Differential gene expression changes and their implication on the disease progression in patients with Chronic Myeloid Leukemia.

The molecular mechanisms responsible for disease progression of CML are not conclusive. The main functional changes associated with disease evolution in CML was high proliferation rate, decreased apoptosis, blockade of differentiation, and strong resistance to chemotherapeutic agents. The current study analyzed the relative expressional profiles of genes related with proliferation, apoptosis, differentiation, and resistance to chemotherapeutic agents such as c-MYC, BAD, BCL-2, C/EBPα/-ß and ABCB1 respectively in different clinical stages of CML by SYBR Green I quantitative real-time (qRT) PCR. We selected a total of 183 CML patients and 30 healthy control samples. The study populations were classified into four groups, including de novo CML-CP (50/183), CML-AP (32/183), CML-BC (51/183) and Imatinib Mesylate or IM resistant CML-CP (50/183) groups. qRT PCR analysis revealed that significant overexpression of c-MYC, ABCB1 and BCL-2 was observed in advanced phases and IM resistant CP of CML compared to healthy controls. Likewise, the mean expression level of BAD, C/EBPα/-ß genes were found to be significantly down regulated. Present study concluded that the complex interplay of several candidate genes like overexpression of c-MYC, ABCB1, BCL-2 and down regulation of BAD, C/EBPα/-ß played a significant role in the disease evolution and development of drug resistant in CML.

Isodicentric Philadelphia Chromosome: A Rare Chromosomal Aberration in Imatinib-Resistant Chronic Myelogenous Leukemia Patients - Case Report with Review of the Literature.

The BCR-ABL1 fusion gene derived from the Philadelphia chromosome, resulting from a classical translocation event t(9;22)(q34.13;q11.23), is responsible for the pathogenesis of chronic myeloid leukemia (CML) in more than 90% of the patients. The isoderivative chromosome 22, ider(22), and relative amplification or duplication of the BCR-ABL1 gene have been considered as one of the major reasons associated with the resistance to chemotherapy with imatinib mesylate, but the data remain unclear. GTG-banding together with FISH were performed to identify the presence of the ider(22) chromosome. Reverse transcription-polymerase chain reaction (RT-PCR) for the detection of BCR-ABL1 fusion transcripts and BCR-ABL1 kinase domain mutation analysis were carried out in this study. Conventional and molecular cytogenetic analysis on metaphase chromosomes confirmed the presence of ider(22) chromosomes in both patients. Molecular characterization revealed the presence of a 210-kDa BCR-ABL1 type b3a2 and lack of mutations at the kinase domain region on the fusion product in both patients. The occurrence of the ider(22) chromosome could be considered as an important marker correlated with the aggressive progression of CML as well as the emergence of drug-resistant cell clones.

A Case Report of Concurrent IDH1 and NPM1 Mutations in a Novel t(X;2)(q28;p22) Translocation in Acute Myeloid Leukaemia without Maturation (AML-M1).

Acute myeloid leukaemia (AML) is one of the fatal haematological malignancies as a consequence of its genetic heterogeneity. At present, the prediction of the clinical response to treatment for AML is based not only on detection of cytogenetic aberrations but also by analysing certain molecular genetic alterations. There are limited in sights into the contribution, disease progression, treatment outcome, and characterisation with respect to the uncommon chromosomal abnormalities leading to AML. Here, we describe the clinical, morphological, cytogenetic, and mutational findings of a 52-year-old female patient with AML without maturation (AML-M1). Conventional karyotyping and spectral karyotyping (SKY) were done on metaphase chromosomes from bone marrow cells at the time of diagnosis. A mutation analysis was performed on the hotspot regions of various genes, including FLT3, CEBPA, NPM1, RAS, c-KIT, IDH1 and IDH2. Cytogenetic and mutation analyses revealed a novel translocation, t(X;2)(q28;p22), with both NPM1 and IDH1 mutations. To the best of our knowledge, the presence of both NPM1 and IDH1 mutations in t(X;2)(q28;p22) is a novel finding in AML.

A novel chromosomal abnormality t (9;14)(p24;q13) in B-acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is a malignant disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow. We describe the clinical, morphologic, immunophenotypic and cytogenetic findings in the case of a 26-year-old man with B-lymphoblastic leukemia. Surface marker analysis revealed that they are positive for CD markers CD10, CD19, CD13, CD34, CD45 and HLA-DR, but negative for CD20, CD33, CD117 and CD11C markers. Cytogenetic analysis established a novel translocation, t (9;14)(p24;q13). Apart from this, spectral karyotyping revealed an additional translocation, t (6p; 14q). This is the first documented case of B-lymphoblastic leukemia with concurrent occurrence of both abnormalities. Further studies are needed to understand the role of this abnormality in carcinogenesis.

Double Philadelphia chromosome: a rare and sole abnormality in pediatric B-acute lymphoblastic leukemia.

The present study describes a 7-year-old male child who had attended the Pediatric Oncology Clinic of the Regional Cancer Centre, Thiruvananthapuram, Kerala, India, and was pathologically confirmed to have B-Acute Lymphoblastic Leukemia (B-ALL). Conventional cytogenetics analysis at diagnosis showed the presence of a double Philadelphia chromosome and the karyotype of the case was 47, XY, t(9;22)(q34;q11.2), + der(22)t(9;22). FISH, done as a molecular confirmation of the translocation, t(9;22)(q34;q11.2), and this case showed an additional fusion signal that confirms the presence of double Ph. As far as we are aware, this represents the initial and only  occurrence of an abnormality report regarding the double Philadelphia chromosome in pediatric B-ALL within India. The double Philadelphia chromosome in B-ALL has a very poor prognosis despite aggressive treatment with chemotherapy. This study reveals the importance of conventional and molecular cytogenetic analysis in risk stratification and prognosis prediction of pediatric B-ALL. The risk stratification based on the conventional and molecular cytogenetic analysis may be taken into consideration for deciding the treatment strategy for each patient.

Acute myeloid leukemia patients with variant or unusual translocations involving chromosomes 8 and 21 - A comprehensive cytogenetic profiling of three cases with review of literature.

Background: t(8;21)(q22;q22) is the most frequent recurrent translocation in acute myeloid leukemia (AML) resulting in an in-frame fusion of RUNX1/RUNX1T1 that regulates various genes involved in the signaling pathways. This leukemogenic alteration is usually associated with a favorable clinical outcome. Variants of t(8;21) can be formed involving a third or fourth chromosome in ~3-4% of t(8;21)-AML. Due to the rarity of variant t(8;21), its clinicopathological features and prognostic significance are still unclear. Here we present three AML cases with cryptic rearrangements of chromosomes 8 and 21 without standard RUNX1/RUNX1T1. Materials and Methods: Conventional karyotyping and fluorescence in situ hybridization and/or spectral karyotyping of the pretreatment bone marrow aspirate of de novo AML patients were performed to delineate chromosomal abnormalities. Results: We identified three cases with novel variants of t(8;21); der(13)t(8;21;13), isodicentric derivative 8 with chromosome 21[,+idicder(8)(q11.1)t(8;21)(q22;q11.1)] and der(21)t(8;12;21)(q22;q?;q22). Conclusion: AML with t(8;21)(q22;q22);RUNX1-RUNX1T1 forms a distinct WHO subcategory and hence the identification of variants or unusual translocations associated with t(8;21) deserves more attention. Contribution to the variant/ unusual t(8;21) database will further refine the risk stratification and may help to significantly advance the current treatment regimen.

Whole Exome Sequencing Identifies Cohesin Component STAG1 Mutation in de novo Acute Myeloid Leukemia (FAB M2): A Pilot Study with Cytogenetics, Clinical and Prognostic Implications.

The clinical implications of cohesin gene complex mutation in acute myeloid leukemia (AML) are not well characterized. In the present study, a cohort of 152 de novo unselected adult AML patients underwent conventional and molecular cytogenetic analysis for chromosomal aberrations. Further, we examined the frequency and clinical implications of mutations in cohesin gene complex STAG1, STAG2, RAD21, SMC1, and SMC3 using whole exome sequencing as a pilot study in 10 de novo patients with AML-FAB M2. Among the 10 cases, we identified a functionally heterozygous mutation in exon16 of STAG1 in one patient (10%), however no mutation was observed in STAG2, RAD21, SMC1, and SMC3. Sanger sequencing analysis for exon 16 of STAG1 in the remaining 142 AML cases did not reveal any further mutations, which underlined the observation that mutations took place throughout the cohesin gene complex without presence of a mutational hot spot region. The present study identified a positive correlation between serum bilirubin, LDH, and hematological parameters such as Hb, WBC, and platelet count with STAG1 mutation. Our data suggest that the cohesin complex may represent an attractive therapeutic target for future preclinical and clinical studies. However, more studies with a larger number of patients should be performed prospectively to determine the pathogenic involvement of STAG 1 mutation in AML patients.

A concise review on the molecular genetics of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults that affects the myeloid lineage. The recent advances have upgraded our understanding of the cytogenetic abnormalities and molecular mutations associated with AML that further aids in prognostication and risk stratification of the disease. Based on the highly heterogeneous nature of the disease and cytogenetic profile, AML patients can be stratified into favourable, intermediate and adverse-risk groups. The recurrent genetic alterations provide novel insights into the pathogenesis, clinical characteristics and also into the overall survival of the patients. In this review we are discussing about the cytogenetics of AML and the recurrent gene alterations such us NPM1, FLT3, CEBPA, TET-2, c-KIT, DNMT3A, IDH, RUNX1, AXSL1, WT1, Ras gene mutations etc. These gene mutations serve as important prognostic markers as well as potential therapeutic targets. AML patients respond to induction chemotherapy initially and subsequently achieve complete remission (CR), eventually most of them get relapsed.

Prognostic Implications of DNA Repair, Ploidy and Telomerase in the Malignant Transformation Risk Assessment of Leukoplakia.

BACKGROUND: Although leukoplakia shows a higher risk for malignant transformation to oral cancer, currently there are no clinically relevant biomarker which can predict the potentially high risk leukoplakia. This study aimed to investigate the genetic alterations such as DNA ploidy, telomerase expression and DNA repair capacity as predictive markers of malignant transformation risk of leukoplakia. METHODS: The study was initiated in September 2005 and patients were followed up to March 2014. Two hundred patients with oral leukoplakia, 100 patients with oral cancer and 100 healthy, age and sex matched adults with normal oral mucosa as controls were recruited. The DNA ploidy content was measured by high resolution flow cytometry, level of telomerase expression was identified by TRAP assay and intrinsic DNA repair capacity was measured by mutagen induced chromosome sensitivity assay of cultured peripheral blood lymphocytes. The Chi-square test or Fisher's Exact test was used for comparison of categorical variables between biomarkers. A p value less than or equal to 0.05 was considered as statistically significant. Analysis was performed with SPSS software version 16. Logistic regression was used to find the association between the dependent and three independent variables. RESULTS: There was significant difference in the distribution of ploidy status, telomerase activity and DNA repair capacity among control, leukoplakia and oral cancer group (p<0.001). When the molecular markers were compared with histological grading of leukoplakia, both DNA ploidy analysis and telomerase activity showed statistical significance (p<0.001). Both aneuploidy and telomerase positivity was found to coincide with high-risk sites of leukoplakia and were statistically significant (p.

Impact of Additional Chromosomal Aberrations on the Disease Progression of Chronic Myelogenous Leukemia.

The emergence of additional chromosomal abnormalities (ACAs) in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukemia (CML), is considered to be a feature of disease evolution. However, their frequency of incidence, impact on prognosis and treatment response effect in CML is not conclusive. In the present study, we performed a chromosome analysis of 489 patients in different clinical stages of CML, using conventional GTG-banding, Fluorescent in situ Hybridization and Spectral Karyotyping. Among the de novo CP cases, ACAs were observed in 30 patients (10.20%) with lowest incidence, followed by IM resistant CP (16.66%) whereas in AP and BC, the occurrence of ACAs were higher, and was about 40.63 and 50.98%, respectively. The frequency of occurrence of ACAs were compared between the study groups and it was found that the incidence of ACAs was higher in BC compared to de novo and IM resistant CP cases. Likewise, it was higher in AP patients when compared between de novo and IM resistant CP cases, mirroring the fact of cytogenetic evolution with disease progression in CML. In addition, we observed 10 novel and 10 rare chromosomal aberrations among the study subjects. This study pinpoints the fact that the genome of advanced phase patients was highly unstable, and this environment of genomic instability is responsible for the high occurrence of ACAs. Treatment response analysis revealed that compared to initial phases, ACAs were associated with an adverse prognostic effect during the progressive stages of CML. This study further portrayed the cytogenetic mechanism of disease evolution in CML.

Genomic amplification of BCR-ABL1 fusion gene and its impact on the disease progression mechanism in patients with chronic myelogenous leukemia.

Identification of BCR-ABL1 fusion gene amplification status is critically important in the effective management of chronic myelogenous leukemia (CML) patients. Earlier reports suggested that overexpression of BCR-ABL1 either through amplification of BCR-ABL1 fusion gene or by the up regulation of BCR-ABL1 transcript level might be an early phenomenon in the establishment of IM resistance and disease evolution in CML. In the current study, we performed dual color dual fusion locus specific BCR/ABL1 FISH analysis along with karyotype analysis using GTG banding (G-banding using trypsin and Giemsa) technique in 489 patients with different clinical stages of CML at diagnosis or during the course of the disease to unravel the spectrum of BCR-ABL1 fusion gene amplification status. Among the study group analyzed, it was found that prevalence of occurrence of BCR-ABL1 fusion gene amplification was significantly higher in advanced stages of disease and in IM resistant CML-CP patients when compared to initial stage of disease, de novo CML-CP. Cytogenetic and metaphase FISH characterization on our study samples revealed that BCR-ABL1 fusion gene amplification was occurred through the formation of extra copies Ph chromosomes and isoderived Ph chromosomes. Current study suggests that unrestrained activity of BCR-ABL1 played a vital role in resistance to targeted therapy and disease evolution in CML. In our study population, patients in progressive stage CML and in IM resistant CP with multiple copies of BCR-ABL1 fusion gene displayed a poor response to targeted treatment with IM. Hence, the early identification of BCR-ABL1 fusion gene amplification using FISH technique will lead to improved interventions and outcome in future CML patients.

Prognostic Implications of Derivative Chromosome 9 Deletions in Patients with Advanced-Stage Chronic Myelogenous Leukemia.

Elucidation of cryptic BCR/ABL1 gene rearrangement is exceptionally important in the clinical diagnosis and prognosis of chronic myelogenous leukemia (CML). Previous reports indicated an adverse prognostic effect of atypical BCR/ABL1 gene rearrangements with submicroscopic ABL1-BCR deletions on derivative chromosome 9 [der(9)] in CML patients. Dual color dual fusion locus-specific BCR/ABL1 fluorescent in situ hybridization (FISH) analysis together with G-banding using trypsin and Giemsa (GTG banding) was performed in 489 patients at different stages of CML to investigate the spectrum of BCR/ABL1 gene rearrangements. Among the study group analyzed, a significantly higher frequency of BCR/ABL1 gene rearrangements that is consistent with der(9) deletion were observed in the blast crisis (BC) phase at 41.67%, followed by the accelerated phase (AP) at 36.84%, the imatinib mesylate (IM)-resistant chronic phase (CP) at 23.08%, and the lowest incidence was found in de novo CP at 16.61%. 1R1G1F (1 red, 1 green, 1 fusion) with concurrent loss of ABL1-BCR fusion gene on der(9) chromosome was the major signal pattern identified in each group. The results from the current study show that this unusual BCR/ABL1 gene rearrangement is one of the steering forces toward disease progression in CML. Patients in AP/BC of CML with der(9) deletion showed poor response to IM therapy; however, patients with der(9) deletion in the early phases of CML responded well to IM treatment. Therefore, the establishment of an atypical FISH signal pattern in CML is of paramount important because it is associated with adverse clinical prognostic implications in advanced stages of the disease.

Novel germline mutations in BRCA2 gene among 96 hereditary breast and breast-ovarian cancer families from Kerala, South India.

PURPOSE: Aim of the present study was to identify the genetic heterogeneity, prevalence and frequency of germline mutations of BRCA2 gene in Hereditary Breast/Ovarian cancer patients from Kerala, South India. METHODS: We analyzed 102 Breast/Ovarian cancer patients from 96 breast and/ovarian cancer families for BRCA2 gene mutations using Conformation-Sensitive Gel Electrophoresis (CSGE) followed by sequencing. RESULTS: Sequence variations in BRCA2 gene were detected in 27 (26.4%) patients. Sixteen distinct sequence variants were detected of which 11 were (69%) in exon 11. We have identified two novel disease-causing frameshift mutations (c.4642delAA and c.4926insGACC) in two unrelated patients. Apart from this, fourteen distinct sequence variants were detected in 25 breast/ovarian cancer patients of which 8 (57%) were also novel. These include nine missense mutations, one silent mutation, one-nonsense mutation and three intronic variants. CONCLUSIONS: The results of this study suggest that germline mutations of BRCA2 gene account for rather small proportion of Hereditary Breast/Ovarian cancer in Kerala, South India.

Characterization of CEBPA Mutations and Polymorphisms and their Prognostic Relevance in De Novo Acute Myeloid Leukemia Patients.

The CCAAT/enhancer-binding protein-alpha (CEBPA) is a transcriptional factor that plays a crucial role in the control of proliferation and differentiation of myeloid precursors. This gene was recognized as the target of genetic alterations and were associated with clinical complexity among AML. We here analyze the frequency and types of CEBPA mutations and polymorphisms in a de novo AML patients from South India and tried to find out associations of these variations with different clinical parameters and the prognostic significance in AML. Study was carried out in 248 de novo AML patients, cytogenetic analysis was performed from the bone marrow samples and was karyotyped. PCR-SSCP analysis and sequencing was performed for the detection of CEBPA gene variations. All the statistical analysis was performed using SPSS 17 (statistical package for social sciences) software. Pearson Chi-square test, Mann-Whitney U test, Kaplan-Meier survival analysis and log rank tests were performed. CEBPA mutations were detected in 18% and CEBPA polymorphisms were detected in 18.9% of AML cases studied. Most of the mutations occured at the C terminal region. Polymorphisms were detected in both N and C terminal region. with most common being, c.584_589dup ACCCGC and c.690G>T. A significant association was not observed for the mutation and polymorphism with respect to clinical and laboratory parameters. Survival advantage was observed for the mutated cases compared to non mutated cases, especially for the normal karyotype groups. Polymorphisms has no effect on the survival pattern of AML patients. CEBPA mutation and polymorphisms were observed with similar frequency and was identified in all the FAB subtypes as well as in cytogenetic risk groups in our study population, but CEBPA mutations alone confer a prognostic value for NK AML patients.

Mutation Analysis of IDH1/2 Genes in Unselected De novo Acute Myeloid Leukaemia Patients in India - Identification of A Novel IDH2 Mutation.

IDH1/2 mutations which result in alternation in DNA methylation pattern are one of the most common methylation associated mutations in Acute myeloid leukaemia. IDH1/2 mutations frequently associated with higher platelet level, normal cytogentics and NPM1 mutations. Here we analyzed IDH1/2 mutations in 200 newly diagnosed unselected Indian adult AML patients and investigated their correlation with clinical, cytogenetic parameters along with cooperating NPM1 mutation. We detected 5.5% and 4% mutations in IDH1/2 genes, respectively. Except IDH2 c.515_516GG>AA mutation, all the other identified mutations were reported mutations. Similar to reported c.515G>A mutation, the novel c.515_516GG>AA mutation replaces 172nd arginine to lysine in the active site of the enzyme. Even though there was a preponderance of IDH1/2 mutations in NK-AML, cytogenetically abnormal patients also harboured IDH1/2 mutations. IDH1 mutations showed significant higher platelet count and NPM1 mutations. IDH2 mutated patients displayed infrequent NPM1 mutations and lower WBC count. All the NPM1 mutations in the IDH1/2 mutated cases showed type A mutation. The present data suggest that IDH1/2 mutations are associated with normal cytogenetics and type A NPM1 mutations in adult Indian AML patients.

Novel t(7;10)(p22;p24) along with NPM1 mutation in patient with relapsed acute myeloid leukemia.

Chromosomal abnormalities/genetic mutations associated with hematological malignancies alter the structure and function of genes controlling cell proliferation and differentiation through multiple and complex pathways, resulting different clinical outcomes. This is a case study of a lady presented with acute myeloid leukemia (AML M1) at our center who relapsed 10 years after the induction therapy. Cytogenetic and molecular analyses were performed in this case at the time of relapse to find out the chromosomal abnormalities and genetic abnormalities like FMS-like tyrosine kinase (FLT3) and nucleophosmin (NPM1) mutation. The cytogenetic analysis of bone marrow established a novel translocation t(7;10) (p22;q24) in 100% of the cells analyzed. Phytohaemagglutinin (PHA)-stimulated blood culture also revealed the same abnormality. Apart from this, the molecular analysis showed NPM1 exon 12 (hot-spot) mutation in this patient. This was the first report of novel chromosomal translocation in this subset of AML in which a new translocation along with NPM1 mutation was discussed.

Contribution of XPD (Lys751Gln) and XRCC1 (Arg399Gln) polymorphisms in familial and sporadic breast cancer predisposition and survival: an Indian report.

The etiology of a significant proportion of familial breast cancers is still poorly understood, with known high penetrance gene mutations accounting for only a small proportion of the cases. The increased risk of breast cancer for the majority of women with a family history likely reflects shared minor low penetrant genetic factors. In the present case-control study undertaken to examine the influence of DNA damage repair gene polymorphisms in familial and sporadic breast cancer susceptibility, 219 Sporadic and 140 familial breast cancer patients and 367 controls were genotyped using PCRRFLP. Odds Ratios (ORs) and 95% Confidence Intervals (95%CIs) were calculated by unconditional logistic regression adjusted to age. Variant genotypes XRCC1 Arg/Gln or Gln/Gln and XPD Lys/Gln or Gln/Gln increased both familial and sporadic breast cancer susceptibility. However, when the intra group risk was compared, the risk due to the XPD polymorphic genotypes Lys/Gln or Gln/Gln was significantly lower among familial breast cancer patients compared to sporadic breast cancer patients [OR = 0.61; 95%CI = 0.39-0.94; p value = 0.024) whereas the risk implied by XRCC1 variant genotype was not significantly different between the familial and nonfamilial groups of breast cancer patients [OR = 0.97; 95%CI = 0.63-1.49; p value = 0.882]. Both these variant genotypes were not associated with the disease characteristics or survival of either familial or sporadic breast cancer patients. This study represents an addition to previous published work on GSTs from the same study population and substantiates the hypothesis that the impact of the low penetrance gene polymorphisms differ by family history of the disease.