q12) chromosomal translocation using the LSI-EWSRI (22q12) dual color probe (Diff-Quik stain and FISH, original magnification ×50 and 1000).

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Figure 3. Fine-needle aspiration biopsy of a case of synovial sarcoma showing relatively bland spindle cells in (A) Papanicolaou-stained direct smear and demonstrating (B) yellow fusion signals by fluorescence in-situ hybridization (FISH) indicating t(x;18) (p11;q11) chromosomal translocation using dual color break apart SYT 18 q11.2 probe (Papanicolaou stain and FISH, original magnification ×50 and 1000).

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There are rare reports of using FNAB in conjunction with ancillary FISH analysis to demonstrate 1p deletion and N-myc amplification for molecular characterization of neuroblastic tumors.27

Molecular analysis to demonstrate c-kit mutations can also be performed using FNAB material for making a diagnosis of primary and recurrent gastrointestinal stromal tumors (GIST). These tumors differentiate along the lines of interstitial cells of Kajal and are positive for c-kit, which is used for diagnosis and for predicting response to therapy with imatinib therapy.28 Immunostaining for c-kit alone might be sufficient to make a diagnosis and predict response to imatinib therapy.29 But in a small percentage of cases with confusing immunophenotypic findings because of overlapping features with other spindle cell tumors or for the diagnosis of rare c-kit-negative cases of GIST, molecular analysis to demonstrate c-kit mutations could be valuable. Molecular analysis can be successfully performed on FNAB samples in selected cases of GIST, as demonstrated by Willmore-Payne et al.30

HEMATOPOIETIC NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

FNAB is often used to diagnose malignant lymphomas. Because of considerable cytomorphologic overlap between reactive hyperplasia and small B-cell and T-cell lymphomas and between the different subtypes of lymphomas, cytomorphology alone is of limited value, particularly for the diagnostic classification of small B-cell and T-cell lymphomas. Ancillary immunophenotyping and molecular analysis are necessary for establishing monoclonality and typing malignant lymphomas using FNAB. Such analyses can be successfully performed in the majority of cases when the quality of the procured aspirate with respect to cellularity is good. Using a multiparametric approach, specifically diagnosing many cases of primary and recurrent lymphoma based on the current (2001) World Health Organization criteria, is possible in many laboratories and is gaining in popularity.31–36 The inability to procure satisfactory aspirate because of fibrosis or necrosis in the involved lymph node can lead to failure in rendering a specific diagnosis. Of note is that cytomorphologic features in conjunction with immunophenotyping of aspirated material using flow cytometry and/or immunocytochemistry (ICC) alone may be sufficient for making a specific diagnosis in many cases when the results are characteristic of a particular type of lymphoma. However, the occurrence of immunophenotypic variability among particularly small B-cell non-Hodgkin lymphoma (B-NHL) subtypes can be a confounding variable in cases that require additional molecular testing. In cases of T-cell lymphoma, molecular testing is usually needed to demonstrate T-cell receptor gene rearrangement because immunophenotyping using flow cytometry or ICC alone may not be very informative. Therefore, in selected cases of malignant lymphoma where immunophenotyping using flow cytometry and/or ICC produces confusing results, ancillary molecular testing using FISH and/or PCR can be effective.

Molecular tests such as Southern blotting, PCR, and FISH can be used in selected cases with FNAB to demonstrate clonality.37–47 Of these, PCR is the most widely used for detection of immunoglobulin (Ig) heavy-chain and T-cell receptor gene rearrangements. PCR has been used also from archival cytologic smears to make a diagnosis of B-cell lymphomas.42 Although the majority of studies have found PCR to be useful for assessing clonality, Stewart et al.44 found ICC and FISH for detection of Ig light-chain mRNA to be more valuable than PCR because PCR produced misleading results in 16% of their cases.

Safley et al.45 used a multiparametric approach that included cytology, flow cytometry, and PCR. Whereas they used flow cytometry and PCR to evaluate clonality, they used FISH and PCR to detect bcl-1 and bcl-2 gene rearrangements in aspirates obtained from 30 consecutive patients with suspected hematolymphoid malignancies. In that study the investigators diagnosed all cases of low-grade follicular lymphoma and mantle cell lymphoma and 50% of the cases of small lymphocytic lymphoma and large B-cell lymphoma using this approach. Venkatraman et al.46 also reported on the use of PCR to detect clonality and specific translocations in the diagnosis of malignant lymphoma using FNAB. They demonstrated a concordance of 78.2% between PCR and ICC in the detection of clonality in that study. Clonality assessment using PCR is most useful for T-cell malignant lymphoma and in cases of B-cell NHL that are morphologically and/or clinically suspicious but lack Ig light-chain restriction by immunophenotyping using flow cytometry and/or ICC. Figure 4 is an illustration of a representative case of T-cell malignant lymphoma showing the result of T-cell receptor gamma chain PCR analysis detected by capillary electrophoresis that clearly shows clonal T-cell receptor gamma rearrangement. Partial involvement of the lymph nodes by malignant lymphoma resulting in the presence of abundant polyclonal cells in aspirate, hypermutation in the rearranged VH region hindering efficient binding of the primers, and t(14;18) translocation of follicular lymphoma including the IgH locus that can render 1 allele unsuitable for VDJ amplification can all be attributed to false-negative results of PCR. False-positive assessment of clonality can occur rarely in human immunodeficiency virus-positive patients. Of note is that the occurrence of monoclonal IgH bands has been reported in 6% of aspirates obtained from patients with reactive lymphoid hyperplasia, which can result in a false-positive diagnosis.46

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Figure 4. Fine-needle aspiration biopsy of a case of T-cell malignant lymphoma showing small lymphoid cells in Diff-Quik-stained (A) direct smear and (B) result of T-cell receptor gamma chain PCR analysis, detected by capillary electrophoresis. The large red peaks represent biallelic clonal T-cell receptor-gamma rearrangement (Diff-Quik stain, original magnification x50).

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Molecular tests such as FISH and PCR can be used to demonstrate the relatively specific chromosomal abnormalities in selected cases of suspected malignant lymphoma using FNAB.46–54 PCR has also been used to demonstrate bcl2 rearrangement for making a diagnosis of follicular lymphoma using archival cytological smears of FNAB of lymphomas.53 The utility of a FISH assay to demonstrate the t(11;14)(q13;q32) translocation in diagnosing mantle cell lymphoma using FNAB samples with the probes at the 14q32 and 11q13 (cyclin D1) loci has been reported by a few investigators.49–51 Shin et al.52 indicated the use of FISH for detection of ALK breakpoints to demonstrate t(2;5)(p23;q35) using aspirated material. Demonstration of this chromosomal translocation can be useful in the identification of a subset of patients with T-null anaplastic large cell lymphoma who may have a favorable prognosis. Similarly, FISH can be used to demonstrate the t(14;18)(q32;q21) translocation in selected cases for diagnosis of follicular lymphoma when cellularity is low to perform immunophenotyping using flow cytometry and/or ICC or if the results are either noncontributory or not classical.54 Illustrations of a representative case of mantle cell lymphoma and follicular lymphoma showing the presence of t(11;14) (q13;q32) and t(14;18) (q32;q21) chromosomal translocations as demonstrated by FISH performed on cytospin preparations obtained from FNAB of lymph nodes is shown in Figures 5 and 6.

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Figure 5. Fine-needle aspiration biopsy of a case of mantle cell lymphoma comprised of small lymphoid cells in (A) Papanicolaou-stained direct smear and demonstrating (B) yellow fusion signals by fluorescence in-situ hybridization (FISH) using cyclin D1 and immunoglobulin heavy chain probes indicating t(11;14)(q13;q32) chromosomal translocation (Papanicolaou stain and FISH, original magnification ×50 and 1000).

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Figure 6. Fine-needle aspiration of a case of follicular lymphoma comprised predominantly of small lymphoid cells in (A) Papanicolaou-stained direct smear and demonstrating (B) yellow fusion signals by fluorescence in-situ hybridization (FISH) using bcl-2 and immunoglobulin heavy chain probes indicating t(14;18)9q32;q21) chromosomal translocation (Papanicolaou stain and FISH, original magnification ×50 and 1000).

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The only study using FNAB of lymph nodes for transcriptional profiling is that of Goy et al.55 Those authors tested the feasibility of performing gene expression profiling using FNAB of lymph nodes obtained from 24 patients with a diagnosis of follicular lymphoma and diffuse large B-cell lymphoma. They identified 72 genes that were differentially expressed between these 2 types of lymphomas. That study indicates the potential of FNAB specimens of lymph nodes for genomic studies with tremendous implications for clinical and research studies.

THYROID NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

FNAB is routinely used to guide management of patients in the preoperative evaluation of thyroid nodules. Although it can be useful for providing a definite preoperative diagnosis, in 5% to 10% of cases the results are inadequate, and in 20% of cases the findings could be indeterminate for malignancy. Patients whose thyroid nodules show indeterminate or suspicious cytologic features in FNAB samples, however, would require thyroidectomy because of a 20% risk of thyroid cancer.56 Few molecular tests have been investigated for possible use in concert with conventional cytologic examination of thyroid FNAB to aid in selecting candidates for thyroidectomy. Of the several genetic alterations, telomerase, RET/PTC rearrangements, and BRAF mutation have been studied with FNAB of the thyroid.

More than half of all papillary thyroid carcinomas (PTC) harbor at least 1 of several chimeric oncogenes, called RET/PTC, which results from gene rearrangements involving the ret proto-oncogene on chromosome 10 resulting in the generation of novel fusion transcripts.57, 58 These transcripts constitute a specific molecular marker for PTC that is identified in almost half of the tumors. Cheung et al.59 studied 73 thyroid aspirates for the presence of common ret/PTC gene rearrangements by RT-PCR and Southern hybridization and they found ret/PTC gene rearrangements in 52% of the cases. Based on the absence of false-positive cases and on the identification of ret/PTC gene rearrangement in 9 of 15 indeterminate and in 2 of 6 inadequate cases, that study supports the usefulness of RET/PTC RT-PCR as an ancillary test to cytology in selected cases for a definitive diagnosis of PTC.

Evaluation of BRAF mutations in thyroid FNAB samples by RT-PCR has also been investigated as an adjunct to cytology for making a definitive diagnosis of PTC. BRAF mutations are found in 70% of PTC cases and they are highly specific; they have not been noted in benign nodules or in other thyroid malignancies.60 Cohen et al.61 examined BRAF mutational status in FNAB samples and in subsequent surgically excised specimens by PCR. Analysis of the PCR products for BRAF mutation was performed using direct automated sequencing (sequence the nucleotides in the BRAF gene) and/or the Mutector assay. The Mutector assay is designed for the detection of any type of known DNA mutation. A detection primer is designed that does not permit primer extension when the target base is not mutated. In that study, BRAF mutations confirmed the preoperative diagnosis of PTC in 72% of cases. BRAF mutations also established the presence of malignancy in 16% of carcinomas that could not be diagnosed conclusively by FNAB alone. Rowe et al.62 also reported that BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. Using BRAF mutation analysis by PCR 15.8% of the FNAB samples could be conclusively diagnosed as PTC by those authors. The use of FNAB in evaluating ret/PTC and BRAF mutations for making a diagnosis of PTC in indeterminate and suspicious cases of thyroid malignancies will require validation in future studies.

Several groups have investigated telomerase activity in thyroid FNAB samples to distinguish malignant from benign lesions. Telomeres are highly conserved hexameric nucleotide repeats at the ends of chromosomes.63 Telomerase activity and hTERT gene expression are noted in malignancy and in such inflammatory conditions as lymphocytic thyroiditis and up to 40% of benign lesions, such as follicular adenomas. The utility of measuring hTERT gene expression in thyroid FNABs has been investigated in several studies. Umbricht et al.64 tested hTERT mRNA by RT-PCR on 100 thyroid FNAB samples that were suspected of but not diagnostic for malignancy. They reported an overall sensitivity of 91% and specificity of 79% after excluding cases of lymphocytic thyroiditis. The low specificity was attributed to hTERT positivity in 35% of lesions that on surgical excision proved to be benign. The false-negative and false-positive results were 10% and 35%, respectively. Siddiqui et al.65 used archival Diff-Quik stained smears of thyroid FNAB for hTERT expression by RT-PCR. Whereas 67% of malignant tumors were positive in that study, 29% hyperplastic nodules and 38% of follicular adenomas also showed hTERT gene expression. Mora and Lerma66 also studied the utility of evaluating telomerase activity on thyroid FNAB samples by using the telomerase PCR enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics, Indianapolis, IN), a method based on the telomere repeat amplification protocol, which uses PCR with colorimetric detection of products. They reported sensitivity of 41.4% and specificity of 100%. FNAB sampling alone produced sensitivity of 85.7%. These studies therefore indicate that although measurement of telomerase activity is a potentially useful adjunct to cytology for distinguishing benign from malignant thyroid nodules when thyroiditis is excluded, the occurrence of telomerase activity in benign tumors decreases the specificity of the test in the preoperative diagnosis of thyroid nodules on FNAB specimens.

Kebebew et al.67 published the only study to use the results of gene expression profiling for FNAB samples to aid in distinguishing benign from malignant lesions in the thyroid. The researchers used extracellular matrix and adhesion molecule cDNA arrays to identify differentially expressed genes that were subsequently confirmed by real-time quantitative RT-PCR. They found several genes to be up-regulated in malignant tumors and by quantitative PCR expression of extracellular matrix protein (ECM1) and transmembrane protease serine 4 (TMPRSS4) mRNA was determined to be an independent predictor of malignant thyroid neoplasm. Testing for these markers by RT-PCR improved the diagnostic accuracy of FNAB in 35 of 38 cases with indeterminate results on cytology. The same authors used a real-time quantitative RT-PCR assay of 6 candidate genes including ECM1, TMPRSS4, angiopoietin 2, tissue metallopeptidase inhibitor 1, ephrin-B2, and epidermal growth factor receptor EGFR in FNABs of 31 thyroid nodules.68 This novel multigene assay provided a sensitivity of 91.0% and specificity 95.0% for distinction of benign from malignant disease. The utility of measuring mRNA of genes such as that described by Kebebew et al. by RT-PCR for the preoperative distinction of benign and malignant thyroid nodules from FNAB samples is promising and should be validated in prospective trials.

PULMONARY NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

The sensitivity of lung FNAB in the diagnosis of bronchogenic carcinoma is around 80%.69 The paucity of diagnostic material and the overlapping cytologic features between reactive and malignant processes leads to results such as “atypical,” “suspicious,” or “indeterminate” for malignancy. The need for ancillary molecular testing with lung FNAB arises in some cases with indeterminate or suspicious cytology findings where corresponding CNB are unavailable or when CNB could not be performed either because of technical reasons or because of the development of pneumothorax. The availability of the multitarget FISH assay (La Vysion, Vysis), which includes probes for chromosome 6p11-q11, 7p12 (EGFR), 8q24 (myc), and 5p15.2-chromosomal loci commonly affected in nonsmall-cell lung carcinoma (NSCLC) can be used to distinguish benign from malignant lesions. Savic et al.70 tested a multitarget FISH assay in destained Papanicolaou (Pap)-stained smears from 101 cases with equivocal respiratory cytology that included mainly exfoliative cases and only 8 transbronchial cases of FNAB. They reported an overall sensitivity and specificity of 79% and 100%, respectively. Because FISH analysis using the multitarget probe can be performed on destained Pap smears, this test has potential in clinical practice for making a definite diagnosis in selected cases of lung FNAB. The c-myc E2F-1/p21 WAF1/Cip1 interactive gene expression index has been used for standardized RT-PCR testing of lung FNAB results by Warner et al.71 with a sensitivity of 100% and specificity of 94%. The contribution of these molecular tests as an adjunct to cytology needs further validation.

In addition to the potential role of molecular tests for the diagnosis of bronchogenic carcinoma in selected cases of FNAB of lung, there also is a role for molecular tests performed on FNAB material to aid in the selection of patients with advanced NSCLC for therapy with epidermal growth factor receptor (EGFR) inhibitors. EGFR has emerged as a leading target for the treatment of patients with NSCLC. EGFR protein expression alone fails to predict response to EGFR inhibitors. Specific mutations in the EGFR gene has been reported to predict good response to tyrosine kinase (TK) inhibitors.72, 73 RT-PCR-based assays that target specific mutations can be easily performed on FNAB material. There are also reports indicating the utility of assessing EGFR gene amplification for selecting patients for EGFR inhibitor therapy.74 Whether gene amplification or detection of mutation in the TK domain should be used for patient selection is not entirely clear.75 Nevertheless, both tests can be performed with FNAB samples.

Lim et al.76 tested the feasibility of performing gene expression profiling of advanced NSCLC using RNA from 46 cases of FNAB of lung and showed that transcriptional profiles generated from FNAB was comparable with that from surgically excised tissue.

METASTATIC TUMOR IN LYMPH NODE

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

PCR for selected markers has been used to explore their potential utility in increasing the sensitivity of conventional cytology for detecting metastasis in FNAB samples from lymph nodes. Most of the investigations pertain to endoscopic ultrasound (EUS)-guided FNAB. Pellise et al.77 used MSP to detect hypermethylation of CpG islands in promoter regions of MGMT, p16 and p14 as markers for micrometastases in EUS-FNAB samples of lymph nodes from 42 patients with gastrointestinal and lung cancer. The researchers reported sensitivity, specificity, and accuracy of conventional cytology as 76%, 100%, and 88%, respectively, compared with 81%, 67%, and 76% for methylation analysis. The usefulness of EUS-FNAB for detecting micrometastasis in lymph nodes by RT-PCR has also been reported by Wallace et al.78 That group initially used RT-PCR to detect levels of human telomerase reverse transcriptase mRNA to identify metastasis in the lymph nodes in patients with NSCLC and reported a high false-positive rate of 57%—attributed to telomerase activity in reactive lymph nodes. In a subsequent study they used quantitative RT-PCR to measure expressions of lung-cancer-associated genes to detect occult metastasis in 87 clinically negative mediastinal lymph nodes in patients with lung cancer.79 In that study, gene transcripts of metastatic disease were present in at least one-third of the cytology-negative cases and the molecular marker with the highest observed sensitivity and specificity for NSCLC was KS1/4, a gene that encodes a glycoprotein that is expressed on epithelial cells and recognized by the monoclonal antibody BerEP4.

Myo et al.80 studied the value of numerical aberrations in cyclin D1 gene copy numbers by FISH on FNAB specimens in patients with previously untreated stage I and II oral squamous cell carcinoma who had not undergone radical neck dissection. They showed that Cyclin D1 numerical aberrations independently predicted late cervical lymph node metastasis and that the results of such an analysis could be a valuable marker for poor prognosis, tumor aggressiveness, recurrence, and for selecting patients for elective cervical lymph node dissection. Voit et al.81 used RT-PCR for tyrosinase, a tissue-specific enzyme that regulates melanin biosynthesis, to detect metastasis of melanoma in FNAB samples from lymph nodes. The sensitivity was not significantly different for cytology and RT-PCR: 90% vs 100%. However, for metastases smaller than 1 cm the sensitivity was 100% for RT-PCR compared with 78% for cytology.

Conventional cytology alone might be sufficient for detecting most macrometastasis in lymph nodes, but ancillary molecular tests can be used to detect small metastases. The impact of detecting small-sized metastasis by molecular techniques in the management of different malignancies is not presently clear and is to be considered currently investigational.

PANCREATIC NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

The sensitivity for EUS-FNA of solid pancreatic masses for the diagnosis of pancreatic adenocarcinoma ranges from 60% to 95%, with specificities nearing 100%.82 The paucity of diagnostic material, the overlapping features of low-grade malignant tumors, and reactive processes such as chronic pancreatitis make definite diagnosis of malignancy difficult if not impossible in some cases. Mutations of K-ras oncogene have been most frequently investigated using FNAB specimens as a possible adjunct to conventional cytology for making a definite diagnosis of pancreatic adenocarcinoma. The K-ras oncogene is activated by point mutations in 75% to 90% of pancreatic adenocarcinoma with typical localization in codon 12. Evans et al.83 used mutant-enriched PCR to detect point mutations in K-ras in pancreatic FNAB samples and reported that 21 of 25 specimens showed K-ras mutations. Tada et al.84 evaluated mutant K-ras gene semiquantitatively by PCR and enzyme linked mini-sequence assay of 34 FNAB specimens obtained from pancreatic masses and found high amounts of mutant gene in 77% of cases with pancreatic carcinoma and absent or low levels in the 8 patients with chronic pancreatitis in that study. Van Heek et al.85 also studied K-ras mutations by PCR in 94 pancreatic aspirates and found 66% of cases that were subsequently proven to be pancreatic adenocarcinoma to harbor mutations. It is notable that K-ras mutations has been described in few proven cases of chronic pancreatitis, which decreases the specificity and therefore the clinical utility of this test in the diagnosis of difficult cases of pancreatic FNABs, particularly the low-grade types where there is maximum need for an ancillary test that can be used in conjunction with conventional cytology.86 Mishra et al.87 prospectively evaluated semiquantitative PCR for assessment of telomerase activity as a possible ancillary test to increase the sensitivity of pancreatic FNAB in 71 patients. The incremental benefit of this test when compared with cytology alone was 13% (from 85–98%). Because telomerase is not always expressed in all pancreatic adenocarcinomas, its role as a molecular marker must be approached with caution because sensitivity can never reach 100%. Kitoh et al.88 successfully performed CGH analysis on 15 cases of pancreatic FNAB samples, indicating that this technique can be used in comprehensive genetic analysis of these types of samples. Comparative genomic hybridization (CGH) enables the study of global chromosomal aberrations in patient tissues without the need to culture the constituent cells. Target mutant DNA and normal DNA are tagged with different fluorescent signals and are mixed and applied to the normal metaphase chromosome preparations. The normal and mutant DNAs compete to hybridize with their complementary chromosomal loci. Digital image scanning is then used to quantify and compare the relative amounts of differentially colored signals indicating loss or gain of DNA in a given chromosomal locus. Laurell et al.89 conducted gene expression profiles of pancreatic adenocarcinoma on surgically obtained tissue to identify differentially expressed genes, which were subsequently validated by RT-PCR in EUS-FNAB. They used 2 of the differentially expressed genes, lipocalin 2 (LCN2) and PLAT (tissue type plasminogen activator or tPA), for validation by RT-PCR in 12 FNAB samples and reported them to be significantly increased in all the samples. Lipocalins are small extracellular proteins with an important role in cell proliferation and differentiation. PLAT is important in tumor angiogenesis and in the development of exdocrine pancreatic cancer, contributing to the invasive phenotype. The utility of evaluating markers, such as lipocalin 2 and PLAT by RT-PCR using FNAB of pancreas for the diagnosis of pancreatic adenocarcinoma, needs further validation in future studies.

RENAL NEOPLASMS

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

Molecular tests have been investigated as potential ancillary aids for making a definite diagnosis of malignancy on renal FNA. Li et al.90 reported on the utility of detecting MN/CA9 gene expression by RT-PCR in FNAB of kidney. MN/CA9 belongs to the carbonic anhydrase family and is up-regulated in many cancers in response to hypoxic conditions and is regulated by hypoxia inducible factor 1-alpha.91 It has been established as a reliable biomarker for RCC and is present in almost all clear RCCs and in about 56% of papillary RCCs, but is absent from chromophobe RCCs, oncocytomas, and normal tissue.92 Li et al. studied 35 patients with solid renal masses that were indeterminate by radiological examination. The overall sensitivity and specificity were 68% and 100%, respectively. Because MN/CA9 protein expression can also be demonstrated by ICC, the clinical utility of RT-PCR analysis of renal masses for making a diagnosis of renal cell carcinoma is doubtful.

Whereas much progress has been made in the field of cytogenetics and molecular genetics of renal tumors, there are, however, very few reports using FNAB for detecting cytogenetic abnormalities of renal neoplasms. Li et al.93 performed conventional cytogenetic analysis from aspirated material of a case of adult Wilms tumor in the kidney. There is a single report of FISH performed on FNAB of kidney tumors. Al-Khafaji et al.94 used centromere-specific probes for chromosome Y, 7, 17, 16, 12, 8, and 3 to study chromosomal alterations in FNA and effusions of primary and metastatic renal cell carcinoma and found numerical aberrations of chromosome 3 to be most frequent in RCC.

INFECTIOUS DISEASE

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES

There are few reports that compare the increased sensitivity of molecular methods for detection of infectious organisms on FNAB samples from lymph nodes with that available from conventional methods, including cytologic examination, histochemical staining, or microbiologic cultures. Most of the reports discuss PCR on FNAB samples from lymph nodes as an adjunct to conventional methods for the diagnosis of tuberculous lymphadenitis. Molecular tests can be used in conjunction with the traditional method of histochemical staining for acid-fast organisms and microbiologic cultures to increase the rate of detection and to identify resistant strains.

Bruijnesteijn et al.95 developed an RT-PCR assay to diagnose and identify the causative agent of suspected mycobacterial lymphadenitis using primers to detect M. avium and M. tuberculosis. This assay detected mycobacterial infections in 71.6% of patients; auramine staining and culture were positive in 46.3% and 41.2% of patients, respectively. Aljafari et al.96 and Gong et al.97 reiterated the promising role of PCR for increasing the rate of detection of mycobacterial infections, particularly in cases where tuberculous lymphadenitis is suspected but no epithelioid granulomas or acellular necrosis are noted in direct smears.

Avidor et al.98 developed a PCR method for the amplification of Bartonella henselae DNA, and showed that the results of this test can be useful for making an accurate diagnosis of cat-scratch disease in FNAB specimens of lymph nodes and primary lesion in comparison to Warthin-Starry silver impregnation stains and culture and can obviate the need for excisional biopsy.

The detection of human papillomavirus (HPV) DNA in metastatic squamous cell carcinoma by molecular methods, including PCR or in-situ hybridization, can be used in selected cases to support a possible anogenital origin of the tumor. Although the exact frequency of HPV DNA in SCC in different anatomical sites is debated, its detection in FNAB samples would be useful if the differential diagnosis includes tumors reported to have a low prevalence of HPV DNA such as from lung, esophagus, and skin in nonimmunocompromised patients as reported by Starac et al.99 The presence of high-risk HPV type in that case would suggest either an anogenital or a head-and-neck origin of the carcinoma.

The reported literature indicates the excellent potential of using material procured from FNAB for almost any type of molecular test. These tests have been used with different organ systems and different objectives, including the detection of cancer cells, diagnosis, distinction of benign and malignant disease, prediction of response to chemotherapy, risk assessment, and selection of patients for targeted therapy. It is notable that except for a few tests such as assessment of HER2/neu for gene amplification in breast cancer, detection of clonality in hematopoietic neoplasms, and specific chromosomal translocations in the former and in the diagnosis of soft-tissue sarcoma, most of the molecular tests using FNAB specimens are currently investigational. However, some of these tests have the potential for clinical use in the coming years. The possible integration of molecular tests with current practice for the distinction of benign from malignant lesions in selected cases, determining the genetic makeup of tumors, and identifying specific molecular targets for typing, diagnosis, determining prognosis, and response to therapy are some of the anticipated uses of molecular tests as applied to FNAB in the near future.

REFERENCES

  1. Top of page
  2. Abstract
  3. BREAST NEOPLASMS
  4. SOFT-TISSUE NEOPLASMS
  5. HEMATOPOIETIC NEOPLASMS
  6. THYROID NEOPLASMS
  7. PULMONARY NEOPLASMS
  8. METASTATIC TUMOR IN LYMPH NODE
  9. PANCREATIC NEOPLASMS
  10. RENAL NEOPLASMS
  11. INFECTIOUS DISEASE
  12. REFERENCES
  • 1
    Rubin I,Yarden Y. The basic biology of HER2. Ann Oncol. 2001; 2: 38.
  • 2
    Slamon DJ,Godolphin W,Jones LA, et al. Studies of the HER2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989; 224: 701712.
  • 3
    Leyland-Jones B. Trastuzumab: hopes and realities. Lancet Oncol. 2002; 3: 187144.
  • 4
    Thor A. HER-2. A discussion of testing approaches in the USA. Ann Oncol. 2001; 12: 101107.
  • 5
    Bofin AM,Ytterhus B,Martin C,O'Leary JJ,Hagmar BM. Detection and quantitation of HER-2 gene amplification and protein expression in breast carcinoma. Am J Clin Pathol. 2004; 122: 110119.
  • 6
    Nizzoli R,Bozzetti C,Crafa P, et al. Immunocytochemical evaluation of HER-2/neu on fine-needle aspirates from primary breast carcinomas. Diagn Cytopathol. 2003; 28: 142146.
  • 7
    Mezzelani A,Alasio L,Bartoli C, et al. c-erbB2/neu gene and chromosome 17 analysis in breast cancer by FISH on archival cytological fine-needle aspirates. Br J Cancer. 1999; 80: 519525.
  • 8
    Patterson AH,McMamus DT,Maxwell P. Detection of chromosomal numerical abnormalities in clinical breast tumor fine-needle aspirations by fluorescence in situ hybridization (FISH): requirement of a method. Br J Biomed Sci. 1998; 55: 27.
  • 9
    Sneige N,Liu B,Yin G,Gong Y,Arun BK. Correlation of cytologic findings and chromosomal instability detected by fluorescence in situ hybridization in breast fine-needle aspiration specimens from women at high risk for breast cancer. Mod Pathol. 2006; 19: 622629.
  • 10
    Tsukamoto F,Miyoshi Y,Koyama H, et al. Detection of chromosomal aneusomy by fluorescence in situ hybridization in fine-needle aspirates from breast tumors: application to the preoperative diagnosis of breast carcinoma. Cancer (Cancer Cytopathol). 2000; 90: 373378.
  • 11
    Magda JL,Minger BA,Rimm DL. Polymerase chain reaction-based detection of clonality as a non-morphologic diagnostic tool for fine-needle aspirations of the breast. Cancer (Cancer Cytopathol). 1998; 84: 262267.
  • 12
    Euhus DM,Maitra A,Wistuba II. Use of archival fine-needle aspirates for the allelotyping of tumors. Cancer (Cancer Cytopathol). 1999; 87: 372379.
  • 13
    Pu RT,Laitala LE,Alli PM,Fackler MJ,Sukumar S,Clark DP. Methylation profiling of benign and malignant breast lesions and its application to cytopathology. Mod Pathol. 2003; 16: 10951101.
  • 14
    Geaonimo C,Costa J,Martins MC, et al. Detection of gene promoter hypermethylation in fine needle washing from breast lesions. Clin Cancer Res. 2003; 9: 34133417.
  • 15
    Assersohn L,Gangi L,Zhao Y, et al. The feasibility of using fine needle aspiration from primary breast cancers for CDNA microarray analysis. Clin Cancer Res. 2002; 8: 794801.
  • 16
    Symmans WF,Pusztai L,Ayers M, et al. RNA yield from needle biopsies for cDNA microarray analysis of breast cancer prior to neoadjuvant chemotherapy. Cancer. 2003; 97: 29602971.
  • 17
    Pusztai L,Ayers M,Stec J. Gene expression profiles obtained from fine-needle aspirations of breast cancer reliably identify routine prognostic markers and reveal large-scale molecular differences between estrogen-negative and estrogen-positive tumors. Clin Cancer Res. 2003; 9: 24062415.
  • 18
    Ayers M,Symmans WF,Stec J, et al. Gene expression profiling of fine needle aspirations of breast cancer identifies genes associated with complete pathologic response to neoadjuvant. Taxol/FAC chemotherapy. J Clin Oncol. 2004; 22: 22842293.
  • 19
    Sotiriou C,Powles J,Dowsett M, et al. Gene expression profiles derived from fine needle aspiration correlate with response to systemic chemotherapy in breast cancer. Breast Cancer Res. 2002; 4: R3.
  • 20
    Dei Tos AP,Dal Cin P. The role of cytogenetics in the classification of soft tissue tumors. Virchows Arch. 1997; 431: 8394.
  • 21
    Nilbert M. Molecular and cytogenetics of soft tissue sarcomas. Acta Orthoph Scand. 1997; 68: 6067.
  • 22
    Fletcher CD. Soft tissue tumors. The impact of cytogenetics and molecular genetics. Verh Dtsch Cres Pathol. 1997; 81: 318326.
  • 23
    Saboorian MH,Ashfaq R,Vardersteenhoven TJ, et al. Cytogenetics as an adjunct in establishing a definitive diagnosis of synovial sarcoma by fine needle aspiration. Cancer (Cancer Cytopathol). 1997; 81: 187192.
  • 24
    Hummel P,Yang GCH,Kumar A, et al. PNET-like features of synovial sarcoma of the lung: a pitfall in the cytologic diagnosis of soft-tissue tumors. Diagn Cytopathol. 2001; 24: 283288.
  • 25
    Sapi Z,Antal I,Papai Z, et al. Diagnosis of soft tissue tumors by fine-needle aspiration with combined cytopathology and ancillary techniques. Diagn Cytopathol. 2002; 26: 232242.
  • 26
    Ferlicot S,Coue O,Gilbert E. et al. Intra-abdominal desmoplastic small round cell tumor. Report of a case with fine needle aspiration, cytologic diagnosis and molecular confirmation. Acta Cytol. 2001; 45: 617621.
  • 27
    Frostad B,Martinsson T,Tani E, et al. The use of fine-needle aspiration cytology in the molecular characterization of neuroblastoma in children. Cancer (Cancer Cytopathol). 1999; 87: 6068.
  • 28
    Letcher CD,Bermar JJ,Corless C, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol. 2002; 33: 459465.
  • 29
    Demetri GD,von Mehron M,Blarke CD, et al. Efficacy and safety imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002; 347: 472480.
  • 30
    Willmore-Payne C,Layfield LJ,Holden JA. c-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens. Cancer (Cancer Cytopathol). 2005; 105: 165170.
  • 31
    Dong HY,Harris NL,Pfeffer FI, et al. Fine-needle aspiration biopsy in the diagnosis and classification of primary and recurrent lymphoma: a retrospective analysis of the utility of cytomorphology and flow cytometry. Mod Pathol. 2001; 14: 472481.
  • 32
    Young NA,Al-Saleem TI,Ehya H, et al. Utilisation of fine needle aspiration cytology and flow cytometry in the diagnosis and sub classification of primary and recurrent lymphoma. Cancer (Cancer Cytopathol). 1998; 84: 252261.
  • 33
    Nicol TL,Silberman M,Rosenthal DL, et al. The accuracy of combined cytopathologic and flow cytometric analysis of fine-needle aspirates of lymph nodes. Am J Clin Pathol. 2000; 114: 1828.
  • 34
    Liu K,Stern RC,Rogers RT, et al. Diagnosis of hematopoietic processes by fine-needle aspiration in conjunction with flow cytometry: A review of 127 cases. Diagn Cytopathol. 2001; 24: 110.
  • 35
    Meda BA,Buss DH,Woodruff RD, et al. Diagnosis and sub classifications of primary and recurrent lymphoma: the usefulness and limitations of combined fine-needle aspiration cytomorphology and flow cytometry. Am J Clin Pathol. 2000; 113: 688699.
  • 36
    Sigstad E,Dong HP,Davidson B, et al. The role of flow cytometric immunophenotyping in improving the diagnostic accuracy in fine-needle aspiration specimens. Diagn Cytopathol. 2004; 31: 159163.
  • 37
    Williams ME,Frierson HFJr,Tabbarah S,Ennis PS. Fine-needle aspiration of non-Hodgkin's lymphoma: Southern blot analysis for antigen receptor, bcl-2, and c-myc gene rearrangements. Am J Clin Pathol. 1990; 93: 754759.
  • 38
    Hu E,Horning S,Flynn S,Brown S,Warnke R,Sklar J. Diagnosis of B cell lymphoma by analysis of immunoglobulin gene rearrangements in biopsy specimens obtained by fine needle aspiration. J Clin Oncol. 1986; 4: 278283.
  • 39
    Katz RL,Hirsch-Ginsberg C,Childs C, et al. The role of gene rearrangements for antigen receptors in the diagnosis of lymphoma obtained by fine-needle aspiration: a study of 63 cases with concomitant immunophenotyping. Am J Clin Pathol. 1991; 96: 479490.
  • 40
    Jeffers MD,McCorriston J,Farquharson MA,Stewart CJ,Mutch AF. Analysis of clonality in cytologic material using the polymerase chain reaction (PCR). Cytopathology. 1997; 8: 114121.
  • 41
    Vianello F,Tison T,Radossi P, et al. Detection of B-cell monoclonality in fine needle aspiration by PCR analysis. Leuk Lymphoma. 1998; 29: 179185.
  • 42
    Kikuchi M,Kitamura K,Nishio Y, et al. Diagnosis of B-cell lymphoma: utility of the polymerase chain reaction for detecting clonality from archival cytologic smears. Acta Cytol. 2002; 46: 349356
  • 43
    Maroto A,Rodriguez-Peralto JL,Martinez MA,Martinez M,de Augustin P. A single primer pair immunoglobulin polymerase chain reaction assay as a useful tool in fine-needle aspiration biopsy differential diagnosis of lymphoid malignancies. Cancer (Cancer Cytopathol). 2003; 99: 180185.
  • 44
    Stewart CJR,Duncan JA,Farquharson M,Richmond J. Fine needle aspiration cytology diagnosis of malignant lymphoma and reactive lymphoid hyperplasia. J Clin Pathol. 1998; 51: 197203.
  • 45
    Safley AM,Buckley PJ,Creager AJ, et al. The value of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis of B-cell non-Hodgkin lymphoma by fine-needle aspiration. Arch Pathol Lab Med. 2004; 128: 13951403.
  • 46
    Venkatraman L,Catherwood MA,Patterson A,Tong LF,McCluggage W,Anderson NH. The role of PCR and immunocytochemistry in cytological assessment of lymphoid proliferations. J Clin Pathol. 2006
  • 47
    Alkan S,Lehman C,Sarago C,Sidawy MK,Karchers DS,Garrett CT. Polymerase chain reaction detection of immunoglobulin gene rearrangement and bcl-2 translocation in archival glass slides of cytologic material. Diagn Mol Pathol. 1995; 4: 2531.
  • 48
    Aiello A,Delia D,Giardini R, et al. PCR analysis of IgH and bc12 gene rearrangement in the diagnosis of follicular lymphoma in lymph node fine-needle aspiration. A critical appraisal. Diagn Mol Pathol. 1997; 6: 154160.
  • 49
    Bentz JS,Rowe LR,Anderson SR,Gupta PK,McGrath CM. Rapid detection of the t(11;14) translocation in mantle cell lymphoma by interphase fluorescence in situ hybridization on archival cytopathological material. Cancer (Cancer Cytopathol). 2004; 102: 124131.
  • 50
    Hughes JH,Caraway NP,Katz RL. Blastic variant of mantle-cell lymphoma: cytomorphologic, immunocytochemical, and molecular genetic features of issue obtained by fine-needle aspiration biopsy. Diagn Cytopathol. 1998; 19: 5962.
  • 51
    Caraway NP,Gu J,Lin P,Romaguera JE,Glassman A,Katz R. The utility of interphase fluorescence in situ hybridization for the detection of the translocation t(11;14)(q13;q32) in the diagnosis of mantle cell lymphoma on fine-needle aspiration specimens. Cancer (Cancer Cytopathol). 2005; 105: 110118.
  • 52
    Shin HJ,Thorson P,Gu J,Katz RL. Detection of a subset of CD30+ anaplastic large cell lymphoma by interphase fluorescence in situ hybridization. Diagn Cytopathol. 2003; 29: 6166.
  • 53
    Shivnarain D,Ladanyi M,Zakowski MF. Detection of BCL2 rearrangement in archival cytological smears of B-cell lymphomas. Mod Pathol. 1994; 7: 915919
  • 54
    Gong Y,Caraway N,Gu J, et al. Evaluation of interphase fluorescence in situ hybridization for the t(14;18)(q32;q21) translocation in the diagnosis of follicular lymphoma on fine-needle aspirates: a comparison with flow cytometry immunophenotyping. Cancer (Cancer Cytopathol). 2003; 99: 38593.
  • 55
    Goy A,Stewart J,Barkoh BA, et al. The feasibility of gene expression profiling generated in fine-needle aspiration specimens from patients with follicular lymphoma and diffuse large B-cell lymphoma. Cancer (Cancer Cytopathol). 2006; 108: 1020.
  • 56
    Gharib H,Goellner JR. Fine needle aspiration biopsy of the thyroid: an appraisal. Ann Intern Med. 1993; 118; 282289.
  • 57
    Jhiang SM,Mazzaferri EL. The ret/PTC oncogene in papillary thyroid carcinoma. J Lab Clin Med. 1994; 123: 331337.
  • 58
    Santoro M,Carlomagno F,Hay ID, et al. Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. J Clin Invest. 1992; 89: 15171522.
  • 59
    Cheung CC,Carydis B,Ezzat S,Bedard YC,Asa SL. Analysis of ret/PTC gene rearrangements refines the fine needle aspiration diagnosis of thyroid cancer. J Clin Endocrinol Metab. 2006; 86: 21872190.
  • 60
    Cohen Y,Xing M,Mambo E, et al. BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst. 2003; 95: 625627.
  • 61
    Cohen Y,Rosenbaum E,Clark DP, et al. Mutational analysis of BRAF in fine needle aspiration biopsies of the thyroid: A potential application for the preoperative assessment of thyroid nodules. Clin Cancer Res. 2004; 10: 27612765.
  • 62
    Rowe LR,Bentz BG,Bentz JS. Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules. Cytojournal. 2006; 3: 10.
  • 63
    Rhyu MS. Telomeres, telomerase and immortality. J Natl Cancer Inst. 1995; 87: 884894
  • 64
    Umbricht CB,Conrad GT,Clark DP, et al. Human telomerase reverse transcriptase gene expression and the surgical management of suspicious thyroid tumors. Clin Cancer Res. 2004; 10: 57625768.
  • 65
    Siddiqui MT,Greene KL,Clark DP, et al. Human telomerase reverse transcriptase expression in Diff-Quik stained FNA samples from thyroid nodules. Diagn Mol Pathol. 2001; 10: 123129.
  • 66
    Mora J,Lerma E.Thyroid Neoplasia Study Group. Telomerase activity in thyroid fine needle aspirates. Acta Cytol. 2004; 48: 818824.
  • 67
    Kebebew E,Peng M,Reiff E,Targ O,Clark OH,McMillan A. ECM1 and TMPRSS4 are diagnostic markers of malignant thyroid neoplasms and improve the accuracy of fine needle aspiration biopsy. Ann Surg. 2005; 242: 353361
  • 68
    Kebebew E,Peng M,Reiff E,McMillan A. Diagnostic and extent of disease multigene assay for malignant thyroid neoplasms. Cancer. 2006; 106: 25922597.
  • 69
    Salazar AM,Westcott JL. The role of transthoracic biopsy for the diagnosis and staging of lung cancer. Clin Chest Med. 1993; 14: 99110.
  • 70
    Savic S,Glatz K,Schoenegg R, et al. Multitarget fluorescence in situ hybridization elucidates equivocal lung cytology. Chest. 2006; 129: 16291635.
  • 71
    Warner KA,Crawford EL,Zaher A, et al. The c-myc x E2F-1/p21 interactive gene expression widen augments cytomorphologic diagnosis of lung cancer in fine needle aspirate specimens. J Mol Diagn. 2003; 5: 176183.
  • 72
    Lynch TJ,Bell DW,Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small cell lung cancer to gefitinib. N Engl J Med. 2004; 350: 21292139.
  • 73
    Takaro T,Ohe J,Sakamoto H, et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small cell lung cancer. J Clin Oncol. 2005; 23: 68296837.
  • 74
    Hirsch FR,Varella-Garcia M,McCoy J, et al. Increased EGFR gene copy numbers detected by FISH associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes. A southwest oncology group study. J Clin Oncol. 2005; 23: 68386845.
  • 75
    Johnson BE,Janne PA. Selecting patients for epidermal growth factor receptor inhibitor treatment: a FISH story or a tale of mutations. J Clin Oncol. 2005; 1: 68136816.
  • 76
    Lim EH,Aggarwal A,Agasthian T, et al. Feasibility of using low-volume tissue samples for gene expression profiling of advanced non-small cell lung cancers. Clin Cancer Res. 2003; 9: 59805987.
  • 77
    Pellise M,Castells A,Gines A, et al. Detection of lymph node micrometastases by gene promoter hypermethylation in samples obtained by endosonography-guided fine-needle aspiration biopsy. Clin Cancer Res. 2004; 10: 44444449.
  • 78
    Wallace MB,Block M,Hoffman BJ, et al. Detection of telomerase expression in mediastinal lymph nodes of patients with lung cancer. Am J Resp Crit Care Med. 2003; 167: 16701675.
  • 79
    Wallace MB,Block MI,Gillanders W, et al. Accurate molecular detection of non-small cell lung cancer metastases in mediastinal lymph nodes sampled by endoscopic ultrasound-guided needle aspiration. Chest. 2005; 127: 430437.
  • 80
    Myo K.Uzawa N,Miyamoto R,Sonota I,Yuki Y,Amagasa T. Cyclin D1 gene numerical aberration is a predictive marker for occult cervical lymph node metastasis in TNM stage 1 an 11 squamous cell carcinoma of the dial oral cavity. Cancer. 2005; 104: 27092716.
  • 81
    Voit C,Schoengen A,Schwurzer M,Weber L,Mayer T,Proebstle TM. Detection of regional melanoma metastases by ultrasound B-scan, cytology or tyrosinase RT-PCR of fine-needle aspirates. Br J Cancer. 80: 1999; 16721677.
  • 82
    Eloubeidi MA,Jhala D,Chhieng DC, et al. Yield of endoscopic ultrasound-guided fine-needle aspiration biopsy in patients with suspected pancreatic carcinoma. Cancer (Cancer Cytopathol). 2003; 99: 285292.
  • 83
    Evans DB,Frazier ML,Charnsangavej C. Molecular diagnosis of exocrine pancreatic cancer using a percutaneous technique. Ann Surg Oncol. 1996; 3: 241246.
  • 84
    Tada M,Komatsu Y,Kawabe T, et al. Quantitative analysis of K-ras gene mutation in pancreatic tissue obtained by endoscopic ultrasonograpy-guided fine-needle aspiration. Clinical utility for diagnosis of pancreatic tumor. Am J Gastroentrol. 2002; 97: 22632270.
    Direct Link:
  • 85
    Van Heek T,Raden AE,Offerhaus JA, et al. K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas. A molecular panel correlates with and supplements cytologic diagnosis. Am J Clin Pathol. 2002; 117: 755765.
  • 86
    Talar-Wajnarowska R,Gasiorowska A,Smolarz B, et al. Clinical significance of K-ras and c-erbB-2 mutations in pancreatic adenocarcinoma and chronic pancreatitis. Int J Gastrointest Cancer. 2005; 35: 3342.
  • 87
    Mishra G,Zhao Y,Sweeney J, et al. Determination of qualitative telomerase activity as an adjunct to the diagnosis of pancreatic adenocarcinoma by EUS-guided fine-needle aspiration. Gastrointest Endosc. 2006; 63: 648654.
  • 88
    Kitoh H,Ryozawa S,Harada T, et. Comparative genomic hybridization analysis for pancreatic cancer specimens obtained by endoscopic ultrasonography guided fine-needle aspiration. J Gastroenterol. 2005; 40: 511517.
  • 89
    Laurell H,Bouisson M,Berthelemy P, et. Identification of biomarkers of human pancreatic adenocarcinomas by expression profiling and validation with gene expression analysis in endoscopic ultrasound-guided fine needle aspiration samples. World J Gastroenterol. 2006; 12: 33443351.
  • 90
    Li G,Cuilleron M,Cottier M, et al. The use of MN/CA9 gene expression in identifying malignant solid renal tumors. Eur Urol. 2006; 49: 401405.
  • 91
    Pastorek J,Pastorekkova S,Callbaut I, et al. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and putative helix-loop-helix DNA binding segment. Oncogene. 1994; 9: 28772887.
  • 92
    Li G,Culleron M,Genlil-Perret, et al. Rapid and sensitive detection of messenger RNA expression for molecular differential diagnosis of renal cell. Carcinoma. 2003; 9: 64416446.
  • 93
    Li P,Perle MA,Scholes JV,Tang GCH. Wilms' tumor in adults: aspiration cytology and cytogenetics. Diagn Cytopathol. 2001; 26: 99103.
  • 94
    Al-Khafaji BM,Pasco-Miller LA,Zhang HZ,Katz RL. Chromosomal aberrations in renal cell carcinoma: a diagnostic and prognostic role for cytopathology. Acta Cytol. 1998; 42: 1217.
  • 95
    Bruijnesteijn Van Coppenraet ES,Lindeboom JA,Prins JM,Peeters MF,Claas ECJ,Kuijper EJ. Real-time PCR assay using fine-needle aspirates and tissue biopsy specimens for rapid diagnosis of mycobacterial lymphadenitis in children. J Clin Microbiol. 2004; 42: 26442650
  • 96
    Aljafari AS,Khalis EAG,Elsiddig KE, et al. Diagnosis of tuberculous lymphaderitis by FNAC, microbiological methods and PCR: a comparative study. Cytopathology. 2004; 15: 4448.
  • 97
    Gong G,Lee H,Kang GH,Shin J,Huh J,Khang SK. Nested PCR for diagnosis of tuberculous lymphadenitis and PCR-SSP for identification of rifampicin resistance in fine needle aspirates. Diagn Cytopathol. 2001; 26: 228231.
  • 98
    Avidor B,Varon M,Marmor S, et al. DNA amplification for the diagnosis of cat-scratch disease in small-quantity clinical specimens. Am J Clin Pathol. 2001; 15: 900909.
  • 99
    Starac D,Brestovac B,Sterrett GF,Smith DW,Frost FA. Can HPV DNA testing on FNA material determine anogenital origin in metastatic squamous cell carcinoma? Pathology. 2005; 37: 197203.