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.

Mutagen sensitivity in oral cancer patients, healthy tobacco chewers and controls.

OBJECTIVE: To analyze chromosomal aberrations (CA) as an index of DNA damage, to measure DNA repair capability using mutagen sensitivity assay and to correlate tobacco exposure with CA. STUDY DESIGN: Oral cancer patients, healthy tobacco chewers and healthy tobacco nonusers were studied for spontaneous and mutagen-induced CA. An arbitrary unit obtained for lifetime tobacco exposure (LTE) was compared with CA. RESULTS: Mean levels of spontaneous and mitomycin-C-induced CA were higher in patients as compared to chewers and controls. DNA repair capability of patients was significantly deficient (p < or = 0.016) as compared to that of chewers. LTE was significantly higher (p = 0.004) in patients than chewers. Chewers having high LTE and spontaneous CA above cutoff levels might be at a greater risk of oral carcinogenesis. CONCLUSION: There is a probable risk of oral carcinogenesis in healthy tobacco consumers having higher CA and LTE. Whether the deficient DNA repair capacity of oral cancer patients is due to the disease process or the tobacco exposure needs to be confirmed with a larger population study.

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.

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.

Atypical D-FISH patterns of BCR/ABL gene rearrangements in 169 chronic myeloid leukemia patients.

The Philadelphia (Ph) chromosome, a hallmark chromosomal anomaly observed in 95 percent of chronic myeloid leukemia (CML) cases, is known to involve the Abelson (ABL) proto-oncogene on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22, producing BCR/ABL mRNA encoding an abnormal tyrosine kinase protein. In the process of generating BCR-ABL fusion, the deletion of residual BCR or ABL occurs in 15-30 percent of CML patients. In addition, some rearrangements are complex, and do not yield the ABL/BCR fusion due to the involvement of a third chromosome in the rearrangement. The possible role of these deletions and complex rearrangements in disease outcome is an ongoing topic of research. We report our results of cytogenetic analysis with GTG banding and fluorescence in situ hybridization using dual color dual fusion probe (D-FISH) from Vysis Inc, USA in 169 (109 male and 60 female) CML patients registered at The Gujarat Cancer and Research Institute (GC and RI) from April 2004 to December 2005. GTG banding was carried out in 123 cases having analyzable metaphases. Of these 123 cases, D-FISH revealed atypical signal patterns in 57 patients (46%), and 12 cases revealed additional complex translocations indicative of disease progression. Out of 57 cases with atypical FISH patterns, 22 included metaphase FISH results, and the rest had only interphase FISH performed. In addition to the hallmark Philadelphia chromosome, other chromosomal aberrations in CML revealed heterogeneity of molecular events. Pooling of more data may lead to identification of new CML sub-groups and hence help in the analysis of clinical trials. Patients enrolled in our prospective study of prognostic significance will be followed up for disease free and overall survival in correlation with ABL-BCR deletion status.