Targeted Resequencing of 30 Genes Improves the Detection of Deleterious Mutations in South Indian Women with Breast and/or Ovarian Cancers

Rajkumar, Thangarajan,Meenakumari, Balaiah,Mani, Samson,Sridevi, Veluswami,Sundersingh, Shirley Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.13

Background: We earlier used PCR-dHPLC for mutation analysis of BRCA1 and BRCA2. In this article we report application of targeted resequencing of 30 genes involved in hereditary cancers. Materials and Methods: A total of 91 patient samples were analysed using a panel of 30 genes in the Illumina HiScan SQ system. CLCBio was used for mapping reads to the reference sequences as well as for quality-based variant detection. All the deleterious mutations were then reconfirmed using Sanger sequencing. Kaplan Meier analysis was conducted to assess the effect of deleterious mutations on disease free and overall survival. Results: Seventy four of the 91 samples had been run earlier using the PCR-dHPLC and no deleterious mutations had been detected while 17 samples were tested for the first time. A total of 24 deleterious mutations were detected, 11 in BRCA1, 4 in BRCA2, 5 in p53, one each in RAD50, RAD52, ATM and TP53BP1. Some 19 deleterious mutations were seen in patients who had been tested earlier with PCR-dHPLC [19/74] and 5/17 in the samples tested for the first time, Together with our earlier detected 21 deleterious mutations in BRCA1 and BRCA2, we now had 45 mutations in 44 patients. BRCA1c.68_69delAG;p.Glu23ValfsX16 mutation was the most common, seen in 10/44 patients. Kaplan Meier survival analysis did not show any difference in disease free and overall survival in the patients with and without deleterious mutations. Conclusions: The NGS platform is more sensitive and cost effective in detecting mutations in genes involved in hereditary breast and/or ovarian cancers.

Characterization of clonal immunoglobulin heavy (IGH) V-D-J gene rearrangements and the complementarity-determining region in South Indian patients with precursor B-cell acute lymphoblastic leukemia

Natarajan Sudhakar,Thangarajan Rajkumar,Kamalalayam Raghavan Rajalekshmy,Nirmala Karunakaran Nancy 대한혈액학회 2017 Blood Research Vol.52 No.1

Background: This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identi-fied age-related predominance in VDJ rearrangements. Methods: IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. Results: Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A so-matic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. Conclusion: The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predom-inantly used in precursor-B-ALL. However, the IGH gene rearrangements in pre-cursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.

Characterization of clonal immunoglobulin heavy (IGH) V-D-J gene rearrangements and the complementarity-determining region in South Indian patients with precursor B-cell acute lymphoblastic leukemia

Natarajan Sudhakar,Thangarajan Rajkumar,Kamalalayam Raghavan Rajalekshmy,Nirmala Karunakaran Nancy 대한혈액학회 2017 Blood Research Vol.52 No.1

Background: This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identi-fied age-related predominance in VDJ rearrangements. Methods: IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. Results: Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A so-matic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. Conclusion: The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predom-inantly used in precursor-B-ALL. However, the IGH gene rearrangements in pre-cursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.

Endo-sulfatase Sulf-1 Protein Expression is Down-regulated in Gastric Cancer

Gopal, Gopisetty,Shirley, Sundersingh,Raja, Uthandaraman Mahalinga,Rajkumar, Thangarajan Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.2

In our recent report on gene expression in gastric cancer we identified the endo-sulfatase Sulf-1 gene to be up-regulated in gastric tumors relative to apparently normal (AN), and paired normal (PN) gastric tissue samples. In the present report we investigate the protein expression levels of Sulf-1 gene in gastric tumors, AN and PN samples using tissue microarray (TMA) and immunohistochemistry. Expression data was collected from two sets of TMA's containing replicate sections of tissue samples. Scoring data from TMA set-1 revealed a significant difference in Sulf-1 immunoreactivity between tumors and "normals" (PN and AN) (p-value = 0.001928). Also, Sulf-1 expression in tumors was also significantly different from either PN (p-value = 0.019) or AN (p-value = 0.006) samples. Similar results were obtained from analysis of scoring data from the second set of arrays. Comparison of mRNA expression and protein expression in gastric tumor tissues revealed that in 6/20 (30%) tumor samples showed up-regulated protein expression concordant with over-expression of mRNA. However, a discord with mRNA being over-expressed relative to down regulated protein expression was observed in majority 14/20 (70%) of tumor samples. Our study indicates down regulation of Sulf-1 protein expression in gastric tumors relative to PN and AN samples which is discordant with mRNA over-expression seen in tumors.

Intragenic DNA Methylation Concomitant with Repression of ATP4B and ATP4A Gene Expression in Gastric Cancer is a Potential Serum Biomarker

Raja, Uthandaraman Mahalinga,Gopal, Gopisetty,Rajkumar, Thangarajan Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.11

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down-regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.

Development and Clinical Evaluation of Dendritic Cell Vaccines for HPV Related Cervical Cancer - a Feasibility Study

Ramanathan, Priya,Ganeshrajah, Selvaluxmy,Raghanvan, Rajalekshmi Kamalalayam,Singh, Shirley Sundar,Thangarajan, Rajkumar Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.14

Human papillomavirus infection (HPV) and HPV related immune perturbation play important roles in the development of cervical cancer. Since mature dendritic cells (DCs) are potent antigen-presenting cells (APC), they could be primed by HPV antigens against cervical cancers. In this study we were able to generate, maintain and characterize, both phenotypically and functionally, patient specific dendritic cells in vitro. A randomized Phase I trial with three arms - saline control (arm I), unprimed mature DC (arm II) and autologous tumor lysate primed mature DC (arm III) and fourteen patients was conducted. According to WHO criteria, grade 0 or grade one toxicity was observed in three patients. One patient who received tumor lysate primed dendritic cells and later cis-platin chemotherapy showed a complete clinical response of her large metastatic disease and remained disease free for more than 72 months. Our findings indicate that DC vaccines hold promise as adjuvant sfor cervical cancer treatment and further studies to improve their efficacy need to be conducted.

Autologous cervical tumor lysate pulsed dendritic cell stimulation followed by cisplatin treatment abrogates FOXP3+ cells in vitro

Hemavathi Dhandapani,Abirami Seetharaman,Hascitha Jayakumar,Selvaluxmy Ganeshrajah,Shirley Sunder Singh,Rajkumar Thangarajan,Priya Ramanathan 대한부인종양학회 2021 Journal of Gynecologic Oncology Vol.32 No.4

Objective: Dendritic cells (DCs) are administered as immunotherapeutic adjuvants after the completion of standard treatment in most settings. However, our Phase I trial indicated that one patient out of four, who received autologous tumor lysate-pulsed dendritic cell (TLDC) also received cisplatin chemotherapy and experienced complete regression of her lung lesion, continuing to be disease free till date. Hence, the objective of our current study is to evaluate the sustenance or augmentation of immune responses when autologous human papillomavirus positive cervical tumor lysate pulsed DC- are combined with cisplatin, using co-culture assays in vitro. Methods: Before treatment, peripheral blood and punch biopsy samples were collected from 23 cervical cancer patients after obtaining an informed consent. DC functionality was confirmed through phenotypic and functional assays using autologous peripheral blood mononuclear cells as responders. For cisplatin experiments, the drug was added at 150, 200 (clinical dose equivalent), and 400 µM concentrations to DCs alone or DC-T cell co- cultures. Phenotypic assessment and functional characterization of DCs was done using flow cytometry. Cytokine enzyme-linked immunosorbent assay and interferon (IFN)-γ enzyme- linked immune absorbent spot assays were also performed. Results: The functionality of TLDCs was not compromised upon cisplatin treatment in vitro even at the highest (400 μM) concentration. Even though cisplatin treatment reduced the secretion of IFN-γ and interleukin (IL)-12p40 in co-cultures stimulated with TLDCs, this effect was not significant (p>0.05). A doubling of IFN-γ secretion following cisplatin treatment was observed in at least one of three independent experiments. Additional experiments showed a reduction in both FOXP3+ regulatory T cells and IL-10 levels. Conclusion: Our results provide evidence that cisplatin treatment may be given after autologous TLDC administration to maintain or improve a productive anti-tumor response in vaccinated patients.