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Context: Chromosomal abnormalities play an important role in diagnosis and prognosis of hematological diseases., Aims: The aim of the present study was to study the pattern and frequency of chromosomal aberrations in acute myeloid leukemia (AML) subgroups from western India., Settings and Design: A retrospective study was conducted through evaluating laboratory proforma which were filled during 2005 to 2014 for diagnosis and treatment of AML subjects., Methods and Material: We have studied chromosomal aberrations in 282 subjects with AML from western India. AML patients were sub-grouped according to FAB classification. Cytogenetic study using conventional cytogenetics (GTG-banding) and Fluorescence in situ hybridization (FISH) was carried out using FISH probes (AML1/ETO, PML/RARA, CBFB)., Statistical Analysis: Student's t test for continuous variables and Pearson's Chi-squared test for categorical variables were used to identify the relationship between variables., Results: Cytomorphological study revealed AML- M3 as most frequent (32.3%) group followed by AML-M2 (25.2%) and AML-M4 (19.9%). Chromosomal abnormalities were identified in 145 (51.42%) of the total AML cases. A high frequency (38.6%) of chromosomal abnormalities was identified in AML-M3 subgroup as compared to AML-M2 (31%) and AML-M4 (20.6%)., Conclusions: Cytogenetic study is important for the diagnosis and management of the AML patients. Our study identified chromosomal abnormalities in AML subgroups with varied frequencies. It is important in diagnosis and monitoring of the disease. As younger AML patients were more affected in our study, etiological factors such as environmental factors need to be studied. Combination of conventional cytogenetics and FISH has an advantage of identifying high frequency of chromosomal aberrations in AML patients.

The ASCO/CAP guidelines for HER2 reporting in breast cancer published in 2007 and were updated in 2013 to assure that the right patient receives the targeted therapy. The updated guidelines have lowered the threshold for HER2 positivity criteria and altered the equivocal category for both IHC and FISH. This first study from India addresses the impact of these updated guidelines in the various reporting categories at a tertiary care centre. We compared the trend of HER2 IHC reporting 1 year before (Period A) and 1 year after (Period B) the implementation of updated 2013 ASCO/CAP guidelines. All HER2 equivocal IHC cases of post 2013 guidelines were reclassified as per 2007 guidelines to detect additional number of cases that have been put into equivocal category. Reflex FISH correlation was also assessed to detect any additional cases eligible for anti HER2 therapy with implementation of these updated guidelines. With implementation of updated 2013 guidelines, there was significant decrease in the number of cases scored as 1+ (from 30.7% to 20.6%; P value: .0001) while significant increase in number of 2+ cases (from 20.2% to 27.3%; P value: .004). Post 2013 guidelines, 39% (64 cases) of tumors were additionally put into the equivocal category which would have been considered as negative (score 1+) as per 2007 guidelines. The reflex FISH testing in these equivocal cases resulted in detection of only 1.5% of additional cases eligible for anti HER2 therapy. With implementation of updated 2013 guidelines, there is no significant increase in HER2 positivity trend. However, there is appreciable increase in IHC equivocal cases which subsequently led to increased reflex FISH testing without significantly contributing to the detection of additional eligible cases for anti HER2 therapy, but resulted in delaying of definite HER2 status along with financial implications., (© 2017 Wiley Periodicals, Inc.)

Objectives: Genetic markers are crucial fort diagnostic and prognostic investigation of hematological malignancies (HM). The conventional cytogenetic study (CCS) has been the gold standard for more than five decades. However, FISH (Fluorescence in Situ Hybridization) testing has become a popular modality owing to its targeted approach and the ability to detect abnormalities in non-mitotic cells. We here aimed to compare the diagnostic yields of a FISH panel against CCS in HMs. Methods: Samples of bone marrow and peripheral blood for a total of 201 HMs were tested for specific gene rearrangements using multi-target FISH and the results were compared with those from CCS. Results: Exhibited a greater diagnostic yield with a positive result in 39.8% of the cases, as compared to 17.9% of cases detected by CCS. Cases of chronic lymphocytic leukaemia (CLL) benefited the most by FISH testing, which identified chromosomal aberrations beyond the capacity of CCS. FISH was least beneficial in myelodysplastic syndrome (MDS) where the highest concordance with CCS was exhibited. Acute lymphocytic leukaemia (ALL) demonstrated greater benefit with CCS. In addition, we found the following abnormalities to be most prevalent in HMs by FISH panel testing: RUNX1 (21q22) amplification in ALL, deletion of D13S319/LAMP1 (13q14) in CLL, CKS1B (1q21) amplification in multiple myeloma and deletion of EGR1/RPS14 (5q31/5q32) in MDS, consistent with the literature. Conclusions: In conclusion, FISH was found to be advantageous in only a subset of HMs and cannot completely replace CCS. Utilization of the two modalities in conjunction or independently should depend on the indicated HM for an optimal approach to detecting chromosomal aberrations., (Creative Commons Attribution License)

Background: Children born with congenital anomalies present a very high rate of perinatal death and neonatal mortality. Cytogenetic analysis is a convincing investigation along with clinical suspicion and biochemical screening tests. The current study was designed to characterize the prevalence and types of chromosomal abnormalities in high risk prenatal samples using different cytogenetic techniques., Methods: This study was conducted on a total of 1,728 prenatal samples (1,324 amniotic fluids, 366 chorionic villi and 38 cord blood samples) from 1994 to 2014 at Institute of Human Genetics, Ahmedabad, India. Conventional karyotyping was conducted with GTG-banding. Molecular approaches were used (fluorescence in situ hybridization = FISH and/ or array-comparative genomic hybridization = aCGH) when indicated to detect karyotypic abnormalities., Results: Abnormal karyotypes were detected in 125/1,728 (7.2%) cases. Trisomy 21 was the most common abnormality detected in 46 (2.7%) followed by trisomy 18 in 11 (0.6%) and trisomy 13 in 2 (0.1%) samples. Besides, structural abnormalities such as reciprocal and Robertsonian translocation were detected in 20 [1.2%] cases. Turner syndrome was diagnosed in seven (0.4%) cases; in six (0.34%) cases there was an inversion in the Y-chromosome. Heteromorphic variants were diagnosed in 22 (1.3%) cases. Finally, small supernumerary marker chromosomes (sSMC) were found in six (0.34%) cases., Conclusion: Conventional GTG-banding along with molecular cytogenetic techniques is useful in detecting genomic alterations and rearrangements. Comprehensive characterization of chromosomal rearrangements like sSMC has the potential to save potentially healthy fetuses from being terminated.

To facilitate efficient handling of large samples, an attempt towards networking of laboratories in India for biological dosimetry was carried out. Human peripheral blood samples were exposed to (60)Co γ-radiation for ten different doses (0-5Gy) at a dose rate of 0.7 and 2Gy/min. The chromosomal aberrations (CA) were scored in Giemsa-stained and fluorescence in-situ hybridization with centromere-specific probes. No significant difference (p>0.05) was observed in the CA yield for given doses except 4 and 5Gy, between the laboratories, among the scorers and also staining methods adapted suggest the reliability and validates the inter-lab comparisons exercise for triage applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Breast carcinoma shows amplification/overexpression of Her-2/neu in ∼20-30% of cases. The determination of Her-2/neu expression accurately is vital in clinical practice as it has significant predictive value and eligibility for anti Her-2/neu therapy. Amplification and overexpression of Her-2/neu gene is traditionally identified by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on tissue sections; only a few studies have evaluated feasibility of these techniques on cytological smears. One hundred cases of breast cancer with fine-needle aspiration cytology (FNAC) samples and corresponding surgically resected specimen were selected. Immunocytochemistry (ICC) and FISH for Her-2/neu was done on FNA smears, whereas IHC was performed on corresponding tissue sections. Diagnostic accuracy of ICC was 99% when compared with IHC. Comparison of FISH results with IHC showed 100% concordance. Unlike many centers in West, FNAC is still routinely performed in developing countries like India where vast majority of breast cancer cases present as palpable lumps. The high rates of accuracy of ICC and FISH for Her-2/neu detection can make FNAC a relevant first line of investigation as a cost effective model with a rapid turn-around time, providing complete information necessary for initial management of breast cancer patients., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The current WHO classification of hematologic malignancies defines distinct entities of myeloid disorders based on the presence of recurrent cytogenetic abnormalities. Diagnostic clonal chromosomal abnormalities provide important prognostic information and are among the most important factors in predicting initial response to chemotherapy, duration of remission and overall survival. , Methods: This study analyzed chromosomal abnormalities in bone marrow aspirates of 50 children diagnosed with acute myeloid leuckemia (AML). , Results: The culture success rate was 94%, clonal chromosomal abnormalities constituted 62% and recurrent chromosomal abnormalities were 56%. In the favorable prognostic category, there were 51.6% of cases with t(8;21)(q22;q22), 16.1% had t(15;17)(q22;q21), and a total of 12.9% had chromosome 16 rearrangement. The adverse risk category showed a low frequency of t(9;11)(p22;q13); t(1;22)(p13;q13); inv(3)( q21q26); add 4(q35) and ring chromosome. According to fluorescent in situ hybridization (FISH) results in 16 cytogenetically normal patients, there were no CBFβ/MYH11 fusion genes observed in chromosome 16 rearrangements. , Discussion: Larger studies of this kind may provide more information about chromosome 16 rearrangements in cytogenetically normal patients. The present analysis suggests that both age and cytogenetics are important strategies for risk stratification (outcome). Additional laboratory parameters should also be considered in childhood AML.

Background: Chronic myelogenous leukemia (CML) is characterised by the t(9;22)(q34;q11.2) which results in the formation of the BCR/ABL1 fusion gene. Occasionally, the t(9;22) may be associated with submicroscopic deletions of chromosomes 9 and/or 22 which appear to be associated with a worse prognosis. Three or four-way variant t(9;22) may also occur. All these changes as well as gain of the Philadelphia chromosome which represents disease progression can be detected by fluorescence in situ hybridization (FISH) analysis. FISH analysis at presentation is used to determine the number of cells with BCR/ABL1 fusion and establish whether the patterns are typical or atypical. Response to therapy can then be monitored by serial testing., Patients and Methods: The study group consisted of all patients diagnosed or suspected to have CML who had interphase FISH analysis at presentation on peripheral blood/bone marrow using a commercially available BCR/ABL1 dual colour, dual fusion probe. The study was performed at a tertiary hospital in India between 2004 and 2010., Results: There were 1076 diagnostic samples which were positive for BCR/ABL1 fusion. Typical dual fusion signals (two fusions, one red and one green, 2F1R1G) were seen in 801 cases (74 %). Atypical signal patterns were seen in 275 cases (26%). These were: 1F1R2G (4%), 1F2R1G (2.5%) and 1F1R1G (11%) representing deletions of the derivative 9 involving chromosome 9 sequences, chromosome 22 sequences, or both respectively; 3F1R1G (6.5%) usually representing gain of an additional Philadelphia chromosome and 1F2R2G (1%) representing a three- or four-way variant translocation. More than one signal pattern was seen in 1%., Conclusions: Our findings were similar to the literature with respect to the distribution of signal patterns except that we had a lower number of patients with variant translocations. While each signal pattern is typically associated with a particular abnormality, there can be more than one explanation for each pattern. Hence, metaphase FISH analysis is the "gold standard" for the interpretation of signal patterns.

Aims: The HER-2/neu proto-oncogene is amplified in 15%-25% of breast cancers. In the current study, we evaluated HER-2/neu status of 396 cases of breast cancer by fluorescence in situ hybridization (FISH), and the results were correlated with immunohistochemistry (IHC) for HER-2/neu protein expression., Results: Overall, HER-2/neu amplification was observed in 38.4% of cases. Concordance between IHC and FISH was 90.4% considering only IHC score 0, 1 (negative), and 3 (positive). However, only 37.3% of the IHC score 2 (equivocal) cases showed HER-2/neu gene amplification. A majority of the discordant cases within the IHC negative (score 0 and 1) and IHC positive (score 3) were high-grade tumors. Polysomy 17 and monosomy 17 was seen in 7.3% of the total cases of each. Furthermore, a majority of FISH positive cases were noted in Intraductal Carcinoma grade III and cases with regional lymph nodal metastasis. Polysomy 17 was seen in 7.9% of the FISH positive cases and in 6.3% of the FISH negative cases. Monosomy 17, however was more preponderant in FISH negative cases., Conclusion: We believe that the FISH test should be considered as the gold standard in the estimation of the HER-2/neu status due to its increased sensitivity and better appreciation of aneusomy 17.

Introduction: HER-2/neu status determines the eligibility for targeted therapy with trastuzumab in breast carcinoma. Evaluation for HER-2/neu protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in situ hybridization (FISH) has become the gold standard., Aims: Since data on HER-2/neu assessment by IHC and FISH and studies regarding concordance between the results of the two techniques are limited, especially from India, we sought to study HER-2 gene amplification status by FISH in equivocal (2+) cases by IHC and also study aberrant signal patterns., Settings and Design: Mastectomies and breast core biopsies, equivocal for HER-2/neu protein expression, were analyzed for HER-2 amplification by FISH., Materials and Methods: IHC (DAKO) and FISH (PathVysion dual-probe system) tests were performed on 68 of 112 (after exclusion) 10% neutral buffered formalin (NBF)-fixed paraffin-embedded tissues and evaluated according to American Society of Clinical Oncology ASCO guidelines., Statistical Analysis Used: Chi-square (χ2) test and the two-tailed P value were applied using Graphpad Quickcels software, version 2006., Results: It was found that 73.5% of the IHC 2+ patients were negative for HER-2/neu amplification, 25% were positive (ratios ranging from 2.3 to 5.6) and 1 patient was equivocal (2.2). Retesting FISH HER-2 equivocal case on another tumor block by IHC demonstrated HER-2 overexpression of protein 3+, thus resolving the equivocal status. Polysomy and HER-2 genetic heterogeneity were seen frequently., Conclusions: The findings reiterate that IHC HER-2 equivocal cases are a heterogeneous group and need FISH for further categorization. Low concurrence (25%) rate between both IHC and FISH results in the equivocal scenario can be attributed to tumors with polysomy 17 and HER-2/neu genetic heterogeneity.

Background: The concordance rate between immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) results for HER2/neu according to clinical performance is controversial. The present prospective study was theerefore conducted in Indian breast cancer patients., Methods: Fifty cases (n=50) of invasive duct cancer of breast tested for HER-2/neu by IHC and scored as 0, 1+, 2+ and 3+ by pathologists were further analyzed by FISH using a commercially available double-color probe, and the findings compared., Results: A total concordance of 82.0% was observed with a Kappa coefficient of 0.640 (P <0.001). A high discordance was observed in 30.0% of the patients with IHC 2+, 7.1% in IHC 3+, 19.2% overall in IHC 0 and 1+., Conclusion: IHC can be used firstly to screen the HER-2/neu status, and FISH can be used as a supplementary role to IHC and 2+ and some negative cases. And only those cases with HER-2/neu status of IHC 3+ or FISH positive should be treated with Herceptin (Trastuzumab).

Background & Objectives: Determination of HER2 status in breast cancer has become important to identify potential candidates for anti-HER2 therapy. In this study we compared fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for the determination of HER2 status in breast cancer patients referred to a tertiary care referral centre., Methods: A total of 200 cases of invasive breast cancer were evaluated for HER2 status using IHC and FISH and results were compared., Results: The IHC 3+ (93.9%) and IHC negative (85.9%) cases showed good concordance with the corresponding FISH results; while 66.6 per cent of IHC 2+ cases showed gene amplification by FISH. In addition, hormone receptor expression and HER2 gene status showed a statistically significant inverse association (P<0.05)., Interpretation & Conclusion: These findings reaffirm IHC as a prudent first-step to screen tissue samples for HER2 status and to determine suitability for technically demanding FISH test and the dual coloured FISH as a gold standard for determination of HER2/neu status in IHC equivocal cases of breast carcinoma.

Previously, cases of systemic lupus erythematosus (SLE) and Klinefelter syndrome (KS) in men have been reported in Western populations. We report the case of a 30-year-old man from southern India with known infertility who was diagnosed to have SLE and KS by fluorescence in-situ hybridization, as routine karyotype cultures failed. The diagnosis has implications in management and highlights the need for strong clinical suspicion and laboratory confirmation of KS by molecular methods when suspected in all men with SLE. Quicker, long-term remission and genetic counseling of such individuals can help in better management and coping with this chronic, potentially fatal disease. Lupus (2010) 19, 870-871.

Split-hand/split-foot malformation (SHFM, also called ectrodactyly) is a clinically variable and genetically heterogeneous group of limb malformations. Several SHFM loci have been mapped, including SHFM1 (7q21), SHFM2 (Xq26), SHFM3 (10q24), SHFM4 (3q27) and SHFM5 (2q31). To date, mutations in a gene (TP63) have only been identified for SHFM4. SHFM3 has been shown by pulsed-field gel electrophoresis to be caused by an approximately 500 kb DNA rearrangement at 10q24. This region contains a number of candidate genes for SHFM3, though which gene(s) is (are) involved in the pathogenesis of SHFM3 is not known. Our aim in this study was to improve the diagnosis of SHFM3, and to begin to understand which genes are involved in SHFM3. Here we show, using two different techniques, FISH and quantitative PCR that SHFM3 is caused by a minimal 325 kb duplication containing only two genes (BTRC and POLL). The data presented provide improved methods for diagnosis and begin to elucidate the pathogenic mechanism of SHFM3. Expression analysis of 13 candidate genes within and flanking the duplicated region shows that BTRC (present in three copies) and SUFU (present in two copies) are overexpressed in SHFM3 patients compared to controls. Our data suggest that SHFM3 may be caused by overexpression of BTRC and SUFU, both of which are involved in beta-catenin signalling., ((c) 2006 Wiley-Liss, Inc.)

We carried out this study to detect ALL-1 gene alterations in Indian childhood leukemias (n-84) using non-radioactive Southern blotting and FISH techniques. 18 (21.4%) patients showed altered ALL-1 gene. All 18 patients with altered ALL-1 gene did not have high WBC count and or typical CD10-/19+ phenotype. 4/18 were infants, while 14 were of 1-12 years of age. 13/18 children were boys. 14/18 expired within 1 year. Karyotyping detected abnormal chromosome 11 only in 4/43 patients and Classical t(4:11) in one AML patient but combination of Painting FISH and LS-FISH confirmed ALL-1 gene alteration in 17/18 cases. In addition, FISH identified nine translocations and multiple copies of ALL-1 gene in three cases which conventional cytogenetics had failed to detect. Our result indicates that a combination of Southern blotting, cytogenetic and FISH techniques are useful to identify ALL-1 gene alterations in childhood leukemias.

Zoo-fluorescence in situ hybridization (FISH) with human whole chromosome-specific paint probes revealed extensive homoeologies between Indian muntjac (2n=6, 7 female, male) and human karyotypes (2n=46). Forty-two conserved syntenic segments, corresponding to all human chromosomes except the Y chromosome, produced a near-complete coverage of the muntjac complement and revealed margins of interspecific segmental homoeology. To test the hypothesis that interstitial satellite DNA loci, illuminated by a Chinese muntjac C5-satellite probe in Indian muntjac chromosome arms, mark ancestral fusion points (Lin CC, Sasi R, Fan YS, Chen Z-Q (1991) New evidence for tandem chromosome fusions in the karyotypic evolution of the Asian muntjacs. Chromosoma 101: 19-24), we combined Zoo-FISH with C5 satellite mapping. Twenty-six interstitial satellite DNA loci were detected in the haploid Indian muntjac genome and were found to co-localize with the margins of conserved human/Indian muntjac syntenic segments. These results were confirmed by two-colour FISH and are in accordance with the tandem fusion hypothesis for Indian muntjac chromosomes. Furthermore, conserved syntenic segment combinations detected in pig, cattle and Indian muntjac Zoo-FISH maps reveal ancestral artiodactyl chromosomes.