Cytogenetic Heterogeneity in Chronic Lymphocytic Leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a malignancy identified by an increase in the number of lymphocytes in the blood. It is one of the most common adult leukemias. It is a heterogeneous clinical disease with changeable progression. Chromosomal aberrations play a significant role in predicting clinical outcomes and survival. Treatment strategies for each patient are determined by chromosomal abnormalities. Cytogenetic methods are sensitive procedures for detecting abnormalities in the genome. The aim of this study was to document the incidence of different genes and gene rearrangements in CLL patients by comparing conventional cytogenetic and fluorescence in situ hybridization (FISH) results and predicting their prognosis. Materials and Methods A total of 23 CLL patients, 18 men and five women with ages ranging from 45-75 years were enrolled in this case series. Interphase fluorescent in situ hybridization (I-FISH) was conducted on peripheral blood or bone marrow samples, whichever were available, and were cultured in growth culture medium. I-FISH was used to detect chromosomal abnormalities such as 11q-, del13q14, 17p-, 6q- and trisomy 12 in CLL patients. Results FISH results showed that there were different chromosomal gene rearrangements including del13q, del17p, del6q, del11q, and trisomy 12. Recurrent chromosomal abnormalities involved trisomy 12, del17p, del13q and novel translocation (8;17) were only seen in one patient. Conclusion Genomic aberrations in CLL are important independent predictors of disease progression and survival. Interphase cytogenetic analysis using FISH revealed chromosomal changes in the majority of CLL samples and is superior to standard karyotype analysis for identifying cytogenetic abnormalities.

Assessment of PTEN Gene Loss as a Possible Prognostic Marker for Prostate Cancer.

OBJECTIVES: Background: Prostate Cancer (PCa) is a leading cause of cancer deaths in older men worldwide. In the phosphatidylinositol 3-kinase (PIK3)/AKT pathway, the PTEN (10q23.3) gene is a negative regulator and a tumor suppressor gene frequently deleted in PCa. Information about the PTEN deletion in the primary tumor, in addition to clinico-pathological parameters, might be of significance for selecting the ideal treatment for a patient. Therefore, the aim of the present study was to determine the frequency of PTEN deletion in prostate cancer using FISH technique. Materials and Method: Histopathologically proven and diagnosed PCa patients were included for a PTEN gene deletion study by FISH technique. FISH was performed on paraffin embedded tissue using ZytoLight SPEC PTEN/CEN10 Dual Color Probe Kit (CytoVision GmbH, Bremerhaven, Germany). Results: A total of 42 histopathologically proven and diagnosed PCa patients were enrolled in the present study. The median age was 65 years. PTEN gene deletion was positive in 24 patients (57%) while 18 (43%) were negative. PTEN gene deletion was significantly higher in advanced stages as compared to those in early advanced stages. PTEN gene was significantly deleted in patients with the presence of positive lymph nodes compared to patients without positive lymph nodes. Conclusion: The present study suggests that PTEN deletion is associated with tumor stage and lymph node status. This study demonstrated that a higher rate of PTEN deletion is associated with advanced stage cancers with a Gleason's score of 7, which explains the poor prognosis associated with its deletion. Detection of PTEN status will help to identify the specific subsets of patients who might benefit from molecular targeted therapies.

Indian Data on HER2 Fluorescence In Situ Hybridization in Invasive Breast Cancer with Immunohistochemically Equivocal Results As Per 2018 ASCO/CAP Guidelines.

Biren ParikhIntroduction Hormonal status and HER2 expression are valuable biomarkers and dictate the management of the patients diagnosed with invasive breast cancer (IBC). It is crucial to identify the patients who truly respond to anti-HER2 targeted therapy. Updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines has recommended certain modifications in HER2 interpretation by fluorescence in situ hybridization (FISH) with concomitant immunohistochemistry (IHC). Objectives We aimed to evaluate HER2 FISH interpretation in IBC with equivocal IHC results as per 2018 ASCO/CAP recommendations and compare FISH results with hormonal receptor status. Materials and Methods FISH results of 502 cases of IBC with equivocal IHC report between January 2016 to January 2022 were reviewed retrospectively. FISH results were categorized according to ASCO/CAP guidelines 2018 into five respective groups. Results FISH testing in IHC equivocal cases showed 219 (43.6%) cases were classic amplified (positive) belonged to group 1, 217(43.2%) cases were classic nonamplified (negative) fell into group 5, 39 (7.8%) and 02 (0.4%) patients were in group 2 (negative) and group 3 (positive), and 25 (5.0%) cases were in group 4 (negative). About 52.1 and 49.3% of cases with estrogen receptor and progesterone receptor positivity were reported as HER2 positive. Among 502 cases, 25 equivocal cases according to the 2013 guidelines were redefined as HER2 negative and 02 (0.4%) cases reported positive were classified negative as per updated 2018 guidelines. Conclusion Revised 2018 guidelines is helpful in accurate identification of HER2 status and in avoiding targeted therapy in unwarranted cases. Updated 2018 guidelines has removed equivocal HER2-FISH category that has eliminated management dilemma in these cases. Only long-term clinical follow-up will establish the validity of the updated guidelines.

A Comprehensive Review of Chronic Myeloid Leukemia: An Indian Perspective.

OBJECTIVES: The use of imatinib has brought a standard shift in the management of chronic myeloid leukemia (CML) during the last two decades. In India, imatinib has been available for more than fifteen years and has been made available all over the country due to patient assistance programs and cheaper generic versions. Despite improvements in survival of CML patients, there are unique challenges in the Indian context. Indian patients present with more advanced disease. Most centers have access to imatinib as first-line therapy, but there is limited availability of molecular monitoring and second-line therapy. Most of the outcome data is retrospective and comparable with that reported in Western centers. Drug adherence is impaired in at least one third of patients and contributes to poor survival. The aim of this review is to highlight the fact that prospective studies and cooperative studies are very much needed to improve the quality of data available on Indian CML patients.

CML in Chronic Phase with Novel Secondary Cytogenetic Abnormalities: A Case Report.

The clonal evolution in t(9;22)-positive Chronic Myelocytic Leukemia (CML) patients is well established. Four major changes occur in more than 70% of patients: +8, i(17q), +19, and an extra Philadelphia chromosome. Here, we present a case with CML-Chronic phase (CML-CP) and novel t(9;13)(q34;q12~13) in addition to t(9;22)(q34;q11.2). Fluorescence in situ hybridization (FISH) using dual color dual fusion probe analysis on interphase and metaphase cells confirmed the t(9;13)(q34;q12~13) as clonal evolution and secondary event to Philadelphia chromosome. This suggests minor route additional chromosomal aberrations which might affect prognosis. Further studies are required to ascertain the expression of genes that play a role in CML-blast crisis in order to to explore its therapeutic significance and prognostic value.

Location of the BCR/ABL Fusion Genes on Both Chromosomes 9 in Ph Negative Young CML Patients: An Indian Experience.

The BCR/ABL gene rearrangement is cytogenetically visualized in most chronic myeloid leukemia (CML) cases. About 5-10 % of CML patients lack its cytogenetic evidence, however, shows BCR/ABL fusion by molecular methods. We describe two CML patients with Philadelphia (Ph) negative (-ve) and BCR/ABL positive by fluorescence in situ hybridization (FISH). Both the cases were in chronic phase at diagnosis. Conventional cytogenetics and different FISH assays were adopted using BCR/ABL probes. Home-brew FISH assay using bacterial artificial clone (BAC) for BAC-CTA/bk 299D3 for chromosomal region 22q13.31-q13.32 was performed in case 1. Both the patients were Ph-ve. In first case, dual color dual fusion (DCDF)-FISH studies revealed 1 Red (R) 2 Green (G) 1 Fusion (F) signal pattern in 80 % of cells indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph and 2G2F signal pattern in 20 % of cells indicating two BCR/ABL fusions on both chromosomes 9q34 on presentation. In second case, FISH studies revealed the 1R1G1F signal pattern indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph in 100 % of cells at presentation. During follow-up, both the patients exhibited 2G2F signal pattern indicating two BCR/ABL fusions on both chromosomes 9q34, which indicated a clonal evolution in 100 % cells. Both the patients did not achieve therapeutic response. Relocation of BCR/ABL fusion sequence on sites other than 22q11 represents a rare type of variant Ph, the present study highlights the hot spots involved in CML pathogenesis and signifies their implications in Ph-ve BCR/ABL positive CML. This study demonstrated the genetic heterogeneity of this subgroup of CML and strongly emphasized the role of metaphase FISH, especially in Ph-ve CML cases, as it detects variations of the classical t(9;22).

Acute Myeloid Leukemia with a Masked Type of Three-Way t(8;11;21) Revealed by Fluorescence In Situ Hybridizations Using AML1-ETO Probe.

The translocation (8;21)(q22;q22) is associated with acute myeloblastic leukemia (AML) with M2 subtype. The accurate detection of this chromosomal rearrangement is vital due to its association with a favorable prognosis. Variants of t(8;21)(q22;q22) involving chromosomes 8, 21 and other chromosomes account for approximately 3% of all t(8;21)(q22;q22) in AML. Variants in some cases present as hidden translocations, and in such cases it is often difficult to confirm the presence of t(8;21)(q22;q22) by conventional cytogenetic analysis alone. The molecular detection of the AML1-ETO fusion gene is possible by reverse transcriptase polymerase chain reaction (RT-PCR) or dualcolor fluorescence in situ hybridization (FISH) using probes specific for AML1 and ETO. The mechanism described for variant formation is one step or two steps. We report a case of AML with a masked variant translocation. Conventional cytogenetics and FISH study was carried out on a bone marrow sample of the patient at diagnosis. Karyotype result at diagnosis revealed t(8;11)(q22;p15) by G-banding. FISH nalysis disclosed a 3-way translocation involving chromosomes 8, 11, and 21 and identified a masked variant t(8;21)(q22;q22) using AML1-ETO probe and whole chromosome paint probes (WCP) 8 and 11 with a one-step mechanism. FISH analysis with the AML1 and ETO probes is extremely valuable in cases of AML-M2 because of its ability to reveal masked t(8;21) (q22;q22) translocations and thus quickly confirm the diagnosis, allowing patients to be assigned to the correct risk group in terms of treatment. Simple variants of the t(8;21) translocation involving chromosome 8 and a chromosome other than number 21 are rare. Our case illustrates the challenge of recognizing complex aberrations that occur with variant t(8;21) and further reinforces the utility of FISH applications on metaphase for more accurate characterization of chromosome abnormalities which can lead to more precise therapeutic stratification.

Multiple Copies of BCR/ABL Fusion Signals and t(3;21) in a Chronic Myeloid Leukemia: Patient with Blast Crisis - A Rare Event with Imatinib Mesylate (Gleevec)-Resistance in an Indian Patient.

Chronic myeloid leukaemia (CML) is characterized by the expression of BCR/ABL fusion gene, a constitutively activated tyrosine kinase that commonly results from the formation of the Philadelphia (Ph) chromosome after a t(9;22)(q34;q11) or variant rearrangement. The duplication of Ph chromosome is a recurring abnormality acquired during disease progression, whereas intrachromosomal amplification of BCR/ABL is a rare phenomenon and has been associated with imatinib mesylate (IM) therapy resistance. In the present study, we used G-banding to identify the presence of identical isochromosomes of the Ph chromosome and t(3;21)(q26;q22) resulted from clonal evolution in IM-resistant patient. Fluorescence in situ hybridization (FISH) using dual color dual fusion probe analysis on interphase and metaphase nuclei confirmed the amplification of the fused BCR/ABL gene. Our study indicated that the progenitor of CML was BCR/ABL dependent through the amplification of Ph chromosome as a mechanism of resistance to IM therapy. The coexistence of BCR/ABL and t(3;21)(q26;q22) with RUNX1 rearrangement might play a pivotal role in the CML blast transformation.

Trisomy 8 in leukemia: A GCRI experience.

Trisomy of chromosome 8 is frequently reported in myeloid lineage disorders and also detected in lymphoid neoplasms as well as solid tumors suggesting its role in neoplastic progression in general. It is likely to be a disease-modulating secondary event with underlying cryptic aberrations as it has been frequently reported in addition to known abnormalities contributing to clinical heterogeneity and modifying prognosis. Here, we share our findings of trisomy 8 in leukemia patients referred for diagnostic and prognostic cytogenetic assessment. Total 60 cases of trisomy 8, as a sole anomaly or in addition to other chromosomal aberrations, were reported (January 2005-September 2008). Unstimulated bone marrow or blood samples were cultured, followed by GTG banding and karyotyping as per the ISCN 2005. Patients with +8 were chronic myeloid leukemia (CML) (36), acute myeloid leukemia (AML) (17), and acute lymphoblastic leukemia (ALL) (7). In 7 patients, trisomy 8 was the sole anomaly, whereas in 6 patients +8 was in addition to normal clone, in 47 patients, the +8 was in addition to t(9;22), t(15;17), and others, including 3 with tetrasomy 8. Only one patient showed constitutional +8. The present study will form the basis of further cumulative studies to correlate potential differential effects of various karyotypic anomalies on disease progression and survival following a therapeutic regime. To unravel the role of extra 8 chromosome, constitutional chromosomal analysis and uniparental disomy will be considered.

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients.

AIMS: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. METHODS: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. RESULTS: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. CONCLUSION: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.