Anaplastic Lymphoma Kinase (ALK) Gene Rearrangement Detection Using Fluorescene In-Situ Hybridization (FISH) In Lung Cancer Prognostication

Lung cancer is a leading cause of cancer related death worldwide. It is increasing at a very fast rate in both men and women. Some significant mutations occurring at molecular level in lung adenocarcinoma, like ALK, EGFR, KRAS, MET, and, ALK (anaplastic lymphoma kinase) gene mutations for an ALK encoded transmembrane receptor tyrosine kinase domain and subsequently participating in the progression of Non-Small Cell Lung Adenocarcinoma (NSCLC). Some fusion partner genes involved in this process are EML-4, KLC1, KIF5B and TFG. The ALK-EML-4 rearrangement is the second most common oncogenic mutation in the nonsmall cell lung adenocarcinoma. There is 3-7% ALK mutation occurring in early or never-smokers in accompanying NSCLC. The NSCLC with ALK gene mutation generally do not have EGFR or KRAS gene mutation which are also molecular markers, which get mutated in cancer. For the detection of ALK mutation in NSCLC, different types of techniques like Fluorescence in situ Hybridization (FISH), Immunohistochemistry (IHC) and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) are being used. On the basis of sensitivity and specificity, FISH is gold standard in detecting the mutation when compared with other methodologies like IHC and RT- PCR. However in the Indian setting, FISH is more expensive and hence not available everywhere. In this review the efficacy of these different techniques in detecting ALK mutation and the detailed interpretation of results obtained with FISH has been discussed. For the treatment of ALK/MET mutated NSCLC patients an orally administered drug, crizotinib drug (tyrosine kinase inhibitor) has been approved by Food and Drug Administration (FDA) of United States. Highly sensitive and specific techniques are used for the detection of ALK gene mutation in NSCLC patients which have to be given for crizotinib treatment.

Chromosome 7 aneuploidy in clear cell and papillary renal cell carcinoma: Detection using silver in situ hybridization technique.

Background: Chromosome 7 aberrations in renal cell carcinoma (RCC) have been reported in papillary renal cell carcinoma (pRCC) and clear cell renal cell carcinoma (ccRCC). However, the implication of these anomalies on prognosis and survival is still unclear. RCC Chromosome 7 aberrations have commonly been detected by fl uorescent in situ hybridization and chromogenic in situ hybridization but not silver in situ hybridization (SISH). Aim: The purpose was to report chromosome 7 aberrations in ccRCC and pRCC using SISH in paraffi nembedded tissues and determine the association between the anomalies with clinical and pathological features. Materials and Methods: Cases of ccRCC and pRCC from University Malaya Medical Centre (2001-2009) were analyzed. Chromosome 7 staining was performed using an automated SISH method and association tests between chromosomal anomalies, clinical features and survival were performed. Results: SISH is a feasible technique to detect chromosome 7 aberration in RCC. Chromosome 7 aberrations with nuclear grading, staging and survival yielded no signifi cant correlation. Surprisingly, there was a signifi cant association between gender and chromosome 7 expressions. Though grade did not reach statistical signifi cance for survival in our RCC cases, there was a signifi cant correlation between overall survival with race and stage. Conclusion: Chromosome 7 aberrations in ccRCC showed no prognostic signifi cance. Nevertheless, staging and grading systems that include prognostic variables could hold better promise.

HER2-Positive Breast Carcinomas with Co-amplification or Gain of Chromosome 17 Centromere Locus: Report of Three Cases and an Impact on HER2 Testing

Recently we experienced three cases of human epidermal growth factor receptor 2 (HER2)-amplified invasive breast carcinomas associated with co-amplification or gain of chromosome 17 centromere (CEP17) in silver-enhanced in situ hybridization (SISH) analysis. These cases revealed 2+ or 3+ staining for HER2 immunohistochemistry and >6 HER2 copies per cell on SISH analyses. However, the calculated HER2/CEP17 ratios were low (6 per cell vs HER2/CEP17 ratio>2.2). We recommend reporting raw SISH or fluorescence in situ hybridization data, including number of cells counted, average numbers of HER2 and CEP17 signals, and the calculated HER2/CEP17 ratio to prevent underreporting of HER2 amplification.

Impact of polysomy 17 of breast cancer on the testing results of human epidermal growth factor receptor 2 (HER-2) and its clinicopathological significance / 肿瘤

Objective:To investigate the impact of polysomy 17 of breast cancer on testing results of human epidermal growth factor receptor 2 (HER2) and its clinicopathologic significance. Methods:Seventy-one patients with primary invasive breast carcinoma were studied. The HER2 gene and chromosome 17 copy numbers were determined by dual-color fluorescence in situ hybridization (FISH). The testing results were expressed by absolute HER2 gene copy number or the ratio of HER2 to chromosome 17. Based on the FISH testing results and HER2 protein expression determined by immunohistochemistry the results were compared between different groups divided by related clinicopathologic parameters.Results:All patients who had doubtable FISH results, either by absolute HER2 copy number (14 of 71 patients; 19.7%) or by the ratio HER2/chromosome 17 (2 of 71 patients, 2.8%), displayed polysomy 17. Polysomy 17-positive patients had no significant difference with HER2-negative patients in tumor grade, lymph node metastasis, and estrogen receptor (ER) expression (all P>0.05); but compared with HER2-positive patients, they showed lower tumor grade (50.0% vs 81.5%, P=0.025), higher rate of negative lymph node (55.6% vs 25.9%, P=0.045), and higher rate of ER positive expression (83.3% vs 41.7%, P=0.005) and progesterone receptor(PR)positive expression (87.5% vs 44.4%, P=0.003).Conclusion:Compared with HER2 gene amplification group, polysomy 17-positive group tends to have negative HER2 gene expression. Polysomy 17 influences the testing results of HER2 and may be the main factor that caused doubtable results in FISH examination.

Numerical Chromosome 17 Aberrations in Tumor Tissue, Tumor-adjacent Mucosa and Normal Bladder Mucosa of the Patients with Bladder Cancer Detected by in situ Hybridization / 대한비뇨기과학회지

PURPOSE: The purpose of this study is to obtain informations about the accumulation of genetic alterations during the urothelial tumorigenetic processes and to define the efficiency of the polysomy of chrosome 17 as a biomarker to predict the risk of tumor recurrences by observing the differences of numerical aberrations of chromosome 17 in tumor tissue, tumor adjacent mucosa and normal bladder mucosa of the patients with bladder cancer. MATERIALS AND METHODS: In 20 patients with the superficial transitional cell carcinoma of the bladder, tumor tissue, mucosa adjacent to the tumor and normal mucosa distant from the tumor were biopsied. The obtained specimen were probed for numerical chromosome aberrations by nonisotopic, in situ hybridization using chrosome-specific centromeric DNA probes for chrosome 17. Normal bladder mucosa obtained from 9 patients with benign prostate hyperplasia were used as controls. Data obtained from the study were analysed according to the grade of tumor and recurrence status. RESULTS: Frequency of numerical aberrations of chromosome 17 and average polysomy counts were 75%, 8.2% in cancer tissue, 40%, 4.5% in tumor adjacent mucosa and 35%, 4.2% in normal mucosa distant from tumor, which were all significantly higher than control(0%, 2.0%). Cancer tissue showed significantly higher polysomy counts in accordance with high grade(100% vs. 61.5%) and recurrence(100% vs. 54.5%) compared to low grade and no recurrence. CONCLUSIONS: In patients with bladder cancer, tumor adjacent mucosa and endoscopically normal looking mucosa showed higher rate of numerical aberrations of chromosome 17. As cancer tissue showed different polysomy status in accordance with high grade and frequent recurrences, in situ hybridization to detect numerical aberrations of chromosome 17 may be used as a good prognostic marker for bladder cancer.

Polysomy of Chromosome 17 Predicting Treatment Failure in Head and Neck Squamous Cell Carcinomas / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The two major biologically distinct patterns of treatment failure following definitive therapy for the patients with head and neck squamous cell carcinoma are the recurrence of primary tumor and the development of second primary tumor. The purpose of this study is to determine whether the polysomy of chromosome 17 has prognostic significance and is associated with the pattern of treatment failure. MATERIAL AND METHODS: We performed nonfluorescent, nonisotopic, in situ hybridization using chromosome-specific centrometric DNA probe for chromosome 17 on formalin-fixed, paraffin-embedded specimens from the tumor tissue and the resection margins of 42 head and neck squamous cell carcinomas were treated with definitive local therapy. RESULTS: In the tumor tissue, the polysomy of chromosome 17 was a significant predictor for recurrence and treatment failure. In the resection margins, the polysomy of chromosome 17 also showed a predictive significance for the treatment failure. Although there was a chromosomal change in the resection margins believed to be negative on light microscopy, it was also related to the treatment failure. CONCLUSION: The polysomy of chromosome 17 may be a valuable marker for identifying individuals who have the high risk of developing recurrence and treatment failure.

Detection of Numerical Chromosomal Aberration in Squamous Cell Carcinoma of the Lung by In Situ Hybridization Using #17 Centromeric Probes

This study was carried out to understand the relationship between specific chromosome changes and their phenotypic consequences at the tissue level of human lung cancers. Then paraffin-embedded human lung squamous cell carcinoma samples were investigated for in evidence of genetic alterations, using chromosome 7 and 17-specific repetitive alpha-satellite DNA probes. In situ hybridization procedure with chromosome-specific DNA probes was optimized for use on formalin-fixed paraffin-embedded lung tissue sections. The chromosome index ranged from 1.10 to 1.88(median, 1.49) for chromosome 7 and 1.20 to 1.98(median, 1.69) for chromosome 17. Normal lymphocytes and stromal cells showed one or two chromosome signals per cell in most cases. All tumors showed three or more chromosome signals per cell with range of 16.0% to 80.6% of cancer cells(median, 50.9%) for chromosome 7 and 32.7% to 84.7%(median, 69.9%) for chromosome 17. The chromosome index did not correlate with the DNA content in most cases. Chromosomes 7 and 17 were either overrepresented or underrepresented when they were compared with corresponding DNA index determined by FCM. An increase in copy number, particularly of chromosome 7 was associated with a less favorable phenotype, including high nuclear grade. In addition, chromosome alterations were differentially expressed in the different areas of the same tissue section, correlating with histologic heterogeneity. These results suggest that chromosome polysomy can be reliably detected in tissue sections using in situ hybridization. There is a strong correlation between genotypic abnormalities and tumor phenotype in human lung cancer. This capability will prove to be an important tool for determining the underlying genetic basis for tumor development, tissue phenotype heterogeneity and progression by allowing genetic determination to be made on paraffin-embedded tissue sections where tumor histologic architecture is preserved.