Nilanjana Bhattacharya

Subtype-specific alterations of the Wnt signaling pathway in breast cancer: clinical and prognostic significance.

The aim of the study is to understand the importance of the Wnt/β‐catenin pathway in the development of breast cancer (BC) and its association with different clinicopathological parameters. Alterations (deletion/methylation/expression) of some Wnt/β‐catenin pathway antagonists like APC, SFRP1/2, CDH1 and activator β‐catenin (CTNNB1) were analyzed in primary BC in Indian patients. High frequencies (65–70%) of overall alterations (deletion/methylation) of the antagonists were seen in the BC samples. Also, 99% (156/158) of the samples showed alterations in any one of the genes, indicating the importance of this pathway in the development of this tumor. Co‐alterations of these genes were observed in 30% of samples, with significantly high alterations in late‐onset (37%) and estrogen receptor (ER)−/progesterone receptor (PR)− (37%) BC compared with early onset (21%) and ER/PR+ (18%) BC samples, respectively. Significantly high (P‐value = 0.001–0.02) alterations of APC and CDH1 genes were seen in ER−/PR− BC compared with ER/PR+ BC. Immunohistochemical analysis showed reduced expression of the Wnt antagonists in BC concordant with their molecular alterations. Nuclear localization of β‐catenin showed significant association with alterations in the antagonists and was also significantly high in the ER−/PR− BC samples. Alterations of SFRP2 coupled with a late clinical stage and low/nulliparity predicted the worst prognosis in BC patients. Therefore, the present study suggests that cumulative alterations in more than one Wnt antagonist along with increased nuclear accumulation of β‐catenin play an important role in the development of BC and have significant clinical as well as prognostic importance. (Cancer Sci 2012; 103: 210–220)

MYC gene amplification reveals clinical association with head and neck squamous cell carcinoma in Indian patients

BACKGROUND Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). This study is focused to analyze the correlation between MYC gene amplification and various clinicopathological features and outcome in a cohort of 49 dysplastic and 187 primary head and neck lesions. METHODS MYC gene amplification was assessed by differential polymerase chain reaction using primer sets from the MYC gene as target locus and DRD2 gene as the control locus. RESULT The MYC gene amplification was detected in a total of 23.7% (56/236) head and neck lesions comprising 14.2% (7/49) dysplastic lesions and 26% (49/187) HNSCC samples. The clinicopathological association study between MYC gene amplification with the different clinical parameters like sex, tumor stage, tumor differentiation, lymph node status, tobacco habit and HPV 16/18 status determined significant association of MYC amplification with tumor progression (P = 0.009). Kaplan Meier analysis revealed MYC gene has no prognostic significance on survival in HNSCC. CONCLUSION In conclusion, our results suggest that MYC gene amplification is associated with tumor progression in HNSCC.

RBSP3 Is Frequently Altered in Premalignant Cervical Lesions: Clinical and Prognostic Significance

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4–9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN → Stage I/II and for ITGA9 from CIN → Stage I/II and also from Stage I/II → Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p‐RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p‐RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (≥5)/early sexual debut (≤19 years) coupled with RBSP3 alterations/RB1 deletion predicted worst prognosis. Thus, inactivation of RBSP3 might be one of the early events in cervical carcinogenesis.

Deletion in chromosome 11 and Bcl-1/Cyclin D1 alterations are independently associated with the development of uterine cervical carcinoma.

Purpose The aim of this study was to understand whether there is any association between specific deleted regions in chromosome 11 (chr.11) and alteration (amplification/rearrangement) of Bcl-1/Cyclin D1 locus, located at 11q13, in uterine cervical carcinoma (CA-CX).Methods The deletion mapping of chr.11 was studied using 17 highly polymorphic microsatellite markers in 65 primary uterine cervical lesions. The Bcl-1/Cyclin D1 alterations were analyzed by Southern blot and/or polymerase chain reaction (PCR) method in respective cervical lesions.Results Chr.11 deletion was found to be significantly associated with progression of CA-CX. High frequency (48–65%) of deletion was found in 11p15.5 (D1), 11q22.3–23.1(D2), and 11q23.3–24.1(D3) regions and significant association was seen among deletions in D2 and D3 regions. Bcl-1/Cyclin D1 locus alteration was observed in overall 27% cervical lesions. Co-amplification of Bcl-1/Cyclin D1 locus was seen in 10% samples. However, no association was found between the deleted regions and Bcl-1/Cyclin D1 locus alterations.Conclusions Our study suggests that there is no co-operativity between the deleted regions (D1- D3) in chr.11 and Bcl-1/Cyclin D1 alterations, but these alterations may provide cumulative effect in progression of the tumor. The D1–D3 regions may harbor candidate tumor suppressor gene(s) (TSGs) associated with the development of CA-CX.

Frequent alterations of LOH11CR2A, PIG8 and CHEK1 genes at chromosomal 11q24.1-24.2 region in breast carcinoma: clinical and prognostic implications.

To understand the importance of frequent deletions at chromosome 11q24.1‐24.2 region in breast carcinoma, alterations (deletion/methylation) of the candidate genes LOH11CR2A, ROBO3, ROBO4, HEPACAM, PIG8 and CHEK1 located in this region were analyzed in 106 breast carcinoma samples. Among these genes, LOH11CR2A showed highest frequency of deletion (56%), followed by PIG8 (35%), CHEK1 (31%) and ROBO3/ROBO4/HEPACAM loci (28%). Comparable frequency of promoter methylation (26–35%) was observed for LOH11CR2A, CHEK1 and PIG8. Overall alterations (deletion/methylation) of these genes were in the following order: LOH11CR2A (60%) > PIG8 (46%) > CHEK1 (41%) and showed significant association with each other. Breast carcinoma samples that were estrogen/progesterone receptor negative showed significantly high deletion and overall alterations than estrogen/progesterone receptor positive samples for LOH11CR2A, CHEK1 and PIG8. The methylation and overall alteration of LOH11CR2A were significantly associated with tumor stages in breast carcinoma. However, in early/late onset and estrogen/progesterone receptor positive/negative breast carcinoma, the overall alterations of LOH11CR2A, PIG8 and CHEK1 were differentially associated with advanced stages, tumor grade and lymph node metastasis. Alterations of PIG8 and CHEK1 were significantly associated with poor prognosis in patients with early age of onset of the disease indicating significant prognostic importance. Quantitative mRNA expression analysis detected reduced expression of the genes in the order LOH11CR2A > CHEK1 > PIG8. Immunohistochemical analysis showed reduced protein expression of PIG8 and CHEK1 that was concordant with their molecular alterations. Thus, our study suggests that LOH11CR2A, PIG8 and CHEK1 are candidate tumor suppressor genes associated with breast carcinoma and have significant clinical as well as prognostic importance.

The stem cell renewal and DNA damage response pathways are frequently altered in fibroepithelial tumors of breast in Indian patients

Genetic and epigenetic alterations in genes associated with distinct cellular pathways were checked in fibroepithelial tumors, including fibroadenomas, benign and malignant phyllode and atypical ductal hyperplasia. A panel of 22 genes associated with different cellular pathways such as stem cell renewal (Wnt and Hedgehog), DNA damage response [homologous recombination (HR), mismatch repair (MMR) and nucleotide excision repair (NER)] and cell proliferation signaling pathway were tested. Alterations (genetic/epigenetic) of the genes associated with Wnt signaling pathway were detected in 100% (20/20) of the breast tumors for at least one out of the six Wnt antagonists tested. Frequent molecular alterations (57-64%) were detected in HR and MMR pathway and low frequency of alterations (8-25%) were seen in cell-proliferation and cell signaling pathways showing a differential pattern of alterations in different tumor types. The patterns of alterations, in particular the epigenetic alterations, differed little from that seen previously in breast carcinoma cells, suggesting epigenetic alterations to be an early event in the development of the tumors. In gene ontology analysis, it was evident that Wnt signaling pathway [GO: 0030111, Kegg: 04310], cell proliferation pathway [GO: 0008285] and pathways in cancer [Kegg: 05200] were significantly enriched by differentially altered genes in fibroadenoma and phyllode tumor types. All these results may provide a new breakthrough in early diagnosis, prognosis and treatment of these tumors.

Association of APC and MCC polymorphisms with increased breast cancer risk in an Indian population

The adenomatous polyposis coli (APC) and mutated in colorectal cancer (MCC) genes are key regulatory genes of the Wnt/β-catenin signaling pathway, which are independently involved in maintaining low levels of β-catenin in the cell. In addition to genetic and epigenetic alterations, some genetic polymorphisms in the genes associated with the Wnt signaling pathway have been reported to be associated with an increased risk of cancer, including breast cancer. In the present study we analyzed the association of genotype and haplotype status of two single nucleotide polymorphisms (SNPs), rs2229992 and rs11283943, in the APC and MCC genes, respectively, with an increased risk of breast carcinogenesis in a breast cancer and control population from eastern India. We observed a significant association of the rs11283943 SNP with increased breast cancer risk. Two specific haplotypes involving the minor allele of rs11283943 were found to be associated with an increased breast cancer risk. Kaplan-Meier curves showed a significant association of the 2–2 genotype (genotype homozygous for the rs11283943 minor allele) with decreased survival (p=0.045) of the breast cancer patients in our study, in particular patients with early-onset BC.

Amplification of c-myc Locus is Independently Associated with the Deletions of Chromosome 8p in Breast Carcinoma

Abstract Attempts have been made in this study is to find out the mechanism of c-myc gene activation in breast carcinoma (CaBr) by analyzing alterations (rearrangement/amplification) in the ~580 Kb surroundings of this gene. The alteration in the c-myc locus was correlated with the deletions in chromosome (chr.) 8p to find if there is any association between the two phenomenons. The c-myc locus alteration was analyzed by Southern hybridization using the pal-1/ c-myc/ mlvi-4 probes. Overall, amplification in the c-myc locus was seen in 26% of the samples with 22% in the pal-1 region, 19% in the c-myc gene and 7% in the mlvi-4 region. This indicates that the c-myc gene activation may occur due to the amplification in the pal-1 region located 550 Kb 5’ and mlvi-4 region located 20 Kb 3’ of c-myc. About 42% of the samples showed loss of heterozygosity (LOH) in ≥ 40% of the microsatellite markers tested. Atleast 21% of the samples showed co-alterations in both arms of chr.8. No significant association was observed between the amplification in the c-myc locus and deletions in chr.8p. Thus the deletions in chr.8p and the amplification in the c-myc locus are independently associated with the development of CaBr.