Frequent alterations of MCPH1 and ATM are associated with primary breast carcinoma: clinical and prognostic implications.

BACKGROUND: MCPH1 is a proximal regulator of DNA damage response pathway that is involved in recruitment of phosphorylated ATM to double-stranded DNA breaks. METHODS: To understand the importance of MCPH1 and ATM in deregulation of DNA damage response pathway in breast carcinoma, we studied m-RNA expression and genetic/epigenetic alterations of these genes in primary breast carcinoma samples. RESULTS: Our study revealed reduced expression (mRNA/protein) and high alterations (deletion/methylation) (96 %, 121 of 126) of MCPH1 and ATM. Mutation was, however, rare in inactivation of MCPH1. In immunohistochemical analysis, reduced protein expression of MCPH1, ATM and p-ATM were concordant with their molecular alterations (P = 0.03-0.01). Alterations of MCPH1 and deletion of ATM were significantly high in estrogen/progesterone receptor-negative than estrogen/progesterone receptor-positive breast carcinoma samples compared to early or late age of onset tumors, indicating differences in pathogenesis of the molecular subtypes (P = 0.004-0.01). These genes also showed differential association with tumor stage, grade and lymph node status in different subtypes of breast carcinoma (P = 0.00001-0.01). Their coalterations showed significant association with tumor progression and prognosis (P = 0.003-0.05). Interestingly, patients with alterations of these genes or MCPH1 alone had poor outcome after treatment with DNA-interacting drugs and/or radiation (P = 0.01-0.05). CONCLUSIONS: Inactivation of MCPH1-ATM-associated DNA damage response pathway might have an important role in the development of breast carcinoma with diagnostic, prognostic and therapeutic implications.

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/ß-catenin pathway in the development of breast cancer (BC) and its association with different clinicopathological parameters. Alterations (deletion/methylation/expression) of some Wnt/ß-catenin pathway antagonists like APC, SFRP1/2, CDH1 and activator ß-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 ß-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 ß-catenin play an important role in the development of BC and have significant clinical as well as prognostic importance.

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 (>or=5)/early sexual debut (

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.

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.

Approximately 580 Kb surrounding the MYC gene is amplified in head and neck squamous cell carcinoma of Indian patients.

Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). However, there are no data concerning whether the amplification is confined to the MYC gene itself or spans distant 5' and/or 3' regions of this gene in HNSCC as seen in different lymphomas, colon carcinoma, and uterine cervical carcinoma (CaCx). In this study, we analyzed the alterations (amplification/rearrangement) in the 580 Kb surrounding of the MYC gene to understand the status of this locus in primary HNSCC of Indian patients. The MYC alterations were analyzed by Southern blot using the pal-1/MYC/MLVI4 probes. The alterations in the MYC locus (adjacent region of the c-myc gene) were then correlated with the various clinicopathological parameters. The overall amplification involving the MYC locus was seen in 46% of the samples. The MYC gene, pal-1 region, and MLVI4 region were amplified in about 38%, 24%, and 20% of the samples, respectively. Some samples showed co-amplification encompassing pal-1-MYC-MLVI4 or pal-1-MYC or MYC-MLVI4 regions. No significant association was observed between the amplification in the MYC locus and the different clinicopathological parameters except for tumor differentiation. Thus, it seems that, similar to other tumors, the MYC gene may be activated by amplification in its surrounding 5' and/or 3' region in HNSCC.

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/ß-catenin signaling pathway, which are independently involved in maintaining low levels of ß-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.

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.