Alterations in candidate genes PHF2, FANCC, PTCH1 and XPA at chromosomal 9q22.3 region: Pathological significance in early- and late-onset breast carcinoma

Breast carcinoma (BC) is the second leading cause of cancer deaths and the most common cancer among women. About 23% of urban women in eastern India are affected by this disease [1]. Depending on age at onset, BC can be early-onset (≤ 40 years) and late-onset (> 40 years) type. However, the cut-off value for early-onset BC varies among investigators, ranging from 35–50 years. Significant differences in clinico-pathological features like large tumor size of higher grade, presence of positive lymph nodes, absence of steroid receptors, and high S-phase fraction in younger women with BC indicated altered biology and pathogenesis between these two groups of BC [2‐4]. Another report showed that in Asian population, BC patients below 40 years have tumors with a poorer prognostic profile. However, this did not translate into a poorer overall survival, and this might be attributable to more aggressive adjuvant treatment of younger patients [5]. All these reports suggested early-onset BC to be a biologically separate disease and independently predict more adverse outcomes. Thus, the molecular analysis of BC in the two age groups is pertinent to understand the differences in pathogenesis, if any. Our earlier studies on chromosomes (chrs.) 1p/q, 9p and 11p/q showed differential pattern of molecular alterations between the two age groups of BC [3‐6]. In order to understand the pathogenesis in detail our aim is to analyze the alterations in other chromosomal regions which showed frequent alterations in BC.

Cytogenetic analyses have revealed a variety of chromosomal aberrations at chr.9q22 in different malignancies including BC [7]. Comparative genomic hybridization (CGH) and flow-cytometric analyses also showed losses at chr.9q in BC [8‐10]. Studies in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) as well as bladder, prostate, esophageal and blood cancer detected frequent loss of heterozygosity (LOH) at chr.9q22.3 [11, 12]. Significant correlation between losses of chr.9q22.3 with lymph node metastasis in BC is reported [13].

Chr.9q22.3 is a relatively broad region (8.71 Mb) harboring several putative tumor suppressor genes (TSGs). We focused primarily on a 4.4 Mb region between the chr.9q22.32-22.33 where DNA-damage repair genes like FANCC (Fanconi anaemia Complementation-group C), XPA (Xeroderma Pigmentosum A) as well as the hedgehog (HH) pathway associated gene PTCH1 (human homologue of Drosophila patched gene) are localized [13‐16]. FANCC localized at chr.9q22.32 (96.89 Mb from p-ter) is associated with reconstitutive and self-renewal potential of haematopoietic stem cells [17, 18]. Its ablation leads to increased sensitivity to DNA crosslinking agents, loss of FANCD2 activation, and impaired homologous recombination due to poor functioning of the BRCA1-BRCA2-NBS1-RAD51 mediated DNA repair [19]. Deletion of FANCC in bladder carcinoma, mutations in young-onset pancreatic cancer and Fanconi Anaemia-C patients, and down-regulation in head and neck squamous cell carcinoma (HNSCC) are reported [16, 20‐22]. PTCH1 (350 Kb downstream to FANCC) is a key regulator in stem cell renewal hedgehog signaling pathway and functions as a tumor suppressor by inhibiting G₁-S and G₂-M phases of cell cycle. PTCH1 has been implicated in the development of medulloblastomas, glioblastomas, rhabdomyosarcomas, pancreatic cancers, and other soft tissue tumors [23]. A recent report suggested loss of PTCH1 in 19% of human breast cancers, and 33% of breast cancer cell lines [9]. Mutations in PTCH1 gene have been reported in nevoid basal cell carcinoma syndrome (NBCCS), familial BC and its loss induced tumor progression in basal cell carcinoma (BCC) [24, 25]. Furthermore, immunohistochemical analysis showed reduced expression of PTCH1 due to promoter methylation in BC [26]. XPA (2.23 Mb down-stream of PTCH1) gene is essential for assembly of the pre-incision complex during the processing of DNA damage via the nucleotide excision repair (NER) pathway [27‐29]. Defects in NER are associated with several human autosomal recessive hereditary disorders, such as xeroderma pigmentosum and a marked predisposition to skin cancer (early BCC, SCC and melanoma); early internal tumors [30]. LOH in sporadic ovarian, colon, lung carcinomas and significant association between oral premalignant lesions and polymorphism of XPA are reported [31, 32]. PHF2, a novel transcriptional regulatory gene associated with hereditary sensory neuropathy type 1 (HSN1) has also been identified at chr.9q22.32, 1.52 Mb upstream of FANCC [33]. However, its role in tumorigenesis is not yet well understood.

Thus, it is evident that alterations of these genes together could cumulatively increase genetic instability and cellular proliferation. Furthermore, study of co-alterations of these genes could be useful in diagnosis and prognosis of a disease. Thus an attempt was made in this study to analyze the alterations (deletion, methylation and expression) of PHF2, FANCC, PTCH1 and XPA in early- and late-onset BC of Indian patients and correlate them with clinico-pathological differences.

Previous studies have shown differences in alteration pattern of various chromosomal regions in the development of early- and late-onset BC [3‐6]. To delineate the molecular pathogenesis of BC in the two age groups in detail, molecular alterations (deletion/methylation) of candidate genes PHF2, FANCC, PTCH1 and XPA within chr.9q22.32-22.33 region were analyzed due to its association with a wide variety of tumors including BC. Nearly 91% samples in both groups presented alterations in any one of the genes. Compared to XPA, overall frequencies of alterations in PHF2, FANCC and PTCH1 were higher (Figure 4). This is the first report of its kind. The overall alterations of these genes were differentially associated in the two age groups and indicated differences in pathogenesis and adverse prognosis in patients.

Significant association between deletion and methylation of FANCC in both groups and PTCH1 in group-A only, suggested them to be candidate TSGs in BC. The concordance between deletion/methylation of these genes with protein expression also supported this fact. Down-regulation in mRNA expression of these genes were also seen in MCF-7 and primary BC samples. Deletion, mutation and down-regulation of FANCC have also been reported in other malignancies [16‐22]. Significant association of FANCC-XPA methylation in group-A and FANCC-BRCA2 deletion in both age groups supported interlink between multiple DNA repair pathways. This warrants further analysis of other members of this repair pathway needs to be done. The association noted between FANCC alterations and adverse patient survival in either age group suggests it as a prognostic marker.

Contrary to previous reports, frequencies of alterations in PTCH1 in our samples were higher [9]. This may be due to differences in etiology, ethnicity and techniques used in our study. Mutations in exon-23 of PTCH1 were reported in BCC but, no such changes were seen in our samples [43]. Significant association between PTCH1 and XPA deletion in both groups as well as between their methylation in group-B suggested some functional relationship between hedgehog dependent stem cell renewal pathway and the NER pathway. Coincident germline mutations in PTCH1 and BRCA1 genes have been seen in familial BC and NBCCS [24]. Similarly, correlation between PTCH1 alterations with BRCA1 deletion in group-A suggested its association with other DNA repair pathways. Poor survival in group-A patients with PTCH1 alterations indicated its prognostic significance. In PHF2 and XPA no association was seen between their deletion and methylation. So, other mechanisms of inactivation such as mutation cannot be ruled out. High deletion along with significant association of PHF2 methylation with FANCC methylation in both age groups and XPA methylation in group-A suggested PHF2 to be a candidate TSGs in BC. Reduced expression of PHF2 mRNA also supported this fact. Further analysis of PHF2 is required to assess its role in tumorigenesis.

Thus it may be concluded that impairment of multiple DNA repair pathways and PTCH1 associated self-renewal pathway are necessary for the development of both groups of BC. However, differential association of these genes with adverse patient outcome in both groups suggests their differences in molecular pathogenesis.