Interphase fluorescence in situ hybridization improves the detection of malignant cells in effusions from breast cancer patients.

In diagnostic evaluation of effusions, difficulties are encountered when atypical reactive mesothelial cells have to be differentiated from malignant cells. We tested the impact of fluorescence in situ hybridization (FISH) to identify metastatic cells in breast cancer effusions by detection of numerical chromosomal changes. Pleural and ascitic fluid samples (n=57) from 41 breast cancer patients were concomitantly evaluated by routine cytology and FISH, using centromere-specific probes representing chromosomes 7, 11, 12, 17 and 18. After setting stringent cut-off levels deduced from non-malignant control effusions (n=9), the rates of cells with true aneuploidy were determined in each effusion sample from breast cancer patients. The occurrence of aneuploid cells, as detected by FISH and indicative of malignancy, was correlated with the cytological findings. Routine cytology revealed malignancy in 60% of effusions. Using FISH, aneuploid cell populations could be observed in 94% of cytologically positive and in 48% of cytologically negative effusions, thus reverting diagnosis to malignancy. To confirm malignancy in cases with a low frequency of aneuploid cells, two-colour FISH was additionally performed and indeed showed heterogeneous chromosomal aneuploidy within single nuclei. We conclude that FISH is a valuable tool in the diagnosis of malignancy and may serve as an adjunct to routine cytological examination, as demonstrated here for breast cancer effusions.     

Cytogenetics and fluorescence in situ hybridization as adjuncts to cytology in the diagnosis of malignant mesothelioma

BACKGROUND:

Virtually all malignant mesotheliomas (MMs) exhibit clonal chromosomal aberrations. Although the chromosome regions affected by these aberration(s) may vary from 1 tumor to another, certain regions are commonly disrupted. These aberrations are absent in benign mesothelial cells, and therefore their presence can be used to confirm a diagnosis of MM. In the current study, the authors investigated the value of karyotyping and fluorescence in situ hybridization (FISH) as adjuncts to conventional cytologic examination in patients with MM.

METHODS:

A retrospective analysis of 48 pleural or peritoneal fluids from patients with histologically confirmed MM was performed. Karyotypic analyses were attempted in all cases. In 27 cases, FISH for deletions of 9p21 (CDKN2A gene) and 22q was also performed because the karyotype was normal or unsuccessful.

RESULTS:

Karyotypes were obtained in 35 (73%) of the specimens. Of these, 15 (43%) were abnormal and 20 (57%) were normal. Thirteen additional abnormal results were detected by FISH in cases for which the karyotypes were normal or unsuccessful. A total of 24 cases (50%) had an associated cytologic interpretation. Karyotyping or FISH was abnormal in 8 cases that were interpreted cytologically as either negative or suspicious.

CONCLUSIONS:

The combination of FISH and karyotyping was found to improve on the diagnostic sensitivity of karyotyping alone in detecting MM in effusions. The authors concluded that karyotyping and FISH together were a more useful adjunct to cytology than FISH or karyotyping alone. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

荧光原位杂交和尿细胞学检查对膀胱癌诊断意义的Meta分析

背景与目的：目前膀胱癌疗效和监测的主要方法是膀胱镜和尿细胞学检查,前者为侵人性检查,令患者感到不适;后者虽无创且特异性高．但敏感性太低,且受主观因素影响大。本研究拟对中、英文有关比较荧光原位杂交（fluorescence in situ hybridization,FISH）和尿细胞学检查诊断膀胱癌研究的结果进行系统分析,以明确FISH对膀胱癌的诊断意义。方法：采用Cochrane系统评价方法,MEDLINE（1966年1月～2008年6月）、EMBASE（1988年1月。2008年6月）、Cochrane图书馆、中国生物医学期刊文献数据库（CMCC,1979年。2008年6月）、CNKI数字图书馆（1979年1月～2008年6月）进行有关FISH和尿细胞学检查诊断膀胱癌文献的检索、质量评价和资料提取,采用MetaDiScl．4软件进行Meta分析。结果：共检索到相关研究242篇,排除230篇,符合纳入标准12篇进入Meta分析,涉及研究对象3430例。异质性检验提示无阈值效应,但存在其它原因导致的异质性。按随机效应模型进行Meta分析．FISH和尿细胞学诊断膀胱癌的准确度指标敏感度、特异度、阳性似然比、阴性似然比以及诊断优势比等汇总及95％C1分别为74％（71％-77％）VS．57％（54％-61％）、88％（86％-90％）VS．85％ （83％-87％）、6．18（3．56～10．73）VS．4．15（2．78～6．20）、0．29（0．19～0．45）VS．0．51（0．41～0．63）及24．17（9．33～62．64）VS．9．59（5．91～15．57）。FISH和尿脱落细胞学检查的敏感度随肿瘤分级、分期的升高而增高。综合受试者工作特征曲线下面积分别为0．8938、0．8247．Q^＊值分别为0．7847、0．7226。结论：FISH诊断膀胱癌的准确度较高,但对高分期的敏感度较细胞学低,目前尚不能取代传统的尿细胞学检查,但可作为膀胱癌术前诊断、术后监测和随访的指标。     

The role of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fine‐needle aspiration

BACKGROUND:

Fine‐needle aspiration (FNA) has been used in the evaluation of lymphadenopathy for a long time and is highly reliable in the identification of metastatic malignancies. However, the role of FNA in the assessment of new lymphoproliferative disorders continues to be a subject of debate. The objective of the current study was to evaluate the role of molecular cytogenetic studies in FNA diagnoses of lymphoproliferative disorders.

METHODS:

A retrospective, computer‐based search for lymph node FNAs from 2006 to 2007 was performed. Cases with either fluorescence in situ hybridization (FISH) and/or polymerase chain reaction (PCR) studies were subjected to further analysis.

RESULTS:

In total, 243 lymph node FNAs were performed during the period, including 104 that were positive/suspicious for metastatic malignancies, 16 that were positive/suspicious for lymphomas, 15 that demonstrated atypical lymphoid proliferation, 73 that were reactive, 14 that were deemed granulomas, and 21 that were determined to be nondiagnostic. Molecular analysis included combined FISH/PCR in 4 cases, FISH only in 7 cases, and PCR only in 4 cases. By using multiplex PCR, 6 cases with atypical/negative flow cytometry results were diagnosed as 4 B‐cell lymphomas, 1 T‐cell lymphoma, and 1 reactive lymph node; and 4 cases that had atypical T cells determined by flow cytometry were diagnosed as reactive. One CD10‐negative follicular lymphoma and 2 cases with suspicious flow cytometry results were positive for t(14;18)(q32;q21) by FISH. Forty‐five cases had follow‐up histology with 3 false‐negative findings and no false‐positive results.

CONCLUSIONS:

In this study, multiplex PCR studies for immunoglobulin heavy‐chain or T‐cell receptor gene rearrangements were useful for demonstrating clonality, and FISH studies were able to detect translocations or gene rearrangements that allowed for the subclassification of B‐cell non‐Hodgkin lymphomas. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Clonality of primary pulmonary lymphoproliferative disorders; using in situ hybridization and polymerase chain reaction for immunoglobulin

Primary pulmonary lymphoproliferative disorders (PLDs) are histologically divided into a neoplastic state of high and low grade malignant lymphoma (ML), and a reactive state of follicular bronchitis/bronchiolitis (FB) and lymphoid interstitial pneumonia (LIP). We reviewed 19 cases with PLDs, including 4 cases each of high and low grade B cell ML, 6 FB cases, and 5 cases of LIP. To clarify the clonality of the proliferating cells, we performed an immunohistochemical examination (IHC), in situ hybridization (ISH) for the immunoglobulin light chain and a polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain gene using DNA obtained from paraffin sections. In addition, a Southern blot analysis was also performed in 6 cases using fresh materials. In IHC, all ML were positive for L26 (CD20), while the monoclonality of the kappa light chain was observed in only one high grade case. However, using ISH we could detect the clonality in three of four high grade ML cases and in one of four low grade ML cases. In FB and LIP, no clonality of immunoglobulin by ISH was observed. In a PCR analysis for the immunoglobulin heavy chain gene, we could detect one or two prominent bands in all 8 cases of high and low grade ML. On the other hand, in all cases of FB and LIP, we could only detect either an oligoclonal or polyclonal population. In summary, the presence of monoclonality of ISH and/or PCR for the immunoglobulin heavy chain gene were limited in the neoplastic state, but not in the reactive state.     

Utilization of microdissection and the polymerase chain reaction for the diagnosis of adrenal cortical carcinoma in fine-needle aspiration cytology.

BACKGROUND: Loss of heterozygosity (LOH) for several tumor suppressor genes (including loci on 3p, 1p, and 17p,) has been documented in surgical specimens of adrenal cortical carcinomas (ACCA) without accompanying losses in benign hyperplastic and adenomatous adrenal cortical lesions (ACL). This disparate pattern of LOH raises the possibility of exploitation of these differences for diagnostic utilization. Cytologic differentiation of benign versus malignant ACL may be impossible based solely on fine-needle aspiration (FNA) material. The authors attempted to extrapolate the genetic findings on surgical specimens to FNA specimens of ACL to determine whether LOH studies could be utilized as a definitive diagnostic tool. METHODS: Microdissection of archival material was performed on FNAs of ten ACCAs (stained with the Papanicolaou and Diff-Quik stains) with corresponding histologic material (stained with hematoxylin and eosin), one FNA of a benign ACL, and three touch preparations of benign adrenal cortex. LOH analysis was performed by polymerase chain reaction (PCR) with flanking markers for the following putative tumor suppressor genes: p53 (17p13; TP53), 1p (1p36; D1S165), and the von Hippel-Lindau gene at 3p25 (D3S1038 and D3S1110). RESULTS: Similar results were obtained with cytologic and histologic material. As expected, benign ACL showed no LOH for the markers examined. Of the informative ACCA cases, 70% showed LOH for at least 1 of the 3 markers tested on both FNA and histologic samples. For all cases with amplifiable DNA, there was a 100% concordance rate for LOH between cytologic and histologic material, with at least 7 of the 10 cytologic samples originating from metastatic lesions and all of the surgical material originating from the primary adrenal neoplasm. CONCLUSIONS: The results of this study suggest that the combination of microdissection and PCR for LOH of p53, 1p, and 3p25 from FNA material has the potential to be utilized to distinguish ACCA from benign ACL in informative cases. It also shows a 100% concordance rate between metastatic and primary ACCAs for the losses observed, a finding that can be extremely useful for the definitive identification of metastatic lesions. Archival cytologic preparations of ACCA are a reliable source of DNA for LOH studies. [See editorial counterpoint on pages 173-5 and reply to counterpoint on pages 176-7, this issue.] Cancer (Cancer Cytopathol) Copyright 1999 American Cancer Society.     

Detection of breast cancer micrometastases in bone marrow using reverse-transcriptase chain reaction and southern hybridization

Thirty-flveto40%ofallpatientswithbreastcarcinoma,includingupt024%ofpatientswithn0evidence0fmetastasesatthetimeofdiagnosis,willrelapseafterprimarytherapy.l"']Themostreliableprognosticindicesofaxillarylymphnodestatusandsizeofprimarytumorcannotpredictinwh0mthediseasewillrecur.Bonemarrowisafrequentandreadilyaccessiblesiteofmetastases.Inup8O%ofpatients,therelaPsedevelopsstariingfrombonemarrowmetastasesats0mepointinthepr0cessoftheirillness.l3]Currentmethodstodetectboneinvolvement,suchasX-rayandbon…     

应用7、8号人染色体着丝粒特异探针和FISH技术检测胸腔积液中的超二倍体肿瘤细胞

目的研究良恶性胸腔积液中间期细胞遗传学方面的差异,以及FISH技术对此种差异的鉴别能力.方法采用7号、8号人染色体着丝粒特异性探针对21例临床诊断明确患者的胸腔积液(11例肿瘤患者,10例非肿瘤患者)进行FISH分析,计数超二倍体细胞的比例,以正常人外周血淋巴细胞作为对照来判定染色体数目的异常,并将结果与临床诊断进行比较.结果在正常对照的淋巴细胞中,7、8号人染色体着丝粒探针各出现2个杂交信号,肿瘤患者胸腔积液细胞中7、8号人染色体数目的变化主要表现为拷贝数增多.在11例恶性胸腔积液中,出现7、8号人染色体数目增多的例数分别为8(72.7%)和9(81.8%),10例良性胸腔积液中7、8号人染色体为正常二倍体的各有9例(90.0%).结论 7、8号人染色体超二倍体改变是肿瘤患者胸腔积液细胞的主要特征,采用染色体着丝粒特异性探针的荧光原位杂交技术能够检测到胸腔积液标本中的超二倍体肿瘤细胞,并且具有较好的性能.     

Detection of Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin's disease using the polymerase chain reaction and in situ hybridization

Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the BamHI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.     

Detection of human papillomavirus infection in penile samples through liquid‐based cytology and polymerase chain reaction

BACKGROUND.

The human papillomavirus (HPV) is strongly related to cervical cancer and its precursor lesions. However, unlike in the case of women, there are limited data regarding HPV infection in men. Analysis of male HPV infection is frequently hindered by the lack of consistency in collection methods, sample adequacy, and low sensitivity of cytologic analysis.

METHODS.

The objective of the current study was to compare the results of liquid‐based cytology and HPV DNA testing through polymerase chain reaction in 99 penile samples collected from men presenting with condyloma acuminate or male partners of HPV‐infected women who had attended a public health service in the city of Belo Horizonte, Minas Gerais, Brazil. Classic and nonclassic cytomorphologic signs were adopted to evaluate the presence of HPV infections in penile smears.

RESULTS.

HPV DNA was detected in 93 (93.9%) of the 99 samples analyzed. Koilocytosis was detected in 1 smear and nonclassic signs were detected in 23 smears, 22 of which were found to be positive for HPV DNA.

CONCLUSIONS.

The cytopathologic detection of HPV infection in penile samples collected for liquid‐based cytology is low, even when cytologic nonclassic signs are applied, and does not appear to improve the diagnosis of HPV infection in men. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.     

Sensitive detection of tumour cells in effusions by combining cytology and fluorescence in situ hybridisation (FISH)

Diagnosis of malignant cells in effusions is important for staging procedures and resulting therapeutic decisions. Cytodiagnostics in effusions is sometimes difficult since reactive mesothelial cells can mimic malignant cells. We used fluorescence in situ hybridisation (FISH) in single-colour or if appropriate in dual-colour evaluation to detect chromosomal aberrations in effusion cells as markers of malignancy, to raise the diagnostic yield. Cytologic and FISH evaluations--by using probes representing several chromosomes always including chromosomes 11 and 17--were performed in 358 effusion fluids. Cytology was positive for malignancy in 44.4% of all effusions, whereas FISH was positive in 53.9% (P=0.0001). The combination of cytology and FISH was diagnostic for malignancy in 60.9% of effusions. Diagnostic superiority of FISH was demonstrated in effusions from breast cancer, lung cancer, pancreatic cancer, and in effusions from the entire group of gynaecological and gastrointestinal carcinomas. In transudates (effusion protein <2.5 g dl(-1)), malignant cells were detectable by cytology, FISH, and combined use of both methods in 18.6, 30, and 37.1% of effusions, respectively, suggesting that cytologic and molecular analysis should be performed also with transudates. In conclusion, FISH in combination with conventional cytology is a highly sensitive and specific diagnostic tool for detecting malignant cells in effusions.