Rare germline structural variants increase risk for pediatric solid tumors.

Pediatric solid tumors are rare malignancies that represent a leading cause of death by disease among children in developed countries. The early age-of-onset of these tumors suggests that germline genetic factors are involved, yet conventional germline testing for short coding variants in established predisposition genes only identifies pathogenic events in 10-15% of patients. Here, we examined the role of germline structural variants (SVs)-an underexplored form of germline variation-in pediatric extracranial solid tumors using germline genome sequencing of 1,766 affected children, their 943 unaffected relatives, and 6,665 adult controls. We discovered a sex-biased association between very large (>1 megabase) germline chromosomal abnormalities and a four-fold increased risk of solid tumors in male children. The overall impact of germline SVs was greatest in neuroblastoma, where we revealed burdens of ultra-rare SVs that cause loss-of-function of highly expressed, mutationally intolerant, neurodevelopmental genes, as well as noncoding SVs predicted to disrupt three-dimensional chromatin domains in neural crest-derived tissues. Collectively, our results implicate rare germline SVs as a predisposing factor to pediatric solid tumors that may guide future studies and clinical practice.

Lung cancer in patients who have never smoked - an emerging disease.

Lung cancer is the most common cause of cancer-related deaths globally. Although smoking-related lung cancers continue to account for the majority of diagnoses, smoking rates have been decreasing for several decades. Lung cancer in individuals who have never smoked (LCINS) is estimated to be the fifth most common cause of cancer-related deaths worldwide in 2023, preferentially occurring in women and Asian populations. As smoking rates continue to decline, understanding the aetiology and features of this disease, which necessitate unique diagnostic and treatment paradigms, will be imperative. New data have provided important insights into the molecular and genomic characteristics of LCINS, which are distinct from those of smoking-associated lung cancers and directly affect treatment decisions and outcomes. Herein, we review the emerging data regarding the aetiology and features of LCINS, particularly the genetic and environmental underpinnings of this disease as well as their implications for treatment. In addition, we outline the unique diagnostic and therapeutic paradigms of LCINS and discuss future directions in identifying individuals at high risk of this disease for potential screening efforts.

Prevalence and Therapeutic Targeting of High-Level ERBB2 Amplification in NSCLC.

INTRODUCTION: ERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody-drug conjugate (ADC) therapeutic strategy. METHODS: Our institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. RESULTS: High-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. CONCLUSIONS: High-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.

Rate of Pathogenic Germline Variants in Patients With Lung Cancer.

PURPOSE: Germline genetic testing (GGT) is now recommended for all patients diagnosed with ovarian or pancreatic cancer and for a large proportion of patients based solely on a diagnosis of colorectal or breast cancer. However, GGT is not yet recommended for all patients diagnosed with lung cancer (LC), primarily because of a lack of evidence that supports a significant frequency of identifying pathogenic germline variants (PGVs) in these patients. This study characterizes GGT results in a cohort of patients with LC. METHODS: We reviewed deidentified data for 7,788 patients with GGT (2015-2022). PGV frequencies were compared to a control cohort of unaffected individuals. GGT results were stratified by genomic ancestry, history of cancer, and PGV clinical actionability per current guidelines. RESULTS: Of all patients with LC, 14.9% (1,161/7,788) had PGVs. The rate was similar when restricted to patients with no cancer family history (FH) or personal history (PH) of other cancers (14.3%). PGVs were significantly enriched in BRCA2, ATM, CHEK2, BRCA1, and mismatch repair genes compared with controls. Patients of European (EUR) genomic ancestry had the highest PGV rate (18%) and variants of uncertain significance were significantly higher in patients of non-EUR genomic ancestry. Of the PGVs identified, 61.3% were in DNA damage repair (DDR) genes and 95% were clinically actionable. CONCLUSION: This retrospective study shows a LC diagnosis identifies patients with a significant likelihood of having a cancer-predisposing PGV across genomic ancestries. Enrichment of PGVs in DDR genes suggests that these PGVs may contribute to LC cancer predisposition. The frequency of PGVs among patients with LC did not differ significantly according to FH or PH of other cancers.

Pneumonitis in Patients Receiving Thoracic Radiotherapy and Osimertinib: A Multi-Institutional Study.

Introduction: Thoracic radiotherapy (TRT) is increasingly used in patients receiving osimertinib for advanced NSCLC, and the risk of pneumonitis is not established. We investigated the risk of pneumonitis and potential risk factors in this population. Methods: We performed a multi-institutional retrospective analysis of patients under active treatment with osimertinib who received TRT between April 2016 and July 2022 at two institutions. Clinical characteristics, including whether osimertinib was held during TRT and pneumonitis incidence and grade (Common Terminology Criteria for Adverse Events version 5.0) were documented. Logistic regression analysis was performed to identify risk factors associated with grade 2 or higher (2+) pneumonitis. Results: The median follow-up was 10.2 months (range: 1.9-53.2). Of 102 patients, 14 (13.7%) developed grade 2+ pneumonitis, with a median time to pneumonitis of 3.2 months (range: 1.5-6.3). Pneumonitis risk was not significantly increased in patients who continued osimertinib during TRT compared with patients who held osimertinib during TRT (9.1% versus 15.0%, p = 0.729). Three patients (2.9%) had grade 3 pneumonitis, none had grade 4, and two patients had grade 5 events (2.0%, diagnosed 3.2 mo and 4.4 mo post-TRT). Mean lung dose was associated with the development of grade 2+ pneumonitis in multivariate analysis (OR = 1.19, p = 0.021). Conclusions: Although the overall rate of pneumonitis in patients receiving TRT and osimertinib was relatively low, there was a small risk of severe toxicity. The mean lung dose was associated with an increased risk of developing pneumonitis. These findings inform decision-making for patients and providers.

Machine learning for genetics-based classification and treatment response prediction in cancer of unknown primary.

Cancer of unknown primary (CUP) is a type of cancer that cannot be traced back to its primary site and accounts for 3-5% of all cancers. Established targeted therapies are lacking for CUP, leading to generally poor outcomes. We developed OncoNPC, a machine-learning classifier trained on targeted next-generation sequencing (NGS) data from 36,445 tumors across 22 cancer types from three institutions. Oncology NGS-based primary cancer-type classifier (OncoNPC) achieved a weighted F1 score of 0.942 for high confidence predictions ([Formula: see text]) on held-out tumor samples, which made up 65.2% of all the held-out samples. When applied to 971 CUP tumors collected at the Dana-Farber Cancer Institute, OncoNPC predicted primary cancer types with high confidence in 41.2% of the tumors. OncoNPC also identified CUP subgroups with significantly higher polygenic germline risk for the predicted cancer types and with significantly different survival outcomes. Notably, patients with CUP who received first palliative intent treatments concordant with their OncoNPC-predicted cancers had significantly better outcomes (hazard ratio (HR) = 0.348; 95% confidence interval (CI) = 0.210-0.570; P = [Formula: see text]). Furthermore, OncoNPC enabled a 2.2-fold increase in patients with CUP who could have received genomically guided therapies. OncoNPC thus provides evidence of distinct CUP subgroups and offers the potential for clinical decision support for managing patients with CUP.

Utilizing Electronic Health Records (EHR) and Tumor Panel Sequencing to Demystify Prognosis of Cancer of Unknown Primary (CUP) patients.

Cancer of unknown primary (CUP) is a type of cancer that cannot be traced back to its original site and accounts for 3-5% of all cancers. It does not have established targeted therapies, leading to poor outcomes. We developed OncoNPC, a machine learning classifier trained on targeted next-generation sequencing data from 34,567 tumors from three institutions. OncoNPC achieved a weighted F1 score of 0.94 for high confidence predictions on known cancer types (65% of held-out samples). When applied to 971 CUP tumors from patients treated at the Dana-Farber Cancer Institute, OncoNPC identified actionable molecular alterations in 23% of the tumors. Furthermore, OncoNPC identified CUP subtypes with significantly higher polygenic germline risk for the predicted cancer type and significantly different survival outcomes, supporting its validity. Importantly, CUP patients who received first palliative intent treatments concordant with their OncoNPC-predicted cancer sites had significantly better outcomes (H.R. 0.348, 95% C.I. 0.210 - 0.570, p-value 2.32 × 10-5). OncoNPC thus provides evidence of distinct CUP subtypes and offers the potential for clinical decision support for managing patients with CUP.

Combining Nelfinavir With Chloroquine Inhibits In Vivo Growth of Human Lung Cancer Xenograft Tumors.

BACKGROUND/AIM: Nelfinavir is a human immunodeficiency virus protease inhibitor that is currently being repositioned as an anticancer drug. Chloroquine, an anti-malarial lysosomotropic drug, inhibits autophagy. It has been reported that the combination of nelfinavir and chloroquine significantly enhances endoplasmic reticulum (ER) stress and induces selective cell death in multiple cell line models (in vitro). MATERIALS AND METHODS: We assessed the effects of the combination of these drugs on human NSCLC cell lines in vitro using cell proliferation assay and performed preclinical treatment studies using cell line-derived xenograft mouse models in vivo. RESULTS: In vitro, this combination enhanced inhibition of NSCLC cell proliferation with increased proteotoxicity, including ER stress, and apoptosis. In vivo, the growth of human NSCLC xenograft tumors was inhibited, which correlated with increased apoptosis and induction of ER stress as well as NSCLC growth in vitro. CONCLUSION: Our findings suggest that the induction of proteotoxicity provides a promising new target for developing anticancer drugs.

Rescue therapy for patients with anti-PD-1-refractory Merkel cell carcinoma: a multicenter, retrospective case series.

Merkel cell carcinoma (MCC) is a rare but clinically aggressive cancer with a high mortality rate. In recent years, antibodies blocking the interactions among PD-1 and its ligands have generated durable tumor regressions in patients with advanced MCC. However, there is a paucity of data regarding effective therapy for patients whose disease is refractory to PD-1 pathway blockade. This retrospective case series describes a heterogeneous group of patients treated with additional immune checkpoint blocking therapy after MCC progression through anti-PD-1. Among 13 patients treated with anti-CTLA-4, alone or in combination with anti-PD-1, objective responses were seen in 4 (31%). Additionally, one patient with MCC refractory to anti-PD-1 and anti-CTLA-4 experienced tumor regression with anti-PD-L1. Our report - the largest case series to date describing this patient population - provides evidence that sequentially-administered salvage immune checkpoint blocking therapy can potentially activate anti-tumor immunity in patients with advanced anti-PD-1-refractory MCC and provides a strong rationale for formally testing these agents in multicenter clinical trials. Additionally, to the best of our knowledge, our report is the first to demonstrate possible anti-tumor activity of second-line treatment with a PD-L1 antibody in a patient with anti-PD-1-refractory disease.

Rac1-stimulated macropinocytosis enhances Gßγ activation of PI3Kß.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kß, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gßγ subunits to the C2-helical domain linker of p110ß, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110ß. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gßγ led to an additive PI3Kß-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kß that cannot bind Gßγ, Gßγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kß was blocked. Surprisingly, PI3Kß-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110ß that cannot bind Rac1. Instead of directly binding to PI3Kß, CA-Rac1 acts by enhancing Gßγ coupling to PI3Kß, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gßγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kß by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gßγ coupling to PI3Kß.

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

Different inhibition of Gßγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gßγ-dependent regulator of PI3Kγ enzymatic activity.

Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

Identification of the Rab5 binding site in p110ß: assays for PI3Kß binding to Rab5.

Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110ß, and p110δ), p110ß has unique properties. Unlike the other family members, p110ß directly binds to Gßγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110ß binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110ß signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110ß, and discusses methods for the analysis of p110ß-Rab5 interactions.

A phase I trial of the HIV protease inhibitor nelfinavir in adults with solid tumors.

Nelfinavir is an HIV protease inhibitor being repurposed as an anti-cancer agent in preclinical models and in small oncology trials, yet the MTD of nelfinavir has not been determined. Therefore, we conducted a Phase Ia study to establish the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of nelfinavir in subjects with advanced solid tumors. Adults with refractory cancers were given oral nelfinavir twice daily with pharmacokinetic and pharmacodynamic analyses. Twenty-eight subjects were enrolled. Nelfinavir was generally well tolerated. Common adverse events included diarrhea, anemia, and lymphopenia, which were mostly mild. The DLT was rapid-onset neutropenia that was reversible. The MTD was established at 3125 mg twice daily. In an expansion cohort at the MTD, one of 11 (9%) evaluable subjects had a confirmed partial response. This, plus two minor responses, occurred in subjects with neuroendocrine tumors of the midgut or pancreatic origin. Thirty-six percent of subjects had stable disease for more than 6 months. In peripheral blood mononuclear cells, Nelfinavir inhibited AKT and induced markers of ER stress. In summary, nelfinavir is well tolerated in cancer patients at doses 2.5 times the FDA-approved dose for HIV management and showed preliminary activity in tumors of neuroendocrine origin.