Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity.

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high ≥10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with "POLE ExoD driver plus POLE Variant" (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the "POLE ExoD driver plus POLE Variant" tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes. SIGNIFICANCE: Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.

BACKGROUND: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. METHODS: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. RESULTS: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. CONCLUSIONS: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

Investigation of the causal etiology in a patient with T-B+NK+ immunodeficiency.

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.

Radiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605.

BACKGROUND: Radiation exposure of tissues is associated with inflammatory cell influx. Myeloperoxidase (MPO) is an enzyme expressed in granulocytes, such as neutrophils (PMN) and macrophages, responsible for active chlorine species (ACS) generation. The present study aimed to: 1) determine whether exposure to γ-irradiation induces MPO-dependent ACS generation in murine PMN; 2) elucidate the mechanism of radiation-induced ACS generation; and 3) evaluate the effect of the synthetic lignan LGM2605, known for ACS scavenging properties. METHODS: MPO-dependent ACS generation was determined by using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and a highly potent MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and confirmed in PMN derived from MPO-/- mice. Radiation-induced MPO activation was determined by EPR spectroscopy and computational analysis identified tyrosine, serine, and threonine residues near MPO's active site. RESULTS: γ-radiation increased MPO-dependent ACS generation dose-dependently in human MPO and in wild-type murine PMN, but not in PMN from MPO-/- mice. LGM2605 decreased radiation-induced, MPO-dependent ACS. Protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSTP) inhibitors decreased the radiation-induced increase in ACS. Peroxidase cycle results demonstrate that tyrosine phosphorylation blocks MPO Compound I formation by preventing catalysis on H2O2 in the active site of MPO. EPR data demonstrate that γ-radiation increased tyrosyl radical species formation in a dose-dependent manner. CONCLUSIONS: We demonstrate that γ-radiation induces MPO-dependent generation of ACS, which is dependent, at least in part, by protein tyrosine and Ser/Thr dephosphorylation and is reduced by LGM2605. This study identified for the first time a novel protein dephosphorylation-dependent mechanism of radiation-induced MPO activation.

Protein modeling to assess the pathogenicity of rare variants of SERPINA1 in patients suspected of having Alpha 1 Antitrypsin Deficiency.

BACKGROUND: Alpha 1 Antitrypsin (AAT) is a key serum proteinase inhibitor encoded by SERPINA1. Sequence variants of the gene can cause Alpha 1 Antitrypsin Deficiency (AATD), a condition associated with lung and liver disease. The majority of AATD cases are caused by the 'Z' and 'S' variants - single-nucleotide variations (SNVs) that result in amino acid substitutions of E342K and E264V. However, SERPINA1 is highly polymorphic, with numerous potentially clinically relevant variants reported. Novel variants continue to be discovered, and without reports of pathogenicity, it can be difficult for clinicians to determine the best course of treatment. METHODS: We assessed the utility of next-generation sequencing (NGS) and predictive computational analysis to guide the diagnosis of patients suspected of having AATD. Blood samples on serum separator cards were submitted to the DNA1 Advanced Screening Program (Biocerna LLC, Fulton, Maryland, USA) by physicians whose patients were suspected of having AATD. Laboratory analyses included quantification of serum AAT levels, qualitative analysis by isoelectric focusing, and targeted genotyping and NGS of the SERPINA1 gene. Molecular modeling software UCSF Chimera (University College of San Francisco, CA) was used to visualize the positions of amino acid changes as a result of rare/novel SNVs. Predictive software was used to assess the potential pathogenicity of these variants; methods included a support vector machine (SVM) program, PolyPhen-2 (Harvard University, Cambridge, MA), and FoldX (Centre for Genomic Regulation, Barcelona, Spain). RESULTS: Samples from 23 patients were analyzed; 21 rare/novel sequence variants were identified by NGS, including splice variants (n = 2), base pair deletions (n = 1), stop codon insertions (n = 2), and SNVs (n = 16). Computational modeling of protein structures caused by the novel SNVs showed that 8 were probably deleterious, and two were possibly deleterious. For the majority of probably/possibly deleterious SNVs (I50N, P289S, M385T, M221T, D341V, V210E, P369H, V333M and A142D), the mechanism is probably via disruption of the packed hydrophobic core of AAT. Several deleterious variants occurred in combination with more common deficiency alleles, resulting in very low AAT levels. CONCLUSIONS: NGS and computational modeling are useful tools that can facilitate earlier, more precise diagnosis, and consideration for AAT therapy in AATD.

Mouse modeling and structural analysis of the p.G307S mutation in human cystathionine ß-synthase (CBS) reveal effects on CBS activity but not stability.

Mutations in the cystathionine ß-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The p.G307S mutation is the most frequent cause of CBS deficiency in Ireland, which has the highest prevalence of CBS deficiency in Europe. Individuals homozygous for this mutation tend to be severely affected and are pyridoxine nonresponsive, but the molecular basis for the strong effects of this mutation is unclear. Here, we characterized a transgenic mouse model lacking endogenous Cbs and expressing human p.G307S CBS protein from a zinc-inducible metallothionein promoter (Tg-G307S Cbs-/-). Unlike mice expressing other mutant CBS alleles, the Tg-G307S transgene could not efficiently rescue neonatal lethality of Cbs-/- in a C57BL/6J background. In a C3H/HeJ background, zinc-induced Tg-G307S Cbs-/- mice expressed high levels of p.G307S CBS in the liver, and this protein variant forms multimers, similarly to mice expressing WT human CBS. However, the p.G307S enzyme had no detectable residual activity. Moreover, treating mice with proteasome inhibitors failed to significantly increase CBS-specific activity. These findings indicated that the G307S substitution likely affects catalytic function as opposed to causing a folding defect. Using molecular dynamics simulation techniques, we found that the G307S substitution likely impairs catalytic function by limiting the ability of the tyrosine at position 308 to assume the proper conformational state(s) required for the formation of the pyridoxal-cystathionine intermediate. These results indicate that the p.G307S CBS is stable but enzymatically inert and therefore unlikely to respond to chaperone-based therapy.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

BACKGROUND: Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. METHODS: MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. RESULTS: LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl-. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. CONCLUSIONS: We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.

Plk2 Loss Commonly Occurs in Colorectal Carcinomas but not Adenomas: Relationship to mTOR Signaling.

Plk2 is a target of p53. Our previous studies demonstrated that with wild-type p53, Plk2 impacts mTOR signaling in the same manner as TSC1, and Plk2-deficient tumors grew larger than control. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk2's tumor suppressor functions in relationship to mTOR signaling. Archival specimens from 12 colorectal adenocarcinomas were stained for markers including Plk2, phosphorylated mTOR (serine 2448) and ribosomal S6 (Serine 235/236). We show that Plk2 is expressed in normal colon, with a punctate staining pattern in supranuclear cytoplasm. In colorectal adenocarcinoma, Plk2 demonstrates complete or partial loss of expression. Strong expression of phosphorylated mTOR is observed in the invasive front. Phosphorylated S6 expression partially correlates with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, in the subset of cases examined. In order to determine whether Plk2 is lost prior to the development of invasive cancer, 8 colon polyps from 6 patients were evaluated for Plk2 expression. All polyps are positive for Plk2. A Cancer Genome Atlas search identified Plk2 mutations to be infrequent in colorectal adenocarcinomas. Neither Plk2 methylation (in the gene body) nor copy number variations correlated with changes in mRNA expression levels. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR and phosphorylated S6 at the invasive front in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression. Plk2 protein expression is lost at the same stage in colorectal carcinogenesis as p53. The p53 dependence of Plk2 loss and tumor suppressor function in relationship to mTOR signaling may have therapeutic implications.

BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations.

Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.

The conformational feasibility for the formation of reaching dimer in ASV and HIV integrase: a molecular dynamics study.

Retroviral integrases are reported to form alternate dimer assemblies like the core-core dimer and reaching dimer. The core-core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit's core and C-terminal domains (CTD), as well as CTD-CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16-18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core-core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core-core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer < HIV reaching dimer < composite core-core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

Biological function derived from predicted structures in CASP11.

In CASP11, the organizers sought to bring the biological inferences from predicted structures to the fore. To accomplish this, we assessed the models for their ability to perform quantifiable tasks related to biological function. First, for 10 targets that were probable homodimers, we measured the accuracy of docking the models into homodimers as a function of GDT-TS of the monomers, which produced characteristic L-shaped plots. At low GDT-TS, none of the models could be docked correctly as homodimers. Above GDT-TS of ∼60%, some models formed correct homodimers in one of the largest docked clusters, while many other models at the same values of GDT-TS did not. Docking was more successful when many of the templates shared the same homodimer. Second, we docked a ligand from an experimental structure into each of the models of one of the targets. Docking to the models with two different programs produced poor ligand RMSDs with the experimental structure. Measures that evaluated similarity of contacts were reasonable for some of the models, although there was not a significant correlation with model accuracy. Finally, we assessed whether models would be useful in predicting the phenotypes of missense mutations in three human targets by comparing features calculated from the models with those calculated from the experimental structures. The models were successful in reproducing accessible surface areas but there was little correlation of model accuracy with calculation of FoldX evaluation of the change in free energy between the wild-type and the mutant. Proteins 2016; 84(Suppl 1):370-391. © 2016 Wiley Periodicals, Inc.

Successes and challenges with retroviral enzymes.

Collaborations between the Wlodawer and Skalka laboratories have covered a period of almost 30 years. During that time our groups have co-authored 18 publications, including several much cited journal articles, book chapters, and scholarly reviews. It has therefore been most rewarding for us to share enthusiasm, insights, and expertise with our Frederick colleagues over the years, and also to enjoy lasting friendships.

Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers.

Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers.

Systematic evaluation of underlying defects in DNA repair as an approach to case-only assessment of familial prostate cancer.

Risk assessment for prostate cancer is challenging due to its genetic heterogeneity. In this study, our goal was to develop an operational framework to select and evaluate gene variants that may contribute to familial prostate cancer risk. Drawing on orthogonal sources, we developed a candidate list of genes relevant to prostate cancer, then analyzed germline exomes from 12 case-only prostate cancer patients from high-risk families to identify patterns of protein-damaging gene variants. We described an average of 5 potentially disruptive variants in each individual and annotated them in the context of public databases representing human variation. Novel damaging variants were found in several genes of relevance to prostate cancer. Almost all patients had variants associated with defects in DNA damage response. Many also had variants linked to androgen signaling. Treatment of primary T-lymphocytes from these prostate cancer patients versus controls with DNA damaging agents showed elevated levels of the DNA double strand break (DSB) marker γH2AX (p < 0.05), supporting the idea of an underlying defect in DNA repair. This work suggests the value of focusing on underlying defects in DNA damage in familial prostate cancer risk assessment and demonstrates an operational framework for exome sequencing in case-only prostate cancer genetic evaluation.

Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Defects in DNA Repair Genes Predict Response to Neoadjuvant Cisplatin-based Chemotherapy in Muscle-invasive Bladder Cancer.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25-50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking. OBJECTIVE: To discover and validate biomarkers predictive of response to NAC for MIBC. DESIGN, SETTING, AND PARTICIPANTS: Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments-certified laboratory. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set. RESULTS AND LIMITATIONS: Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p=0.024) and validation (p=0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p<0.001; 87% sensitivity, 100% specificity) and better overall survival (p=0.007). This test remained predictive for pathologic response in the validation set (p=0.033), with a trend towards better overall survival (p=0.055). These results require further validation in additional sample sets. CONCLUSIONS: Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin. PATIENT SUMMARY: Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.

Cancer Signature Investigation: ERBB2 (HER2)-Activating Mutation and Amplification-Positive Breast Carcinoma Mimicking Lung Primary.

Next-generation sequencing of primary and metachronous metastatic cancer lesions may impact patient care. We present a case of relapsed metastatic breast cancer with a dominant pulmonary lesion originally identified as lung adenocarcinoma. A 72-year-old, never-smoker woman with a protracted cough was found to have a large lung mass and regional lymphadenopathy on a chest CT. Lung mass biopsy showed adenocarcinoma with focal TTF-1 (thyroid transcription factor 1) positivity, favoring a lung primary. In addition to stereotactic brain radiation for cerebral metastases, she was started on carboplatin/pemetrexed. As part of the workup, the tumor was analyzed by a 50-gene targeted mutation panel, which detected 3 somatic mutations: ERBB2 (HER2) D769H activating missense mutation, TP53 Y126 inactivating truncating mutation, and SMARCB1 R374Q missense mutation. Of note, the patient had a history of stage IIA triple-negative grade 3 invasive ductal carcinoma of the left breast 1.5 years ago and received neoadjuvant chemotherapy and adjuvant radiation, and underwent a lumpectomy. Further analysis of her primary breast tumor showed a mutational profile identical to that of the lung tumor. Fluorescence in situ hybridization revealed HER2 amplification in the lung tumor, with a HER2/CEP17 ratio of 3.9. The patient was diagnosed with recurrent HER2-positive metastatic breast carcinoma with a coexisting ERBB2 (HER2) activating mutation. Chemotherapy was adjusted to include dual HER2-targeted therapy containing trastuzumab and pertuzumab, resulting in an ongoing partial response. This case demonstrates that a unique genetic mutational profile can clarify whether a tumor represents a metastatic lesion or new malignancy when conventional morphological and immunohistochemical methods are indeterminate, and can directly impact treatment decisions.