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Background: Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. Methods: Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. Results: Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. Conclusions: These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

MAF file for copy number variations identified in the cohort

Treatment history for patients enrolled on the PancSeq protocol.

Supplementary Figure S1: Overview of workflow and data generation. Supplementary Figure S2: Analysis of neoplastic cellularity. Supplementary Figure S3: Recurrent copy number alterations. Supplementary Figure S4: Mutational signature analysis. Supplementary Figure S5: Analysis of PDAC gene expression signatures. Supplementary Figure S6: Clinically relevant alterations in the cohort.

Supplementary Experimental Methods

MAF file for mutations identified in the cohort

The United States Medical Licensing Examination (USMLE) consists of Step 1, Step 2 Clinical Knowledge, Step 2 Clinical Skills, and Step 3. To be licensed to practice medicine in the United States, medical students must pass all parts of the USMLE. However, in addition to that pass/fail grade, students are currently given a numerical score for Step 1, Step 2 Clinical Knowledge, and Step 3. Residency program directors have come to use the Step 1 score to efficiently screen a growing number of residency applicants. As a result, a deleterious environment in undergraduate medical education has been created, given the importance of Step 1 to medical students matching to their preferred residency program. It was announced in February 2020 that the score-reporting protocol for Step 1 would be changed from a 3-digit numerical score to pass/fail only, beginning no earlier than January 1, 2022. This decision will undoubtedly impact medical students, medical schools, and residency program directors. Here, the authors discuss the impact that the change to Step 1 scoring will have on these key stakeholder groups, from their perspective as students at MD-granting medical schools in the United States. They also call attention to outstanding issues with the USMLE that must be addressed to improve undergraduate medical education for all stakeholders, and they offer advice for further improvements to the residency application process.

Brain metastases from lung adenocarcinoma (BM-LUAD) frequently cause patient mortality. To identify genomic alterations that promote brain metastases, we performed whole-exome sequencing of 73 BM-LUAD cases. Using case-control analyses, we discovered candidate drivers of brain metastasis by identifying genes with more frequent copy-number aberrations in BM-LUAD compared to 503 primary LUADs. We identified three regions with significantly higher amplification frequencies in BM-LUAD, including MYC (12 versus 6%), YAP1 (7 versus 0.8%) and MMP13 (10 versus 0.6%), and significantly more frequent deletions in CDKN2A/B (27 versus 13%). We confirmed that the amplification frequencies of MYC, YAP1 and MMP13 were elevated in an independent cohort of 105 patients with BM-LUAD. Functional assessment in patient-derived xenograft mouse models validated the notion that MYC, YAP1 or MMP13 overexpression increased the incidence of brain metastasis. These results demonstrate that somatic alterations contribute to brain metastases and that genomic sequencing of a sufficient number of metastatic tumors can reveal previously unknown metastatic drivers.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with predisposition to developing leukemia. We found that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations inEIF6orTP53.EIF6mutations cause functional compensation for the germline deficiency by alleviating the SDS ribosome joining defect, improving translation, and reducing p53 activation.TP53mutations decrease checkpoint activation without affecting ribosome assembly. We link development of leukemia with acquisition of biallelicTP53alterations. Our results define distinct pathways of clonal selection driven by germline fitness constraint and provide a mechanistic framework for clinical surveillance.

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC. Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096–111. ©2018 AACR. See related commentary by Collisson, p. 1062. This article is highlighted in the In This Issue feature, p. 1047

Background: Shwachman-Diamond Syndrome (SDS) is a bone marrow failure disorder caused by impaired removal of EIF6 from the nascent 60S ribosome subunit, resulting in defective ribosome assembly. SDS patients have a high risk of myeloid neoplasms (MN) and the prognosis of those that develop MN is poor. Knowledge of the kinetics and functional consequences of somatic mutation acquisition in SDS may offer insight into mechanism of transformation and the potential for therapuetic intervention. Methods: We performed whole exome sequencing of 45 samples from 30 patients, and validated recurrent somatically mutated genes using targeted sequencing with error suppression in prospectively collected samples from 110 patients in the North American SDS Registry. We correlated mutation status with clinical outcome and performed functional studies to understand the consequence of somatic mutations in SDS. Results: We detected somatic mutations in 74 of 98 (76%) patients with germline biallelic SBDS mutations (median 2 mutations/patient, range 0-21). We found no mutations in patients with SDS-like disease; those who have clinical features of SDS without disease defining mutations. Of the 83 patients with SDS without a MN diagnosis, 60 (72%) had detectable clonal hematopoiesis (CH), 40 of whom had more than one mutation (median 3, range 1-21). The most frequently mutated genes were EIF6 (60/98, 61%),TP53 (44/98, 45%), PRPF8 (12/98, 12%), and CSNK1A1 (6/98, 6%). Among SDS patients with TP53 mutated CH, 90.9% (30 of 33) had concurrent EIF6 mutations. To determine whether EIF6 and TP53 mutations occur in the same or different clones, we performed single cell DNA sequencing. Among the 47 clones identified with either EIF6 or TP53 mutations, 24 had a sole EIF6 mutation, and 21 had a sole TP53 mutation, showing that these mutations arise in separate clones. To study the functional consequences of EIF6 missense mutations, we cloned 7 patient-derived mutations and generated cell lines expressing wild-type or mutant EIF6 cDNA. We found six mutants (I13N, R67W, G69S, P73R, A194T, G196R) reduced levels of EIF6 protein compared with wild type EIF6, despite comparable abundance of mRNA. The most common recurrent mutation, N106S, was found in 20% of patients and, by contrast to others listed above, did not change protein expression. This mutation is located at the EIF6/60S protein interface and disrupted the interaction of N106S-EIF6 with the 60S subunit as measured by polysome profiling followed by western blotting. To compare the effects of EIF6 versus TP53 somatic mutations in context of SDS deficient translation, we measured ribosome maturation and translation in SDS cells containing shRNAs targeting EIF6 or TP53. EIF6 knockdown ameliorated the SDS defect, reflected by improved ribosome joining (normalization of the 80:60s ratio) and enhanced protein translation (increased O-propargyl-puromycin incorporation), whereas TP53 knockdown had no effect. Knockdown of EIF6 in SDS deficient cells decreased p53 pathway activation as demonstrated by decreased CDKN1A expression. TP53 mutations were significantly associated with MN diagnosis (p=0.023), but were also common in SDS CH and typically stable over time. To identify the characteristics associated with transformation, we analyzed exomes from 7 patients with TP53 mutated myeloid malignancy for allelic imbalances at the TP53 locus and found that all 7 had biallelic alteration of TP53. Using single cell DNA sequencing from serial samples, we observed that TP53 LOH can precede transformation by several years and can distinguish pre-leukemic clones from indolent clones with monoallelic TP53 alterations. Somatic EIF6 mutations were not found in the leukemic clones. These results suggest early detection of TP53 LOH may distinguish clones with leukemic potential. Conclusions: In SDS, impairment of ribosome maturation drives selection of clones with somatic EIF6 or TP53 mutations. EIF6 mutations promote competitive fitness by rescuing the SDS ribosome defect and decreasing p53 pathway activation, and do not contribute to malignant transformation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. These results provide genetic evidence that germline SBDS deficiency causes a global, disease-specific HSC fitness constraint that drives parallel development of somatic CH and provides a mechanistic rationale for clinical surveillance. Disclosures Dale: Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Research Funding; X4 Pharmaceuticals: Honoraria. Gansner:Alnylam Pharmaceuticals: Current Employment, Current equity holder in private company. Edwards:Jazz Pharmaceuticals: Consultancy, Honoraria. Fleming:DISC Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding.

Although lung adenocarcinomas frequently metastasize to the brain, treatment options for lung adenocarcinoma brain metastases are limited. We discovered novel candidate drivers of progression by using case-control analyses to compare whole-exome sequencing data from a cohort of 73 lung adenocarcinoma brain metastases to a control cohort of 503 primary lung adenocarcinomas. We identified 3 genomic regions with significantly more frequent amplifications in brain metastases compared to the control cohort: MYC (12% vs 6%), YAP1 (7% vs 0.8%) and MMP13 (10% vs 0.6%). We also identified CDKN2A/B as a region deleted at a significantly greater frequency in brain metastases compared to primary lung adenocarcinomas (27% vs 13%, respectively). We confirmed frequent amplifications of MYC and YAP1/MMP13 in an independent validation cohort of 105 lung adenocarcinoma brain metastasis samples using fluorescence in situ hybridization. We further validated that MYC, YAP1 and MMP13 can drive brain metastases in a patient-derived xenograft mouse model. We found a higher incidence of metastases to the brain in mice receiving intracardiac injections of tumor cells expressing the candidate drivers compared to tumor cells expressing LacZ as a control. These results indicate that somatic alterations can drive lung adenocarcinomas to metastasize to the brain. The candidate brain metastasis drivers that we identified may serve as therapeutic targets in patients with lung adenocarcinomas who develop this devastating complication.

Glial tumors have been heavily studied and sequenced, leading to scores of findings about altered genes. This explosion in knowledge has not been matched with clinical success, but efforts to understand the synergies between drivers of glial tumors may alleviate the situation. We present a novel molecular classification system that captures the combinatorial nature of relationships between alterations in these diseases. We use this classification to mine for enrichment of variants of unknown significance, and demonstrate a method for segregating unknown variants with functional importance from passengers and SNPs.

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030. Most patients present with advanced disease and die within 12 months of diagnosis. Recent genomic studies of primary pancreatic cancer resection specimens have identified several molecular alterations and genomic subtypes of the disease that may guide precision medicine approaches to clinical management. However, the molecular landscape of metastatic PDAC has been less well characterized. Moreover, biopsy-driven studies in metastatic PDAC have been historically very challenging due to the aggressive course of this disease as well as the low-volume and heterogeneous nature of biopsies that makes deep molecular characterization difficult. Insufficient genomic analysis of a patient’s tumor early in their disease course is a major barrier to enrollment on clinical trials of targeted therapies. To address these limitations, we have implemented a multi-disciplinary clinical and research biopsy protocol to enable real time comprehensive molecular characterization of metastatic PDAC biopsy specimens. We have performed core needle biopsies of metastatic lesions in the liver or peritoneal cavity in 42 patients at the time of initial presentation. A low rate of complications was observed, with only a single patient having a self-limited hemorrhagic complication after liver biopsy. On average, 4-6 separate biopsy specimens were collected from each patient for histopathology and genomic analysis. Whole exome sequencing (WES) was performed in a CLIA-certified laboratory and a comprehensive molecular report of somatic alterations and selected pathogenic germline variants was returned to the referring clinician with a typical turn-around time of 3-5 weeks. We observed a striking incidence of recurrent germline and somatic alterations in DNA-damage repair genes, such as BRCA2, ATM and CHEK2. We also observed alterations in genes with known therapeutic implications, such as BRAF, RNF43, STK11 and ROS, and in select cases, these results guided choice of second or third line therapy. In parallel to WES, we performed RNA sequencing on bulk tumor tissue and readily identified expression signatures defining multiple subtypes of tumor and stroma that may have prognostic or therapeutic implications for tumor- or stroma-directed therapies. Collectively, these results demonstrate the feasibility and value of real-time genomic characterization of metastatic PDAC and provide a path forward for improved stratification and enrollment of PDAC patients on molecularly defined clinical trials. Citation Format: Andrew J. Aguirre, Scott Carter, Nicholas Camarda, Arezou Ghazani, Jonathan Nowak, Annacarolina Da Silva, Lauren Brais, Dorisanne Ragon, Devin McCabe, Lori Marini, Kristin Anderka, Karla Helvie, Nelly Oliver, Ana Babic, Paul Shyn, Douglas Rubinson, Anuj Patel, James Cleary, Nadine McCleary, Matthew Kulke, Thomas Clancy, Leona Doyle, Jason Hornick, Christine Ardito-Abraham, Ruth Yu, Michael Downes, Ronald Evans, Richard A. Moffitt, Jen Jen Yeh, William C. Hahn, Charles Fuchs, Robert Mayer, Nikhil Wagle, David Tuveson, Levi A. Garraway, Brian M. Wolpin. Real-time genomic characterization of metastatic pancreatic cancer to enable precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2017-3036