Absence of loss of heterozygosity of BRCA1 in a renal tumor from a BRCA1 germline mutation carrier

Abstract

BRCA1 functions as a tumor suppressor gene and germline and somatic mutations in this gene have been shown to be associated with many types of cancer. We report the first tumor study of renal cell carcinoma in a carrier of the deleterious BRCA1 mutation-c.68_69delAG.

Introduction

Mutations in BRCA1 are associated with 60-90% lifetime risk of developing breast cancer and 50% for ovarian cancer [1]. BRCA1 is a tumor suppressor [2], and is moderately expressed in kidney tissue and in renal cell carcinoma [3]. Thus, it is plausible that germline BRCA1 mutations could contribute to the development of renal cell carcinoma. To investigate this hypothesis, we examined the renal tumor removed from a patient who carried a deleterious BRCA1 germline mutation.

Case identification and history

To identify germline BRCA1 and BRCA2 mutation carriers, who had been diagnosed with renal cancer, we reviewed the charts of 1260 BRCA1 or BRCA2 mutation-positive patients counselled by the Clinical Genetics Service at Memorial Sloan-Kettering Cancer Center (43% of these are of Ashkenazi Jewish Ancestry). Of this cohort, only three patients had a history of renal cancer (not significantly different from number of patients with kidney cancer in non-carriers in our BRCA1/2 tested cohort, P=0.1). All three patients were females, of Ashkenazi Jewish descent, and carried the BRCA1 Ashkenazi Jewish founder mutation, c.68_89delAG. Each had been diagnosed with unilateral kidney cancer at 36, 57, and 59 years of age, respectively. Clinical data and tumor tissue were available for one patient (59 years old at diagnosis) who presented with right flank pain of one month duration, and was then found to have a 6.3 cm right renal mass on magnetic resonance imaging done for the back pain work up that was suspicious for renal cell carcinoma. She also had multiple pulmonary nodules that were suspicious of metastasis. Nephrectomy was performed and pathologic examination confirmed the diagnosis of renal cell carcinoma - clear cell type (nuclear grad III/IV). Given her cancer diagnosis and family history of breast cancer related to BRCA1 mutations (c.68_69delAG), she was tested and was found to be carrier of c.68_69delAG as well. Her family history was significant for breast cancer, but not for kidney cancer.

Experiment and results

To investigate the potential mechanisms of tumorigenesis, we analyzed the renal tumor from the case of interest for loss of heterozygosity (LOH) for the BRCA1 wild-type allele and looked for altered BRCA1 protein expression. Frozen tumour tissue was obtained through IRB-approved protocols. A frozen section of the sample was cut, and stained with hematoxylin-eosin. A pathologist experienced in the evaluation of urologic tumors examined the section and mapped areas containing >90% viable tumor cells. These areas were dissected from the frozen tissue sample, and DNA was extracted from this tissue using DNAeasy Blood and Tissue kit (cat# 69504. QIAGEN Inc., Valencia CA). Sanger sequencing of the BRCA1 c.68_69delAG mutation was performed to test for LOH, and Immunohistochemistry (IHC) was performed to test for BRCA1 protein, using a BRCA1 antibody (clone MS110, Cat # OP92. EMD Millipore Corp., Billerica MA), expression. The results of the Sanger sequencing negated our hypothesis of LOH of BRCA1 in the tumor tissue as the tumor tissue was heterozygous for mutant and wild type allele (Fig. 1A). IHC showed expression of BRCA1 in the tissue decreasing the likelihood that other mechanisms of loss of expression of BRCA1 could be involved in tumor development in our patient (Fig. 1B).

Figure 1.

A) Histologic features of renal tumor - typical appearances of clear cell renal cell carcinoma, characterized by nests of epithelial cells with clear cytoplasm separated by a delicate vascular network (hematoxylin and eosin, ×100). Inset - immunohistochemistry for BRCA1 shows nuclear staining in tumor cells(×100).B) 1- Results of Sanger sequencing of the kidney cancer tissue from BRCA1 mutation carrier - electropherogram shows persistence of frameshif indicating no loss of the wild type allele. 2- Results of Sanger sequencing of germline DNA from the same BRCA1 mutation carrier - electropherogram confirming the presence of BRAC1c.68- 69delAG

Discussion

Kidney cancer has well known hereditary components and is characterized by inactivation of the VHL gene (encoding the von Hippel-Lindau protein). VHL is located on chromosome 3p25, and is a two-hit tumor suppressor gene. In clear cell kidney cancer, one allele of VHL is typically inactivated through a point mutation (or indel), and the other is inactivated through a large deletion[4]. Also, it was recently reported that LOH in BAP1, another tumor suppressor gene, characterized a new subtype of clear cell tumors and was associated with high grade tumors [5]. BRCA1 is a tumor suppressor gene that was associated with multiple malignancies including breast, ovarian, and prostate tumors [1, 6]. Using the model of VHL and BAP1, it is possible that BRCA1 mutations could contribute to the development of kidney tumors, or be associated with adverse pathologic characteristics. There are two previous reports of germline mutations of BRCA1 in kidney cancer patients, but neither reported testing of tumor tissue [7, 8]. In fact, in the study by Thompson et al, the hazard ratio estimates predicted a tendency towards protective effect of BRCA1 mutations against kidney cancer (Confidence interval included 1). However, The comparison group in that study was the expected number of kidney cancer cases from large cancer registries and the hazard ratio estimates may have changed if the comparison group was test confirmed BRCA1 non-carriers [8]. Also, previous literature indicated an absence of LOH of BRCA1 in kidney cancer cell lines[9], but to our knowledge, this is the first report on the status of BRCA1 mutations in a patient's renal tumor tissue. We did not find LOH of BRCA1 suggesting that aberrations of BRCA1 may not be causally associated with renal tumors. This finding is consistent with the lack of reported mutations in the COSMIC mutation database. The lack of somatic evidence suggestive of a germline mechanism of tumor susceptibility could also be explained by the small sample size and the dominance of sporadic renal tumors, the other types of possible “second-hits” in BRCA1 and their different biologic effects, and intratumor heterogeneity. Finally, these finding have therapeutic implications. BRCA1 is necessary for the homologous repair pathway that corrects DNA breaks encountered during cell replication [10]. Cells deficient in BRCA1 were shown to be highly sensitive to PARP1 inhibition. PARP1 functions in the same repair pathway as BRCA1, and inhibition of PARP1 leads to accumulation of DNA breaks and apoptosis[11]. Although such agents could be potentially used in treating other cancers in patients with BRCA1 mutations, these findings suggest that these targeted therapeutic strategies may not be relevant for kidney cancer.