Polysomy Of Chromosome Research Articles

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

BackgroundThe role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.MethodsThe protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.ResultsMembrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage.ConclusionIn conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.

Up‐Regulation of Replication Factor C‐40 (RFC40) in estrogen positive as well as negative breast cancer

BackgroundCurrently most therapies for breast cancer are targeted against growth factor and endocrine receptors. Identification of non receptor based molecules that will target receptor negative breast cancers is of paramount importance. Replication Factor C 40 (RFC40) protein required for DNA replication and it influences the cell cycle from DNA replication to cell division. RFC40 is over‐expressed in choriocarcinoma, acute and chronic myeloid leukemia, nasopharyngeal cancer and glioblastomas, suggesting that it may be directly responsible for unrestricted DNA replication and cell proliferation. However, its role in breast cancer has not been explored.MethodsWe determined the expression of RFC40 message and protein by qRT‐PCR and Western blot analyses in normal, estrogen positive and negative breast cancer cell lines and performed immunohistochemistry for RFC40 protein in patient breast tumor microarrays (BTMAs). We performed fluorescent in situ hybridization analyses with BTMAs to determine amplification of the RFC40 gene copy numbers in breast cancer.ResultsWe found that RFC40 message and protein is significantly up‐regulated in all the breast cancer cell lines and in the patient BTMAs. We observed RFC40 gene amplification and polysomy of chromosome 7 in BTMAsConclusionsRFC40 is up‐regulated in breast cancer accompanied by either gene amplification or polysomy of chromosome 7.

Correlation of Ataxia-Telangiectasia-Mutated (ATM) gene loss with outcome in head and neck squamous cell carcinoma

Ataxia-Telangiectasia-Mutated (ATM) gene loss has been associated with poor prognosis and treatment resistance in head and neck squamous cell carcinomas (HNSCC). We investigated the relationship between ATM loss detected by fluorescence in-situ hybridisation (FISH) with patient outcome, and its relationship with Human Papillomavirus (HPV)/p16(INK4A) status. Copy number of the ATM gene and chromosome 11 were determined by FISH and HPV status was determined using p16(INK4A) immunohistochemistry in 87 paraffin embedded tumour samples from patients with HNSCC treated with chemoradiation at a single institution. ATM loss was correlated with patient outcome as both a continuous and dichotomous variable. Of 73 evaluable patients, 44 (60.3%) demonstrated loss of the ATM gene. There was no correlation between ATM loss (defined as a mean ratio of ATM: chromosome 11<0.75) and overall survival (OS, p=0.67) or time to locoregional failure (TTLRF, p=0.72). Similarly, when evaluated as a continuous variable there was no significant relationship between ATM loss and patient outcome (OS, p=0.89; TTLRF, p=0.21). No significant relationship was found between p16(INK4A) status and ATM loss, for patient outcome. We found 35.6% (n=26) of patients demonstrated polysomy of chromosome 11 (defined as the presence of a mean >2.5 copies of chromosome 11) which was significantly associated with p16(INK4A) negative status (p=0.0004), but did not influence outcome. ATM loss is a frequent event in HNSCC, however it does not impact outcome after treatment with chemoradiation. Polysomy of chromosome 11 was significantly associated with p16(INK4A) negative status but also lacks prognostic significance.

Disseminated Carcinoma Ex Pleomorphic Adenoma in an Adolescent Confirmed by Application of PLAG1 Immunohistochemistry and FISH for PLAG1 Rearrangement

A 16-year-old previously asymptomatic boy presented with complaints of fatigue, weight loss, and back pain for several months. Imaging studies revealed a large superior mediastinal mass, numerous bilateral pulmonary nodules, and multiple lytic bone lesions. A needle biopsy from a sternal lesion showed a poorly differentiated carcinoma, immunoreactive for cytokeratins and EMA and immunonegative for various organ/tissue-specific markers.His past medical history was significant for excision of aparotid gland tumor 5 years earlier. Histologic review of the salivary gland tumor revealed a pleomorphic adenoma containing a microscopic focus of invasive carcinoma(carcinoma ex pleomorphic adenoma). By immunohistochemistry, both the salivary gland tumor and the disseminated carcinoma expressed PLAG1 with a strong nuclear pattern.Fluorescence in situ hybridization (FISH), using dual-color, break-apart probes for PLAG1, showed rearrangement of the gene in both the salivary gland and the disseminated tumors.FISH demonstrated additional cytogenetic aberrations in the carcinoma, including polysomy for chromosome 8 (in both the primary salivary gland and the metastatic tumors) and PLAG1 amplification (in the metastatic tumor). We conclude that in the proper clinicopathologic setting, application of PLAG1 immunohistochemistry and FISH for PLAG1 gene rearrangement may be valuable in establishing the diagnosis of carcinoma ex pleomorphic adenoma as the source of a cancer of unknown primary site.

Polysomy 8 in Egyptian patients with de-novo acute myeloid leukemia

Background Tetrasomy, pentasomy, and hexasomy of the entire chromosome 8 (polysomy 8) are relatively rare compared with trisomy 8, which is one of the most common recurring aberrations in myeloid hematologic malignancies. Although polysomy 8 appears to adversely influence outcome, little is known about the prognostic significance of polysomy 8 in patients with acute myeloid leukemia (AML). Aim of the work The aim of the present work was to study the numerical aberrations of chromosome 8 in Egyptian patients with AML and to clarify its role as a prognostic marker. Patients and methods The present study was carried out on 76 newly diagnosed adult patients with AML. They were attending the Hematology Department of Ain Shams University Hospitals. They were subjected to conventional cytogenetic analysis using G-banding and fluorescence in-situ hybridization using a fluorophore-labeled centromeric probe for chromosome 8. Results Metaphase and interphase fluorescence in-situ hybridization for detection of polysomy 8 was successfully performed on 72 samples. Patients were divided into two groups: group A comprised patients associated with polysomy 8 (20 patients; 27.8%) and group B comprised patients without polysomy 8 (52 patients; 72.2%). Group A patients had a statistically significant younger age compared with group B (P=0.04). A statistically significant association was seen between polysomy 8 status and hepatosplenomegaly, fever, and lymph node infiltration (P=0.03, 0.05). The presence of hepatosplenomegaly, skin infiltration, lymph node infiltration, and French–American–British subtype was shown to have a significant impact on patient outcome (P=0.05, 0.03, 0.01, and 0.04, respectively). Patients with skin infiltration, CD34 expression, HLA-DR expression, high/intermediate cytogenetic risk, and presence of polysomy 8 had significantly shorter overall survival. Conclusion and recommendations From the current study we conclude that polysomy 8 occurs in 27.8% of de-novo AML cases; 35% of these cases showed tetrasomy of chromosome 8. Polysomy of chromosome 8 was associated with shortened overall survival. Further studies with a larger number of patients are recommended for proper assessment of polysomy 8, especially tetrasomy 8, among Egyptian patients with AML.

Aggressive B-Cell Lymphomas

Aggressive B-cell lymphomas (ABLs) are a group of B-cell malignancies, that is, a relatively common form of cancer across the world. This issue on aggressive B-cell lymphomas compiles 12 exciting manuscripts, most of which are very meticulously performed reviews of the available current literature. Three areas of lymphoma pathology and biology that are leading to a greater understanding of relatively recently defined entities are discussed. These include anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (ALK+ LBCL), and the so-called grey zone lymphomas or lymphomas with intermediate features and the posttransplant lymphoproliferative disorders. ALK+ LBCL are characterised by gene rearrangement involving clathrin and ALK genes (t(2;17)(p23;q23)). Grey zone lymphomas include two unclassifiable B-cell lymphomas, one with features intermediate between diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL) and the second with features intermediate between DLBCL and Burkitt lymphoma (BL). The biology of common lymphomas is addressed in two manuscripts. M. Rossi et al. review the current literature on molecular subsets of DLBCL, the distinction between DLBCL and BL, and gene expression characteristics of the grey zone lymphomas. This paper is accompanied by M. Cacciatore et al.'s manuscript that explores the importance of the microenvironment in lymphoma biology. Two of the manuscripts deal with those aspects of endemic BL pathogenesis that have hitherto not been explored in great depth. The paper by S. Mannucci et al. explore the possibility of Euphorbia tirucalli acting as a cofactor in BL. They show that, in lymphoblastoid cell lines, E. tirucalli can modulate the EBV latency genes, result in polysomy for chromosome 8, and cause upregulation of c-MYC. Furthermore, Dr. C. van den Bosch raises question about a possible role for RNA viruses in the pathogenesis of BL. Dr. I. Magrath reviews the evolution of BL treatment in Africa—from cyclophosphamide monotherapy to multiagent combination chemotherapy including central nervous system prophylaxis using combinations of cyclophosphamide, vincristine, prednisone, and methotrexate. He also highlights the benefits of collaboration between resource-rich and resource-poor settings. P. Vishnu and D. M. Aboulafia review how the treatment of HIV-associated lymphomas has evolved since the introduction of highly active antiretroviral therapy (HAART). The use of rituximab, in conjunction with multiagent chemotherapy, and where required high-dose chemotherapy (HDCT) and autologous stem cell rescue (ASCT) have all contributed to the success of treating HIV lymphomas. Treatment of ABL is further reviewed in two paper—one on ASCT in DLBCL and the other on novel therapies by K. Foon et al. To complete the issue, S. Donnou reviews the currently available murine models useful for studying lymphomagenesis, the lymphoma microenvironment, and the efficacy of new therapies. By compiling these paper, we hope to enrich our readers and researchers with respect to these particularly common, yet usually highly treatable aggressive B-cell malignancies. Kikkeri N. Naresh Ian Magrath Martine Raphael Lorenzo Leoncini

EBV Reactivation and Chromosomal Polysomies: Euphorbia tirucalli as a Possible Cofactor in Endemic Burkitt Lymphoma.

Burkitt lymphoma is endemic in the Equatorial Belt of Africa, its molecular hallmark is an activated, MYC gene mostly due to a chromosomal translocation. Especially in its endemic clinical variant, Burkitt lymphoma is associated with the oncogenic Epstein-Barr virus (EBV), and holoendemic malaria acts as an amplifier. Environmental factors may also cooperate in Burkitt lymphomagenesis in the endemic regions, such as plants used as traditional herbal remedies. Euphorbia tirucalli, a plant known to possess EBV-activating substances, has a similar geographical distribution to endemic Burkitt's Lymphoma and is used as a hedge, herbal remedy and toy in the Lymphoma BeltI. In this study we aimed at determining if exposure to Euphorbia tirucalli could contribute to lymphomagenesis, and at which extent. Lymphoblastoid and cord blood-derived cell lines were treated with plant extracts, and the expression of EBV-coded proteins was checked, to assess EBV reactivation. The occurrence of chromosomal translocations was then investigated by FISH. Our preliminary results suggest that E. tirucalli is able to reactivate EBV and determine chromosomal alterations, which leads to c-MYC altered expression. The existence of genomic alterations might determine the accumulation of further genetic alteration, which could eventually lead to a transformed phenotype.

49, XXXXY syndrome: an Italian child

The 49, XXXXY syndrome is a rare sex chromosome polysomy, first described by Fraccaro and colleagues in 1960. The approximate incidence of this disorder is 1 in 85,000 male births. To date, >100 cases had been published in the literature. Patients with 49, XXXXY syndrome show some peculiar clinical features, such as mental retardation, facial dysmorphism, ambiguous genitalia, and multiple skeletal and cardiac defects. We report a new case of 49, XXXXY syndrome; the first Italian case to our knowledge.

Gain of Chromosomes 1, 2, 7, 10, 13 and 17 in an Acquired Cystic Kidney Disease Associated Renal Cell Carcinoma

The acquired cystic disease of the kidney-associated renal cell carcinoma (ACDK-RCC) in the current study occurred in a kidney with multiple cysts and was composed of cells with eosinophilic cytoplasm and prominent nucleoli. There were extensive calcium oxalate deposits in both non-neoplastic cysts and tumor. The tumor cells were positive for RCC Ma, CD10, and EMA, focally positive for CK7, negative for vimentin. Interphase in situ hybridizations (FISH) were performed for chromosome 1, 2, 7, 10, 13 and 17. No chromosomal abnormality was observed in the non-neoplastic cysts. Polysomies of chromosomes 1, 2, 7, 10, 13, 17 were observed in the tumor. Trisomy 13 was first reported in this type of tumor, which warranted further study.

P1-12-17: Fluorescent In Situ Hybridization Evaluation of HER2 Status in Tumors with Chromosome 17 Polysomy.

Abstract Introduction: According to the American Society of Clinical Oncology-College of American Pathologists recommendations for HER2 testing, a positive fluorescent in situ hybridization (FISH) result is defined as &amp;gt;6 HER2 gene copies/nucleus (for test systems with no internal control probe [single-color]) or as HER2 gene/chromosome 17 centromere (CEP17) ratio &amp;gt; 2.2 (for systems with an internal control probe [dual-color]). Although an increase in CEP17 copy number (average ≥ 3.0 copies/nucleus) is commonly considered to represent polysomy of chromosome 17, it can also be a result of gains of 17q with centromere involvement, or amplification of the centromeric region. The classification of HER2−positive tumors according to the HER2/CEP17 ratio may therefore misclassify a fraction of truly amplified cases as polysomic. We prospectively evaluated tumors with chromosome 17 polysomy but no HER2 amplification to assess HER2 status using the above two FISH classifications and immunohistochemistry (IHC). Materials and methods: Tumors were tested for gene amplification by FISH with probes to HER2/neu and CEP17 using the PathVysion HER-2 DNA Probe Kit (Vysis). Classification was based on the HER2/CEP17 ratio (amplified when &amp;gt; 2.2) and average HER2 gene copy number/nucleus (amplified when &amp;gt; 6 copies). Both polysomic and equivocal cases (HER2/CEP17 ratio 1.8 - 2.2) were further studied by IHC using the HercepTest (Dako) with 0–3 scoring system (overexpression when 3+). Results: From March 2010 to May 2011 we evaluated 31 primary breast cancers showing chromosome 17 polysomy. Median HER2/CEP17 ratio was 1.3 (range 0.5−1.9), median HER2 copy number was 5.4 (range 2.6−13.8), and median CEP17 copy number was 4.2 (range 3.2−8.0). Thirteen (42%) had an average HER2 gene copy number &amp;gt; 6/nucleus (median 6.8, range 6.1−13.8) and would therefore be considered as amplified if classified according to the absolute HER2 gene copy number. Nine (75%) of these were 3+ at IHC and the remaining 4 were 2+, whereas among the 18 cases with an average HER2 gene copy number &amp;lt; 6/nucleus, one was 2+, ten were 1+, and 7 scored 0. Twenty-nine cases showed negative HER2/CEP17 ratios (&amp;lt; 1.8) and three cases equivocal HER2/CEP17 ratios (between 1.8 and 2.2). Using HER2/CEP17 ratio as first assessment and IHC only in equivocal cases, only one of the 31 “polysomic” cases would have been classified as HER2−positive. However, 8 polysomic cases with HER2/CEP17 ratio &amp;lt; 1.8 showed 3+ immunostaining (all with average HER2 gene copy number &amp;gt; 6/nucleus), while 3 other cases had an average HER2 gene copy number &amp;gt; 6/nucleus with 2+ immunostaining. Conclusions: Our results show that both FISH evaluation criteria and IHC can modify the percentage of polysomic tumors classified as HER2−positive. However, the number of gene copies/nucleus appears more frequently associated with 3+ IHC than the HER2/CEP17 ratio. The predictive impact of the former method on response to anti-HER2 treatments warrants further investigation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-17.

Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures

The diagnosis of indeterminate biliary strictures is limited because of the low sensitivity of cytology. However, an accurate diagnosis of malignancy is critical in the management of patients with suspected biliary malignancy. Testing for chromosomal aneuploidy by fluorescence in situ hybridization (FISH) may increase the yield. To evaluate the diagnostic accuracy of FISH in indeterminate biliary strictures and the additional value of including deletion of 9p21 (p16) in the diagnostic criteria of malignant biliary strictures. Retrospective review. Academic medical center. This study involved 76 consecutive patients who were seen for the evaluation of indeterminate strictures at our institution. These patients were screened, and 50 patients with either a final pathologic diagnosis or ≥ 12 months' conclusive follow-up were included in the analysis. Sensitivity, specificity, and area under the curve (AUC) analysis of cytology alone compared with the presence of FISH polysomy versus FISH polysomy and 9p21 deletion. The presence of increased copy numbers (polysomy) of chromosome 3, 7, or 17 by FISH increased the sensitivity of brush cytology from 21% to 58%, and when the presence of 9p21 deletion was included, the sensitivity increased to 89%. The specificity of FISH was 97% (vs 100% for cytology). The accuracy of cytology combined with FISH polysomy (AUC = 0.93) or p16 deletion was significantly greater than the accuracy of cytology alone (AUC 0.6; P < .001) or even cytology combined with FISH polysomy (AUC = 0.77; P ≤ .05). Sample size. There is a relatively high incidence of malignant biliary strictures in the entire cohort but low incidence among primary sclerosing cholangitis patients, and the majority of cancers are cholangiocarcinomas (as opposed to pancreatic). FISH significantly improves the diagnostic accuracy of brush cytology in indeterminate biliary strictures. In our series, the addition of 9p21 deletion to FISH polysomy and cytology further improved sensitivity. This suggests that 9p21 deletion may be added to the diagnostic criteria in indeterminate strictures.

Monosomy of chromosome 8 could be considered as a primary preneoplastic event in breast cancer: A preliminary study

This pilot study analyzed and compared the presence of chromosome 8 aneusomy in Mexican women with breast cancer and adjacent, intraductal, proliferative lesions. To determine the chromosome 8 copy number, we performed fluorescence in situ hybridization in nine patients (1800 cells) who underwent mastectomy. We selected two tissue samples from each patient, one corresponding to the invasive ductal carcinoma (IDC) and the other adjacent to the intraductal proliferative lesion (IPL). Breast tissue from 17 autopsy samples (1700 cells) was used as a control. The number of cells with monosomy, disomy and polysomy per subject and type of tissue were compared among the three groups of tissue with the RxC statistical software package using 50,000 total replicates. Chromosome 8 aneusomy was found in 66 and 67% of cells from the IDC and IPL samples, respectively. Monosomy was detected significantly more frequently in IPL compared with IDC samples (49.11 vs. 27.11%; p=0.0000), whereas polysomy was significantly more frequent in IDC compared with IPL samples (40.11 vs. 16.99%; p=0.00000). Control cells showed 92.3% disomy. These findings suggest that polysomy of chromosome 8 is more frequently observed in IDC and that monosomy is more frequent in tissue of IPL. Therefore, monosomy may be considered as a primary preneoplastic event. Future studies should be performed to increase the amount of breast tissue with ductal proliferative changes and with cancer, in order to support the results of this pilot study.

Chromophobe renal cell carcinoma with prominent lymph node metastasis and polysomy of chromosome 21: poorly differentiated form or “presarcomatoid” form?

Lymph node metastasis of chromophobe renal cell carcinoma (RCC) is extremely rare. It has been recently reported that sarcomatoid chromophobe RCC frequently show polysomy of chromosomes 1, 2, 6, 10, and 17. In this article, we report an unusual case of chromophobe RCC. A 42-year-old Japanese woman presented with hematuria and complained of inguinal pain 2 months after the initial symptoms. Radical nephrectomy and renal hilar lymphadenectomy were performed. The tumor was 8 cm in greatest diameter; its cut surface was beige in color. Large metastasis to the renal hilar lymph node was identified. Histological examination of the right renal tumor met the criteria of chromophobe RCC. In addition to histological findings of typical chromophobe RCC, small cell foci, comedo-like necrosis, trabecular growth pattern, and sclerosing stroma were observed. However, no sarcomatoid foci were identified anywhere, despite extensive tumor sampling including lymph node lesions. Immunohistochemically, neoplastic cells were positive for E-cadherin and CD117 (c-kit). Ultrastructurally, tumorous cells contained abundant mitochondria and cytoplasmic microvesicles. Fluorescence in situ hybridization showed monosomy of chromosomes 2 and 10 and polysomy of chromosome 21. Finally, we suggest that this tumor may show the poorly differentiated or presarcomatoid form of chromophobe RCC.

EUS-FNA with rescue fluorescence in situ hybridization for the diagnosis of pancreatic carcinoma in patients with inconclusive on-site cytopathology results

Detection of chromosomal abnormalities by fluorescence in situ hybridization (FISH) analysis has not been well-studied in FNA samples of pancreatic masses. Selective use of FISH in patients with inconclusive on-site cytopathology results may improve the sensitivity of EUS for malignancy. To determine the sensitivity and specificity of FISH analysis in patients with inconclusive on-site cytopathology results. Consecutive patients with suspected pancreatic malignancy, nonrandomized cohort study. Final diagnosis was based on either surgical biopsy or disease progression on extended follow-up or death. Academic center, tertiary-care referral cancer center. A total of 212 EUS examinations were performed in 206 patients for solid pancreatic lesions over a 24-month period (January 2009-December 2010). FISH analysis was done for 69 patients with inconclusive or nonavailable on-site cytology results. EUS-guided FNA (EUS-FNA) of solid pancreatic masses with cytology and FISH analysis for polysomy of chromosomes 3, 7, and 17 and deletion of 9p21. Sensitivity/specificity of cytology, FISH, and a composite of cytology and FISH. Patients with positive on-site cytology (110), neuroendocrine tumors (22), insufficient follow-up (1), FISH not obtained (3), and renal cancer with pancreatic metastasis (1) were excluded. Sixty-nine patients comprised the study cohort, 54 with malignancy and 15 with benign disease. Sensitivity for malignancy of cytology, FISH analysis, and the combination were 61%, 74%, and 85%, respectively (P = .009). FISH detected an additional 13 cases of pancreatic adenocarcinoma missed by cytology. There was no false-positive FISH analysis in 15 patients with benign disease. No major complications occurred from EUS-FNA. Single center, selected patients underwent FISH analysis, limited number of patients with benign disease. In patients with suspected pancreatic cancer, FISH analysis can detect additional cases missed by cytology without compromising specificity. FISH analysis to detect polysomy of chromosomes 3, 7, and 17 and deletion of 9p21 should be considered when cytology is negative for malignancy in patients with a known pancreatic mass.

EUS-FNA with rescue FISH for the diagnosis of pancreatic carcinoma in patients with inconclusive on-site cytopathology.

e14641 Background: Endoscopic ultrasound guided biopsy and cytology (EUS-FNA) is commonly used in the diagnosis of pancreatic cancer (PC) with a sensitivity (Sn) of 60%-90%. This study is to determine the Sn and specificity (Sp) of FISH analysis in patients with inconclusive on-site cytopathology (OC). Methods: Retrospective analysis of patients undergoing diagnostic EUS-FNA for solid pancreatic masses in a single University Tertiary Cancer Center from January 2009 to December 2010. Non-diagnostic OC samples were sent for cytology and FISH (UroVysion probe, Abbott Molecular, Des Plaines, IL) to detect aneuploidies of chromosomes 3, 7, 17 and deletions of 9p21. Positive FISH for malignancy was defined as ≥5 cells with ≥3 signals in at least two chromosomes (polysomy), >5 cells with 9p21 deletion, extra copy of chromosome 7 in > 10 cells (trisomy). Confirmation of cancer was based on pathologic diagnosis on surgical specimen, disease progression on extended follow-up or autopsy. Sn, Sp, positive and negative predictive values of cytology and FISH were obtained based on binomial distribution. The Fisher exact test was utilized to compare sensitivity between cytology, FISH and cytology plus FISH. A p value < 0.05 was considered statistically significant. Results: 212 EUS-FNA were performed in 206 patients over a 24 month period. The diagnosis of carcinoma was obtained in 164 patients. OC established the diagnosis in 110 patients that were excluded in addition to patients with neuroendocrine tumor (22), insufficient follow-up (1), FISH not obtained (3) and pancreatic metastasis (1). Sixty-nine patients with inconclusive or non-available OC comprised the study cohort and had FISH analysis, of which 15 patients had benign diseases. The Sn for the diagnosis of PC by cytology, FISH and the combination were 61%, 74% and 85% respectively (p=0.009). FISH detected an additional 13 cases missed by cytology with no false positives in benign diseases. Conclusions: FISH to detect polysomy of chromosomes 3, 7, 17 and deletion of 9p21 should be considered when cytology is negative for malignancy in patients with pancreatic mass. Rescue FISH can detect additional cases missed by cytology without compromising specificity.

Abstract 3211: FGFR1 and FGFR2 gene copy number variations in prostate cancer

Abstract Patients with hormone refractory prostate cancer (HRPC) have very few clinical options. A bottle neck in development of new therapies is the poor understanding of the molecular basis of hormone independency. A pathway thought to be important in this progression is FGFR. Amplification of FGFR1 has been observed in breast cancer and has led to the hypothesis that it is involved in Tamoxifen resistance (Turner et al., 2010). Here we evaluated the copy number variations (CNV) of the gene loci of FGFR1 (8p12) and FGFR2 (10q26) by dual color FISH in FFPE tissue of recurrent PC patients (n=28), non-recurrent PC patients (n=15), PC patients undergoing palliative RPE (n=15), patients with benign prostate hyperplasia (n=17) and tumor free tissue samples (n=12). We analyzed CNV between different clinical patient subgroups. Two types of CNV for the gene loci of FGFR1 and FGFR2 were observed. Either the gene dose of the target gene locus occurred to be amplified or the gene dose was elevated due to polysomy of the respective chromosome. Furthermore patients with CNV of FGFR1 or FGFR2 exhibited in 40-50% intra tumor heterogeneity by presenting normal cells and cells with CNV. FGFR1 gene dose elevations (either due to gene locus amplification or polysomy of chromosome 8) were observed in 7% (1/15) of patients without recurrence but in 49% (17/35) of the patients with recurrence. Also, in patients undergoing palliative RPE 75% (12/16) of the patients exhibited FGFR1 gene dose elevation. In the case of FGFR2, gene dose elevations (gene locus amplification or polysomy of chromosome 10) were observed in none of the patients without recurrence but in 13% of the patients with recurrence and in 20% of patients undergoing palliative RPE. In the control groups such variations were not found. Interestingly, the gene dose variations differed in the prostate cancer patients depending on their hormone sensitivity. 47% (7/15) of hormone refractory patients and 30% (6/20) of the hormone sensitive patients showed elevated FGFR1 gene dose, for FGFR2, 30% and 20% of the respective subgroups were affected. In summary we conclude that this is first evidence that CNV of FGFR1 and FGFR2 might have implications for hormone resistance in PC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2011-3211

Abstract 4177: Copy number variations of FGFR1 and 2 in NSCLC; implications for targeted drug development

Abstract NSCLC is the cancer with highest death rate; it is typically diagnosed at a late stage and has limited therapeutic options. FGFR signaling is a potential oncogenic driver in many cancers, including NSCLC. In this study we evaluated the copy number variations (CNVs) of FGFR1 and FGFR2 by dual color FISH in specimens from 139 NSCLC patients (78 SCC, 46 ADC, 15 other). CNVs were observed for both FGF receptors. However, FGFR1 was more frequently affected by CNV than FGFR2 (45% vs. 17%). Only a small fraction of patients exhibited CNV of both receptors. In general, the level of gene amplification was higher for FGFR1 as compared with FGFR2. Interestingly, FGFR2 CNV exhibited a high degree of intra-tumor heterogeneity. FGFR1 gene dose elevations were observed due to gene locus amplification (27%) as well as polysomy of chromosome 8 (17%). The degree of gene locus amplification was classified from low (gene ratio 1.5 – 2.5) to high (&amp;gt;4.5). An analysis of the two most common histologies (i.e. Squamous Cell Carcinoma (SCC) and Adenocarcinoma (ADC)) indicated that gene locus amplifications were more frequent in SCC (38%) as compared with ADC (7%), while polysomy of chromosome 8 was observed at similar rates (18-19%). Lack of FGFR1 rearrangements was confirmed using a break-apart FISH probe. FGFR2 gene dose elevations show gene locus amplifications and polysomy of chromosome 10. Locus amplifications of FGFR2 were observed in 11% of SCC, but were not found in ADC. Similar rates for polysomy of FGFR1were observed in SCC (7%) and ADC (5%). Overall, gene dose elevation of FGFR1 and FGFR2 were found more frequently in SCC. Of higher relevance is the fraction of patients with gene dose elevation due to gene locus amplification. Approximately 40% of the SCC patients possess gene locus amplification of at least one of the two probed FGF-receptors. However, only ∼9% of the SCC patients have both loci amplified. In conclusion here we present for the first time evidence for a potential genetic addiction of NSCLC, specifically on FGFR1 gene locus amplification. Therefore targeting FGFR in combination with proper patient stratification, may be an attractive opportunity to develop improved clinical options for treatment of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4177. doi:10.1158/1538-7445.AM2011-4177

Expression and Amplification of Topoisomerase-2α in Type 1 and Type 2 Papillary Renal Cell Carcinomas and Its Correlation with HER2/neu Amplification

The current study was undertaken to investigate chromosomal and genetical aberrations leading to overexpression of Topoisomerase-2α (TOP2α) and to reveal the possible association of these aberrations with HER2/neu overexpression and gene amplification, and to search for the relationship between TOP2α and HER2/neu status with prognostical biomarkers in papillary renal cell carcinoma (RCC), a group of tumors with diverse molecular, chromosomal and clinical features. Archival cases of papillary RCC obtained from Departments of Pathology of Pamukkale, Ege and Dokuz Eylul Universities were studied in two groups (type 1 and type 2) each containing 20 cases. The level of TOP2α and HER2/neu expression by tumor cells were determined immunohistochemically. A multicolor FISH probe was used to define both amplification of HER2/neu and TOP2α genes, and polysomy 17. The ratio of cells expressing TOP2α in type 1 and type 2 papillary RCC were 24.29% and 6.89%, respectively. The difference was statistically significant comparing the average or median values of groups separately (p = 0.002). The expression levels of TOP2α and HER2/neu were also correlated. TOP2α and HER2/neu were co-amplified in both groups. Immunohistochemical expression was not observed in 15 of 23 cases with HER2/neu amplification. The most frequent finding detected by FISH method was polysomy of chromosome 17. We had contradictory results compared with the findings reported in the limited numbers of literature. It shows us that papillary RCC constitute a heterogenous group of tumors with various cytogenetic features and morphological classification of these tumors may not be compatible with their molecular characteristics.

Chromosomal abnormalities in renal cell carcinoma variants detected by Urovysion fluorescence in situ hybridization on paraffin-embedded tissue

Urovysion fluorescence in situ hybridization (UVFISH) identifies malignant cells in urine by detecting specific urothelial carcinoma-related chromosomal abnormalities. Some renal carcinomas (RCCs) share overlapping chromosomal aberrations with urothelial carcinoma. Malignant renal cells that are shed in urine can potentially cause a positive UVFISH result. We evaluated UVFISH in RCCs to determine its potential applicability to the diagnosis and grading of RCCs. Paraffin blocks from 39 RCCs (25 clear cell, 9 papillary, 2 chromophobe, and 3 sarcomatoid) and 15 controls (5 renal oncocytomas and 10 urothelial carcinomas) were tested. Of the RCCs, 15 (40%) were UVFISH-positive (9/25 [40%] clear cell, 3/9 [30%] papillary, 1/2 [50%] chromophobe, and 2/3 [67%] sarcomatoid carcinoma) and 24 (60%) were negative. Of the 15 controls, 8 (∼50%) were UVFISH-positive (2/5 [40%] oncocytomas and 6/10 [60%] urothelial carcinomas) and 7 (∼50%) were UVFISH-negative. Polysomy of chromosome 17 showed a statistically significant correlation with RCC subtype, being absent in most of the clear cell RCCs (P = .0096) compared with other RCCs. Polysomy of chromosome 7 was more frequent in high-grade than low-grade RCC (P = .0197) and more likely in high-grade clear cell than low-grade clear cell RCC (P = .0120). In conclusion, we showed that RCC has overlapping chromosomal abnormalities with urothelial carcinoma and can cause a positive UVFISH result. This has implications for the interpretation of Urovysion in patients whose urine contains malignant cells but who have negative cystoscopy and a concomitant renal mass. The chromosomal abnormalities observed in RCC are not distinct from those in urothelial carcinoma; therefore, UVFISH cannot distinguish these tumor types, nor can it type or grade RCC.

CDC28 protein kinase regulatory subunit 1B (CKS1B) expression and genetic status analysis in oral squamous cell carcinoma.

CKS1B is a member of the highly conserved cyclin kinase subunit 1 (CKS1) protein family which interacts with cyclin-dependent kinases and plays a critical role in cell cycle progression. In oral squamous cell carcinoma (OSCC), as in other malignancies, CKS1B overexpression has been correlated with reduced survival. To our knowledge, no studies evaluating the genetic status of CKS1B gene in OSCC have been reported. Herein, genetic and protein status of CKS1B were analyzed by immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) techniques in a series of primary OSCC (n=51) and lymph node OSCC metastases samples (n=14). The observed results were compared with those obtained in either inflammatory (oral lichen planus [OLP]) (n=13) and premalignant oral mucosal lesions (oral leukoplakia) (n=16). A significant CKS1B overexpression was observed in OSCC and lymph node metastases samples than in OLP and oral leukoplakia (mean 70% vs 35%, p<0.001). CKS1B overexpression correlated with p27 loss of expression (p=0.0013) and SKP2 overexpression (p<0.00). FISH study disclosed statistical differences in both gene amplifications and gains between samples corresponding to OSCC and metastases from those of OLP and leukoplakia (p<0.001). Amplifications were present in 53% of OSCC samples and 33% of lymph node metastases vs 14% of oral leukoplakia and 0% of OLP biopsy specimens (p=0.002). Polysomies of chromosome 1 were seen in 46% of OSCC, 33% of ganglionar metastases, 14% of oral leukoplakia and 10% of OLP (p=0.036). Correlation of CKS1B over-expression and gains (both polysomies and amplifications) determined by FISH was statistically significant (p<0.001). Our results indicate that a high CKS1B expression is a common finding in primary OSCC which correlates with p27 low expression and SKP2 overexpression. This phenomenon may be due either to numerical (chromosome 1 polysomy) or structural (amplifications) CKS1B genetic abnormalities. This phenotypical and cytogenetic profile is not observed in premalignant or inflammatory oral mucosal lesions.