Benjamin H. Lok

RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination.

Synthetic lethality is an approach to study selective cell killing based on genotype. Previous work in our laboratory has shown that loss of RAD52 is synthetically lethal with BRCA2 deficiency, while exhibiting no impact on cell growth and viability in BRCA2-proficient cells. We now show that this same synthetically lethal relationship is evident in cells with deficiencies in BRCA1 or PALB2, which implicates BRCA1, PALB2 and BRCA2 in an epistatic relationship with one another. When RAD52 was depleted in BRCA1- or PALB2-deficient cells, a severe reduction in plating efficiency was observed, with many abortive attempts at cell division apparent in the double-depleted background. In contrast, when RAD52 was depleted in a BRCA1- or PALB2-wildtype background, a negligible decrease in colony survival was observed. The frequency of ionizing radiation-induced RAD51 foci formation and double-strand break-induced homologous recombination (HR) was decreased by 3- and 10-fold, respectively, when RAD52 was knocked down in BRCA1- or PALB2-depleted cells, with minimal effect in BRCA1- or PALB2-proficient cells. RAD52 function was independent of BRCA1 status, as evidenced by the lack of any defect in RAD52 foci formation in BRCA1-depleted cells. Collectively, these findings suggest that RAD52 is an alternative repair pathway of RAD51-mediated HR, and a target for therapy in cells deficient in the BRCA1–PALB2–BRCA2 repair pathway.

Molecular pathways: understanding the role of Rad52 in homologous recombination for therapeutic advancement.

The Rad52 protein was largely ignored in humans and other mammals when the mouse knockout revealed a largely “no-effect” phenotype. However, using synthetic lethal approaches to investigate context-dependent function, new studies have shown that Rad52 plays a key survival role in cells lacking the function of the breast cancer type 1 susceptibility protein (BRCA1)–BRCA2 pathway of homologous recombination. Biochemical studies also showed significant differences between yeast and human Rad52 (hRad52), in which yeast Rad52 can promote strand invasion of replication protein A (RPA)–coated single-stranded DNA (ssDNA) in the presence of Rad51 but hRad52 cannot. This results in the paradox of how is hRad52 providing Rad51 function: presumably there is something missing in the biochemical assays that exists in vivo, but the nature of this missing factor is currently unknown. Recent studies have suggested that Rad52 provides back-up Rad51 function for all members of the BRCA1–BRCA2 pathway, suggesting that Rad52 may be a target for therapy in BRCA pathway–deficient cancers. Screening for ways to inhibit Rad52 would potentially provide a complementary strategy for targeting BRCA-deficient cancers in addition to poly (ADP-ribose) polymerase (PARP) inhibitors. Clin Cancer Res; 18(23); 6400–6. ©2012 AACR.

PARP Inhibitor Activity Correlates with SLFN11 Expression and Demonstrates Synergy with Temozolomide in Small Cell Lung Cancer

Purpose: PARP inhibitors (PARPi) are a novel class of small molecule therapeutics for small cell lung cancer (SCLC). Identification of predictors of response would advance our understanding, and guide clinical application, of this therapeutic strategy. Experimental Design: Efficacy of PARP inhibitors olaparib, rucaparib, and veliparib, as well as etoposide and cisplatin in SCLC cell lines, and gene expression correlates, was analyzed using public datasets. HRD genomic scar scores were calculated from Affymetrix SNP 6.0 arrays. In vitro talazoparib efficacy was measured by cell viability assays. For functional studies, CRISPR/Cas9 and shRNA were used for genomic editing and transcript knockdown, respectively. Protein levels were assessed by immunoblotting and immunohistochemistry (IHC). Quantitative synergy of talazoparib and temozolomide was determined in vitro. In vivo efficacy of talazoparib, temozolomide, and the combination was assessed in patient-derived xenograft (PDX) models. Results: We identified SLFN11, but not HRD genomic scars, as a consistent correlate of response to all three PARPi assessed, with loss of SLFN11 conferring resistance to PARPi. We confirmed these findings in vivo across multiple PDX and defined IHC staining for SLFN11 as a predictor of talazoparib response. As temozolomide has activity in SCLC, we investigated combination therapy with talazoparib and found marked synergy in vitro and efficacy in vivo, which did not solely depend on SLFN11 or MGMT status. Conclusions: SLFN11 is a relevant predictive biomarker of sensitivity to PARP inhibitor monotherapy in SCLC and we identify combinatorial therapy with TMZ as a particularly promising therapeutic strategy that warrants further clinical investigation. Clin Cancer Res; 23(2); 523–35. ©2016 AACR.

Long-term regional control in the observed neck following definitive chemoradiation for node-positive oropharyngeal squamous cell cancer

Traditionally, patients treated with chemoradiotherapy for node‐positive oropharyngeal squamous cell carcinoma (N+ OPSCC) have undergone a planned neck dissection (ND) after treatment. Recently, negative post‐treatment positron‐emission tomography (PET)/computed tomography (CT) imaging has been found to have a high negative predictive value for the presence of residual disease in the neck. Here, we present the first comprehensive analysis of a large, uniform cohort of N+ OPSCC patients achieving a PET/CT‐based complete response (CR) after chemoradiotherapy, and undergoing observation, rather than ND. From 2002 to 2009, 302 patients with N+ OPSCC treated with 70 Gy intensity‐modulated radiation therapy and concurrent chemotherapy underwent post‐treatment clinical assessment including PET/CT. CR was defined as no evidence of disease on clinical examination and post‐treatment PET/CT. ND was reserved for patients with

The relative prognostic utility of standardized uptake value, gross tumor volume, and metabolic tumor volume in oropharyngeal cancer patients treated with platinum based concurrent chemoradiation with a pre-treatment [18F] fluorodeoxyglucose positron emission tomography scan

OBJECTIVES This study compared the relative prognostic utility of the Gross Tumor Volume (GTV), maximum Standardized Uptake Value (SUVmax), and Metabolic Tumor Volume (MTV) in a uniform cohort of oropharyngeal squamous cell carcinoma (OPSCC) patients treated with platinum-based concurrent chemoradiation therapy (CCRT). METHODS AND MATERIALS One-hundred OPSCC with a pretreatment [(18)F] fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) were treated with CCRT. Kaplan-Meier curves and Cox proportional hazard models were generated. RESULTS When dichotomized by the median, a smaller MTV correlated with improved 5year locoregional control (LRC) (98.0% versus 87.0%, p=0.049), freedom from distant metastasis (FDM) (91.7% versus 65.0%, p=0.005), progression-free survival (PFS) (80.3% versus 56.7%, p=0.015), and overall survival (OS) (84.1% versus 57.8%, p=0.008), whereas a smaller GTV correlated with improved PFS (80.3% versus 57.4%, p=0.040) and OS (82.1% versus 60.1%, p=0.025). SUVmax failed to correlate with any outcome. On multivariate analysis, when adjusted for GTV, T-stage, and N-stage a smaller MTV remained independently correlated with improved FDM, PFS, and OS. GTV failed to reach significance in the multivariate model. CONCLUSIONS A smaller MTV correlates with improved LRC, FDM, PFS, and OS in OPSCC patients undergoing platinum-based CCRT.

Unravelling the biology of SCLC: implications for therapy

Small-cell lung cancer (SCLC) is an aggressive malignancy associated with a poor prognosis. First-line treatment has remained unchanged for decades, and a paucity of effective treatment options exists for recurrent disease. Nonetheless, advances in our understanding of SCLC biology have led to the development of novel experimental therapies. Poly [ADP-ribose] polymerase (PARP) inhibitors have shown promise in preclinical models, and are under clinical investigation in combination with cytotoxic therapies and inhibitors of cell-cycle checkpoints.Preclinical data indicate that targeting of histone-lysine N-methyltransferase EZH2, a regulator of chromatin remodelling implicated in acquired therapeutic resistance, might augment and prolong chemotherapy responses. High expression of the inhibitory Notch ligand Delta-like protein 3 (DLL3) in most SCLCs has been linked to expression of Achaete-scute homologue 1 (ASCL1; also known as ASH-1), a key transcription factor driving SCLC oncogenesis; encouraging preclinical and clinical activity has been demonstrated for an anti-DLL3-antibody–drug conjugate. The immune microenvironment of SCLC seems to be distinct from that of other solid tumours, with few tumour-infiltrating lymphocytes and low levels of the immune-checkpoint protein programmed cell death 1 ligand 1 (PD-L1). Nonetheless, immunotherapy with immune-checkpoint inhibitors holds promise for patients with this disease, independent of PD-L1 status. Herein, we review the progress made in uncovering aspects of the biology of SCLC and its microenvironment that are defining new therapeutic strategies and offering renewed hope for patients.

Challenges and opportunities in primary CNS lymphoma: A systematic review

BACKGROUND Historically, high-dose methotrexate (HD-MTX) plus consolidation chemotherapy and/or whole brain radiotherapy (WBRT) has been the gold standard on Primary Central Nervous System Lymphoma (PCNSL) management. We sought to examine and summarize the data, on clinical trial (CT) setting, investigating multi-modality treatment to PCNSL. METHODS We performed a systematic review of electronic databases (Medline, EMBASE, Cochrane Database and clinicaltrials.gov) and a manual search to identify original PCNSL phase 2 and phase 3 CT from the last 10years. After a 4stage Prisma based selection process, 32 published (3 Randomized CT and 29 phases 2 CT) studies ultimately were selected for review. Four ongoing clinical trials found on clinicaltrial.gov were reviewed. Two investigators reviewed titles, abstracts, and articles independently. Two investigators abstracted data sequentially and evaluated each study independently. FINDINGS Treatment of PCNSL requires a multidisciplinary approach. HD-MTX represents the most accepted standard of care induction therapy for newly diagnosed PCNSL. When HD-MTX is given with WBRT for consolidation delayed neurotoxicity can be an important complication, particularly in elderly patients. Studies have suggested that WBRT may be deferred until relapse without compromising survival and deferring WBRT may be the best approach in elderly patients. Results from dose-reduced WBRT and consolidative HD-Ara-C are encouraging. High-dose chemotherapy in combination with autologous stem cell transplantation (HDC-ASCT) as chemotherapy alone has emerged as an important consolidative treatment for selected population. The optimal salvage therapy is still to be defined. CONCLUSION WBRT for consolidation is a well-studied modality; however emerging options to selected population such as HDC-ASCT, dose-reduced WBRT or chemotherapy alone are associated with similar survival outcome and less neurotoxicity in selected series. Ongoing and future clinical trials will better define the best approach on this rare disease.

Intensity-modulated radiotherapy for head and neck surgeons

The development of intensity‐modulated radiotherapy (IMRT) has played a major role in improving outcomes and decreasing morbidity in patients with head and neck cancer. This review addresses this vital modality with a focus on the important role of the head and neck surgeon. The technique as well as its benefits and points of caution are outlined, the definitions of tumor and treatment volumes are discussed, and the dose and fractionation are detailed. Following this are several sections dedicated to the role of the head and neck surgeon in the planning of both definitive and postoperative radiotherapy to the primary site and neck. There is a focus throughout on anatomic and surgical considerations; commonly encountered situations are illustrated. With a deeper understanding of this technique and their own pivotal contribution to target delineation, head and neck surgeons will be poised to expand their role and improve cancer care for their patients.

Genomic analysis of exceptional responders to radiotherapy reveals somatic mutations in ATM

Radiation therapy is a mainstay of cancer treatment, yet the molecular determinants of clinical response are poorly understood. We identified exceptional responders to radiotherapy based on clinical response, and investigated the associated tumor sequencing data in order to identify additional patients with similar mutations. Among head and neck squamous cell cancer patients receiving palliative radiotherapy at our institution, we identified one patient with documented complete metabolic response. Targeted sequencing analysis of the tumor identified a somatic frame-shift mutation in ATM, a gene known to be associated with radio-sensitivity in the germline. To validate the association of somatic ATM mutation with radiotherapy response, we identified eight patients with ATM truncating mutations who received radiotherapy, all of whom demonstrated excellent responses with a median local control period of 4.62 years. Analysis of 22 DNA repair genes in The Cancer Genome Atlas (TCGA) data revealed mutations in 15.9% of 9064 tumors across 24 cancer types, with ATM mutations being the most prevalent. This is the first study to suggest that exceptional responses to radiotherapy may be determined by mutations in DNA repair genes. Sequencing of DNA repair genes merits attention in larger cohorts and may have significant implications for the personalization of radiotherapy.

Factors influencing the utilization of prophylactic cranial irradiation in patients with limited-stage small cell lung cancer

Purpose Brain metastases are common in patients with limited-stage small cell lung cancer (LS-SCLC) due to the inability of most chemotherapeutics to penetrate the blood–brain barrier. Prophylactic cranial irradiation (PCI) is therefore recommended for use in patients with a good response to concurrent chemoradiotherapy. However, PCI is not always delivered; therefore, we investigated the reasons for PCI omission in patients who underwent therapy with curative intent. Methods and materials We retrospectively reviewed all patients with LS-SCLC who were treated with curative intent at our institution. Overall survival and cumulative incidence of brain metastasis were estimated by the Kaplan-Meier method. The Pearson χ2 test and Mann-Whitney U test were used to examine factors associated with PCI use, and prognostic factors were analyzed with Cox proportional hazards modeling. Results We examined 208 patients who were treated for LS-SCLC at our institution. A total of 115 patients (55%) received PCI. The most common documented reason for PCI omission was patient refusal due to neurotoxicity concerns (38%). Physician assessment of being medically unfit (33%) and of advanced age (8%) were the second and third most common reasons, respectively. Karnofsky performance status and clinical American Joint Committee on Cancer stage but not PCI were significantly associated with overall survival. Only clinical stage remained an independent factor on multivariate analysis. Conclusions Approximately half of patients with LS-SCLC ultimately receive PCI, generally for guideline-recommended reasons. The most common reason for PCI omission was patient concerns regarding neurotoxicity. Efforts to decrease PCI neurotoxicity, including hippocampal-sparing radiation and memantine use, may increase the use of this survival-improving intervention in eligible patients with LS-SCLC.