Lisa McPherson

Clinical Evaluation of a Multiple-Gene Sequencing Panel for Hereditary Cancer Risk Assessment

PURPOSE Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a representative clinical sample. METHODS Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at -80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants. RESULTS In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes. CONCLUSION Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.

Krüppel-Like Transcription Factor 13 Regulates T Lymphocyte Survival In Vivo

Krüppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Krüppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13−/− mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13−/− thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13−/− thymocytes is due in part to increased expression of BCL-XL, a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8− single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-XL promoter and results in decreased Bcl-XL promoter activity, making KLF13 a negative regulator of BCL-XL.

Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia

Childhood leukemia, which accounts for >30% of newly diagnosed childhood malignancies, is one of the leading causes of death for children with cancer. Genome-wide studies using microarray chips to identify copy number changes in human cancer are becoming more common. In this pilot study, 45 pediatric leukemia samples were analyzed for gene copy aberrations using novel molecular inversion probe (MIP) technology. Acute leukemia subtypes included precursor B-cell acute lymphoblastic leukemia (ALL) (n=23), precursor T-cell ALL (n=6), and acute myeloid leukemia (n=14). The MIP analysis identified 69 regions of recurring copy number changes, of which 41 have not been identified with other DNA microarray platforms. Copy number gains and losses were validated in 98% of clinical karyotypes and 100% of fluorescence in situ hybridization studies available. We report unique patterns of copy number loss in samples with 9p21.3 (CDKN2A) deletion in the precursor B-cell ALL patients, compared with the precursor T-cell ALL patients. MIPs represent an attractive technology for identifying novel copy number aberrations, validating previously reported copy number changes, and translating molecular findings into clinically relevant targets for further investigation.

Dynamic Interplay of Transcriptional Machinery and Chromatin Regulates “Late” Expression of the Chemokine RANTES in T Lymphocytes

ABSTRACT The chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is expressed “late” (3 to 5 days) after activation in T lymphocytes. In order to understand the molecular events that accompany changes in gene expression, a detailed analysis of the interplay between transcriptional machinery and chromatin on the RANTES promoter over time was undertaken. Krüppel-like factor 13 (KLF13), a sequence-specific DNA binding transcription factor, orchestrates the induction of RANTES expression in T lymphocytes by ordered recruitment of effector molecules, including Nemo-like kinase, p300/cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, and Brahma-related gene 1, that initiate sequential changes in phosphorylation and acetylation of histones and ATP-dependent chromatin remodeling near the TATA box of the RANTES promoter. These events recruit RNA polymerase II to the RANTES promoter and are responsible for late expression of RANTES in T lymphocytes. Therefore, KLF13 is a key regulator of late RANTES expression in T lymphocytes.

The role of the retinoblastoma/E2F1 tumor suppressor pathway in the lesion recognition step of nucleotide excision repair.

The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb-/-, p107-/-, p130-/- MEFs repaired both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1-/- MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1-/- cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair.

Interaction of PRP4 with Krüppel-Like Factor 13 Regulates CCL5 Transcription

Activation of resting T lymphocytes initiates differentiation into mature effector cells over 3–7 days. The chemokine CCL5 (RANTES) and its major transcriptional regulator, Krüppel-like factor 13 (KLF13), are expressed late (3–5 days) after activation in T lymphocytes. Using yeast two-hybrid screening of a human thymus cDNA library, PRP4, a serine/threonine protein kinase, was identified as a KLF13-binding protein. Specific interaction of KLF13 and PRP4 was confirmed by reciprocal coimmunoprecipitation. PRP4 is expressed in PHA-stimulated human T lymphocytes from days 1 and 7 with a peak at day 3. Using an in vitro kinase assay, it was found that PRP4 phosphorylates KLF13. Furthermore, although phosphorylation of KLF13 by PRP4 results in lower binding affinity to the A/B site of the CCL5 promoter, coexpression of PRP4 and KLF13 increases nuclear localization of KLF13 and CCL5 transcription. Finally, knock-down of PRP4 by small interfering RNA markedly decreases CCL5 expression in T lymphocytes. Thus, PRP4-mediated phosphorylation of KLF13 plays a role in the regulation of CCL5 expression in T lymphocytes.

Poly (ADP-ribose) polymerase inhibitor LT-626: Sensitivity correlates with MRE11 mutations and synergizes with platinums and irinotecan in colorectal cancer cells

Some colorectal cancers (CRC) display microsatellite instability (MSI) leading to mutations in genes such as MRE11. The aim of this study was to determine whether MSI or MRE11 mutational status correlates with sensitivity to the PARP inhibitor LT-626 and whether LT-626 synergizes with DNA-damaging chemotherapeutic agents. CRC cells harboring biallelic MRE11 mutations were more sensitive to LT-626 and stable overexpression or knock-down of MRE11 in cell lines correlated with sensitivity. Synergism was evident between LT-626 and cisplatin, oxaliplatin and SN-38 suggesting that PARP inhibitors in combination with DNA damaging agents may be a successful strategy for treatment of CRC.

Genetic Analysis of a Papillary Thyroid Carcinoma in a Patient with MEN1

Background: MEN1 is an inherited tumor syndrome characterized by the development of tumors of the parathyroid, the anterior pituitary and the pancreatic islets. Tumors of these endocrine glands in MEN1 patients demonstrate loss of heterozygosity (LOH) at the locus of the MEN1 tumor suppressor gene. Menin, the protein encoded by the MEN1 gene, is ubiquitously expressed in endocrine tissue, and less commonly these patients can present with tumors of other endocrine tissues, including thyroid and adrenal. We hypothesize that MEN1 gene mutation may be involved in the oncogenesis of other less common tumors.Methods: We report a MEN1 patient who was found to have metastatic papillary thyroid cancer at the time of neck exploration for hyperparathyroidism. Genetic analysis of tumor tissue was performed using one intragenic (D11S4946) and two flanking (D11S4945 and D11S4940) polymorphic markers.Results: Two of the markers were informative. Consistent with previous studies, there was LOH in the parathyroid adenoma identified with the intragenic marker D11S4946. However, the papillary cancer was found to be heterozygous at two informative markers.Conclusions: The lack of obvious LOH of the MEN1 locus in the papillary cancer suggests that, in contrast to parathyroid adenoma, deletion of the MEN1 tumor suppressor gene is not etiologically related to the oncogenesis of the papillary cancer in this patient.

Abstract 5490: Poly (ADP-ribose) polymerase inhibitor LT-626 sensitizes lung and pancreatic cancers to fractionated radiation therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of stereotactic body radiation therapy (SBRT) has revolutionized radiation therapy for lung cancers and is an emerging treatment option for pancreatic cancers. However, there are many questions on how to optimize its use in chemoradiotherapy. The most relevant addition to radiotherapy regimens are inhibitors of DNA repair and DNA damage response pathways. One such class of agents are small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP). PARP inhibitors target DNA base excision repair and radiosensitize cells through impaired DNA repair. In this study we examined the effects of the PARP inhibitor LT-626 (BioMarin) together with ionizing radiation in lung and pancreatic cancers, in vitro and in vivo. Clonogenic assays showed that lung cancer cells H1299 and H460 and pancreatic cancer cells Miapaca2 and PDA were sensitive to LT-626 (IC50 from 0.1-5.8 µM) and irradiation (IC50 from 2.2-5.9 Gy). Next, we found the combination of LT-626 and irradiation effective for inhibiting growth in lung and pancreatic cancer cells. Our study showed that both 1 and 10 µM concentrations of LT-626 were highly synergistic with 2, 4 and 6 Gy of irradiation in lung and pancreatic cell lines. Furthermore, in a fractionated radiation regimen study pretreatment with LT-626 followed by irradiation for three days significantly decreased cell survival as studied by clonogenic assay compared to LT-626 or radiation alone. Lung and pancreatic cancer cells treated with LT-626 and irradiation also exhibited DNA damage as evident by increased γH2AX and Rad51 foci formation. However, DNA damage caused by irradiation peaked by 2-4 h following treatment compared with LT-626 treated cells which exhibited maximum DNA damage around 24 h after treatment. Our in vitro study clearly demonstrated that PARP inhibition enhanced the efficacy of irradiation; we therefore decided to study the efficacy LT-626 and radiation therapy in vivo, using two lung xenograft (H1299 and H460) cancer models. After tumor implantation mice were treated with either drug alone (10mg/kg or 20 mg/kg) or radiation alone (2 Gy) or a combination of drug and radiation for five consecutive days after which they were followed until the end of the study. We found that combination of radiation plus LT-626 significantly decreased tumor burden in both H1299 and H460 xenograft models compared to drug alone or radiation alone group. Moreover, the median survival in combination treated animals more than doubled compared to vehicle treated group. Overall our in vitro and in vivo studies proved that PARP inhibitor LT-626 acts synergistically with fractionated irradiation in lung and pancreatic cancers. Citation Format: Kedar Hastak, Lisa McPherson, Yuqiao Shen, Renate Parry, James M. Ford. Poly (ADP-ribose) polymerase inhibitor LT-626 sensitizes lung and pancreatic cancers to fractionated radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5490. doi:10.1158/1538-7445.AM2014-5490

Evaluation of a cancer gene sequencing panel in a hereditary risk assessment clinic.

7 Background: Multiple-gene sequencing panels are entering clinical practice. We report on research testing with a custom sequencing panel comprising 43 genes and 32 cancer-associated variants among patients referred for assessment of hereditary breast and ovarian cancer risk. METHODS Patients referred to the Stanford Cancer Genetics Program for clinical BRCA1 and BRCA2 (BRCA1/2) mutation testing from 2002-2012 were invited to donate a blood sample for research on an Institutional Review Board-approved protocol. Blood samples were frozen at -80 degrees, and DNA extracted after <1-10 years. The entire coding region, exon-intron boundaries (+/- 10bp) and all known pathogenic variants in other regions were sequenced for 43 genes that have published associations with risk of breast, ovarian and other cancers. An additional 32 cancer-associated variants were also sequenced. Clinically significant results were disclosed to patients. RESULTS Germline DNA samples were sequenced from 199 women: 141 had breast cancer, and 57 carried known BRCA1/2 mutations. Analytic results for BRCA1/2 sequencing and pathogenicity interpretations were concordant with prior clinical testing for all patients. Twenty variants designated as pathogenic, either based on published literature or due to a novel truncating or splice donor/acceptor effect, were observed in genes other than BRCA1/2, including ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4. Thirteen patients had pathogenic variants warranting a change in cancer screening or risk reduction based on practice guidelines; they were invited for confirmatory clinical testing and counseling. One 53-year old patient with a personal history of breast and endometrial cancers was found to carry a pathogenic MLH1 mutation; she underwent risk-reducing salpingo-oophorectomy and colonoscopy, with removal of a tubular adenoma. CONCLUSIONS Among patients referred for BRCA1/2 mutation testing, a comprehensive sequencing assay identified 20 [10.1%, 95% confidence interval (CI) 6.5%-15.1%] pathogenic mutations in other genes, of which 13 (6.5%, CI 3.8%-11%) prompted a change in care. Disclosure of research results to participants was feasible and well-tolerated.