Integrative Analysis of Germline Rare Variants in Clear and Non-clear Cell Renal Cell Carcinoma.

Background and objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types. Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events. Key findings and limitations: Clear cell RCC patients (n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p = 4.95e-05). Non-clear cell RCC patients (n = 380) carried enrichment of PVs in FH (OR: 77.9, p = 1.55e-08) and MET (OR: 1.98e11, p = 2.07e-05). In a CHEK2-focused analysis with European participants, clear cell RCC (n = 906) harbored nominal enrichment of low-penetrance CHEK2 variants-p.Ile157Thr (OR: 1.84, p = 0.049) and p.Ser428Phe (OR: 5.20, p = 0.045), while non-clear cell RCC (n = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p < 0.0001), and more than half had secondary somatic events affecting the same gene (n = 10/15, 66.7%). Conversely, CHEK2 PV carriers exhibited a similar age of onset to patients without germline PVs (mean: 60.1 vs 60.2 yr, p = 0.99), and only 30.4% carried somatic events in CHEK2 (n = 7/23). Finally, pathogenic germline cryptic splice variants were identified in SDHA and TSC1, and pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. Conclusions and clinical implications: This analysis supports the existing link between several RCC risk genes and RCC risk manifesting in earlier age of onset. It calls for caution when assessing the role of CHEK2 due to the burden of founder variants with varying population frequency. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants. Patient summary: In this study, we carefully compared the frequency of rare inherited mutations with a focus on patients' genetic ancestry. We discovered that subtle variations in genetic background may confound a case-control analysis, especially in evaluating the cancer risk associated with specific genes, such as CHEK2. We also identified previously less explored forms of rare inherited mutations, which could potentially increase the risk of kidney cancer.

Historical perspective and future directions: computational science in immuno-oncology.

Immuno-oncology holds promise for transforming patient care having achieved durable clinical response rates across a variety of advanced and metastatic cancers. Despite these achievements, only a minority of patients respond to immunotherapy, underscoring the importance of elucidating molecular mechanisms responsible for response and resistance to inform the development and selection of treatments. Breakthroughs in molecular sequencing technologies have led to the generation of an immense amount of genomic and transcriptomic sequencing data that can be mined to uncover complex tumor-immune interactions using computational tools. In this review, we discuss existing and emerging computational methods that contextualize the composition and functional state of the tumor microenvironment, infer the reactivity and clonal dynamics from reconstructed immune cell receptor repertoires, and predict the antigenic landscape for immune cell recognition. We further describe the advantage of multi-omics analyses for capturing multidimensional relationships and artificial intelligence techniques for integrating omics data with histopathological and radiological images to encapsulate patterns of treatment response and tumor-immune biology. Finally, we discuss key challenges impeding their widespread use and clinical application and conclude with future perspectives. We are hopeful that this review will both serve as a guide for prospective researchers seeking to use existing tools for scientific discoveries and inspire the optimization or development of novel tools to enhance precision, ultimately expediting advancements in immunotherapy that improve patient survival and quality of life.

Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Integrative Analysis of Germline Rare Variants in Clear and Non-Clear Cell Renal Cell Carcinoma.

IMPORTANCE: RCC encompasses a set of histologically distinct cancers with a high estimated genetic heritability, of which only a portion is currently explained. Previous rare germline variant studies in RCC have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification that may significantly impact the interpretation and discovery of certain candidate risk genes. OBJECTIVE: To evaluate the enrichment of germline PVs in established cancer-predisposing genes (CPGs) in clear cell and non-clear cell RCC patients compared to cancer-free controls using approaches that account for population stratification and to identify unconventional types of germline RCC risk variants that confer an increased risk of developing RCC. DESIGN SETTING AND PARTICIPANTS: In 1,436 unselected RCC patients with sufficient data quality, we systematically identified rare germline PVs, cryptic splice variants, and copy number variants (CNVs). From this unselected cohort, 1,356 patients were ancestry-matched with 16,512 cancer-free controls, and gene-level enrichment of rare germline PVs were assessed in 143 CPGs, followed by an investigation of somatic events in matching tumor samples. MAIN OUTCOMES AND MEASURES: Gene-level burden of rare germline PVs, identification of secondary somatic events accompanying the germline PVs, and characterization of less-explored types of rare germline PVs in RCC patients. RESULTS: In clear cell RCC (n = 976 patients), patients exhibited significantly higher prevalence of PVs in VHL compared to controls (OR: 39.1, 95% CI: 7.01-218.07, p-value:4.95e-05, q-value:0.00584). In non-clear cell RCC (n = 380 patients), patients carried enriched burden of PVs in FH (OR: 77.9, 95% CI: 18.68-324.97, p-value:1.55e-08, q-value: 1.83e-06) and MET (OR: 1.98e11, 95% CI: 0-inf, p-value: 2.07e-05, q-value: 3.50e-07). In a CHEK2-focused analysis with European cases and controls, clear cell RCC patients (n=906 European patients) harbored nominal enrichment of the previously reported low-penetrance CHEK2 variants, p.Ile157Thr (OR:1.84, 95% CI: 1.00-3.36, p-value:0.049) and p.Ser428Phe (OR:5.20, 95% CI: 1.00-26.40, p-value:0.045) while non-clear cell RCC patients (n=295 European patients) exhibited nominal enrichment of CHEK2 LOF germline PVs (OR: 3.51, 95% CI: 1.10-11.10, p-value: 0.033). RCC patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset compared to patients without any germline PVs in CPGs (Mean: 46.0 vs 60.2 years old, Tukey adjusted p-value < 0.0001), and more than half had secondary somatic events affecting the same gene (n=10/15, 66.7%, 95% CI: 38.7-87.0%). Conversely, patients with rare germline PVs in CHEK2 exhibited a similar age of disease onset to patients without any identified germline PVs in CPGs (Mean: 60.1 vs 60.2 years old, Tukey adjusted p-value: 0.99), and only 30.4% of the patients carried secondary somatic events in CHEK2 (n=7/23, 95% CI: 14.1-53.0%). Finally, rare pathogenic germline cryptic splice variants underexplored in RCC were identified in SDHA and TSC1, and rare pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. CONCLUSIONS AND RELEVANCE: This systematic analysis supports the existing link between several RCC risk genes and elevated RCC risk manifesting in earlier age of RCC onset. Our analysis calls for caution when assessing the role of germline PVs in CHEK2 due to the burden of founder variants with varying population frequency in different ancestry groups. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants, such as cryptic splice variants and CNVs.

Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

Integrative clinical and molecular characterization of translocation renal cell carcinoma.

Translocation renal cell carcinoma (tRCC) is a poorly characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKIs) are worse than those treated with immune checkpoint inhibitors (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8+ T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

Refractory alveolar rhabdomyosarcoma in an 11-year-old male.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

Defining an embryonal rhabdomyosarcoma endotype.

Rhabdomyosarcoma (RMS) is the most common childhood soft-tissue sarcoma. The largest subtype of RMS is embryonal rhabdomyosarcoma (ERMS) and accounts for 53% of all RMS. ERMS typically occurs in the head and neck region, bladder, or reproductive organs and portends a promising prognosis when localized; however, when metastatic the 5-yr overall survival rate is ∼43%. The genomic landscape of ERMS demonstrates a range of putative driver mutations, and thus the recognition of the pathological mechanisms driving tumor maintenance should be critical for identifying effective targeted treatments at the level of the individual patients. Here, we report genomic, phenotypic, and bioinformatic analyses for a case of a 3-yr-old male who presented with bladder ERMS. Additionally, we use an unsupervised agglomerative clustering analysis of RNA and whole-exome sequencing data across ERMS and undifferentiated pleomorphic sarcoma (UPS) tumor samples to determine several major endotypes inferring potential targeted treatments for a spectrum of pediatric ERMS patient cases.

ATM Loss Confers Greater Sensitivity to ATR Inhibition Than PARP Inhibition in Prostate Cancer.

Alterations in DNA damage response (DDR) genes are common in advanced prostate tumors and are associated with unique genomic and clinical features. ATM is a DDR kinase that has a central role in coordinating DNA repair and cell-cycle response following DNA damage, and ATM alterations are present in approximately 5% of advanced prostate tumors. Recently, inhibitors of PARP have demonstrated activity in advanced prostate tumors harboring DDR gene alterations, particularly in tumors with BRCA1/2 alterations. However, the role of alterations in DDR genes beyond BRCA1/2 in mediating PARP inhibitor sensitivity is poorly understood. To define the role of ATM loss in prostate tumor DDR function and sensitivity to DDR-directed agents, we created a series of ATM-deficient preclinical prostate cancer models and tested the impact of ATM loss on DNA repair function and therapeutic sensitivities. ATM loss altered DDR signaling, but did not directly impact homologous recombination function. Furthermore, ATM loss did not significantly impact sensitivity to PARP inhibition but robustly sensitized to inhibitors of the related DDR kinase ATR. These results have important implications for planned and ongoing prostate cancer clinical trials and suggest that patients with tumor ATM alterations may be more likely to benefit from ATR inhibitor than PARP inhibitor therapy. SIGNIFICANCE: ATM loss occurs in a subset of prostate tumors. This study shows that deleting ATM in prostate cancer models does not significantly increase sensitivity to PARP inhibition but does sensitize to ATR inhibition.See related commentary by Setton and Powell, p. 2085.

Undifferentiated small round cell sarcoma in a young male: a case report.

CIC-rearranged sarcomas (CRSs) have recently been characterized as a distinct sarcoma subgroup with a less favorable prognosis compared to other small round cell sarcomas. CRSs share morphologic features with Ewing's sarcoma and prior to 2013 were grouped under undifferentiated sarcomas with round cell phenotype by the WHO classification. In this report, whole-genome sequencing and RNA sequencing were performed for an adolescent male patient with CRS who was diagnosed with undifferentiated pleomorphic sarcoma (UPS) by three contemporary institutions. Somatic mutation analysis identified mutations in IQGAP1, CCNC, and ATXN1L in pre- and post-treatment tissue samples, as well as a CIC-DUX4 fusion that was confirmed by qPCR and DUX4 immunohistochemistry. Of particular interest was the overexpression of the translation factor eEF1A1, which has oncogenic properties and has recently been identified as a target of the investigational agent plitidepsin. This case may provide a valuable waypoint in the understanding and classification of CRSs and may provide a rationale for targeting eEF1A1 in similar soft tissue sarcoma cases.

Volasertib preclinical activity in high-risk hepatoblastoma.

Relapsed and metastatic hepatoblastoma represents an unmet clinical need with limited chemotherapy treatment options. In a chemical screen, we identified volasertib as an agent with in vitro activity, inhibiting hepatoblastoma cell growth while sparing normal hepatocytes. Volasertib targets PLK1 and prevents the progression of mitosis, resulting in eventual cell death. PLK1 is overexpressed in hepatoblastoma biopsies relative to normal liver tissue. As a potential therapeutic strategy, we tested the combination of volasertib and the relapse-related hepatoblastoma chemotherapeutic irinotecan. We found both in vitro and in vivo efficacy of this combination, which may merit further preclinical investigation and exploration for a clinical trial concept.

Challenges in Drug Discovery for Neurofibromatosis Type 1-Associated Low-Grade Glioma.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that results from germline mutations of the NF1 gene, creating a predisposition to low-grade gliomas (LGGs; pilocytic astrocytoma) in young children. Insufficient data and resources represent major challenges to identifying the best possible drug therapies for children with this tumor. Herein, we summarize the currently available cell lines, genetically engineered mouse models, and therapeutic targets for these LGGs. Conspicuously absent are human tumor-derived cell lines or patient-derived xenograft models for NF1-LGG. New collaborative initiatives between patients and their families, research groups, and pharmaceutical companies are needed to create transformative resources and broaden the knowledge base relevant to identifying cooperating genetic drivers and possible drug therapeutics for this common pediatric brain tumor.