Morphologic and molecular analysis of early-onset gastric cancer.

BACKGROUND: Evidence suggests that early-onset gastric cancers are distinct from traditional gastric cancers; however, detailed genomic and morphologic characterization of these cancers has not been performed. METHODS: Genomic analysis was performed for 81 patients with gastric cancer who were 50 years old or younger; pathology slides were available for 53 of these patients, and they were re-reviewed to perform a morphologic-molecular correlation analysis. The results were compared with corresponding cBioPortal data and The Cancer Genome Atlas (TCGA) analysis, which represent traditional gastric cancers. The TP53 molecular signature was established to determine the pattern of somatic mutational damage. Variants of potential germline origin were also identified from next-generation sequencing data. RESULTS: A higher rate of CDH1 mutations (22.2% of early-onset gastric cancers vs 11.4% of traditional gastric cancers; P = .0042) but a similar rate of TP53 mutations (63% of early-onset gastric cancers vs 56.6% of traditional gastric cancers; P = .2674) were seen in early-onset cancers in comparison with traditional gastric cancers. The diffuse/mixed types correlated with the TCGA genomically stable type, and the remaining Lauren types correlated with the TCGA chromosomal instability type. Diffuse and indeterminate histologic types (overall survival, 26.25 months for the intestinal type, 20.5 months for the mixed type, 12.62 months for the diffuse type, and 9 months for the indeterminate type; P = .027) and the presence of a CDH1 gene mutation (overall survival, 9 months for mutant CDH1 and 22 months for wild-type CDH1; P = .013) significantly correlated with worse survival. The TP53 gene frequently showed transition mutations (65.5%) involving the CpG sites (49%). Variants of potential germline origin were seen in high-penetrance genes (CDH1 and APC) and moderate-penetrance genes (ATM, NBN, and MUTYH) in 9.9% of cancers. CONCLUSIONS: Early-onset gastric cancer has distinct genomic alterations, such as CDH1 mutations, but shares with traditional gastric cancers a high frequency of TP53 mutations and the TP53 mutagenic signature. Diffuse and indeterminate histologic types and the presence of a CDH1 mutation are associated with worse overall survival. Endogenous factors leading to cytosine deamination and potential germline alterations in moderate-penetrance cancer susceptibility genes may be implicated in the pathogenesis of these cancers.

Unique aspects of sequence variant interpretation for inborn errors of metabolism (IEM): The ClinGen IEM Working Group and the Phenylalanine Hydroxylase Gene.

The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase (PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG-AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG-AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype-specific guidelines. Our validation of PAH-specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH-specific ACMG-AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG-AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes.

Socioeconomic determinants of the biology and outcomes of acute lymphoblastic leukemia in adults.

ABSTRACT: Various socioeconomic and biologic factors affect cancer health disparities and differences in health outcomes. To better characterize the socioeconomic vs biologic determinants of acute lymphoblastic leukemia (ALL) outcomes, we conducted a single-institution, retrospective analysis of adult patients with ALL treated at the University of Chicago (UChicago) from 2010 to 2022 and compared our outcomes with the US national data (the Surveillance, Epidemiology, and End Results [SEER] database). Among 221 adult patients with ALL treated at UChicago, BCR::ABL1 was more frequent in patients with higher body mass index (BMI; odds ratio [OR], 7.64; 95% confidence interval [CI], 1.17-49.9) and non-Hispanic Black (NHB) ancestry (59% vs 24% in non-Hispanic White (NHW) and 20% in Hispanic patients; P = .001). In a multivariable analysis, age (hazard ratio [HR], 6.93; 95% CI, 2.27-21.1) and higher BMI at diagnosis (HR, 10.3; 95% CI, 2.56-41.5) were independent predictors of poor overall survival (OS). In contrast, race or income were not predictors of OS in the UChicago cohort. Analysis of the national SEER database (2010-2020) demonstrated worse survival outcomes in Hispanic and NHB patients than in NHW patients among adolescent and young adults (AYAs) but not in older adults (aged >40 years). Both AYA and older adult patients with higher median household income had better OS than those with lower income. Therefore, multidisciplinary medical care coupled with essential supportive care services offered at centers experienced in ALL care may alleviate the socioeconomic disparities in ALL outcomes in the United States.

Acute Lymphoblastic Leukemia with Myeloid Mutations Is a High-Risk Disease Associated with Clonal Hematopoiesis.

Myeloid neoplasms arise from preexisting clonal hematopoiesis (CH); however, the role of CH in the pathogenesis of acute lymphoblastic leukemia (ALL) is unknown. We found that 18% of adult ALL cases harbored TP53, and 16% had myeloid CH-associated gene mutations. ALL with myeloid mutations (MyM) had distinct genetic and clinical characteristics, associated with inferior survival. By using single-cell proteogenomic analysis, we demonstrated that myeloid mutations were present years before the diagnosis of ALL, and a subset of these clones expanded over time to manifest as dominant clones in ALL. Single-cell RNA sequencing revealed upregulation of genes associated with cell survival and resistance to apoptosis in B-ALL with MyM, which responds better to newer immunotherapeutic approaches. These findings define ALL with MyM as a high-risk disease that can arise from antecedent CH and offer new mechanistic insights to develop better therapeutic and preventative strategies. SIGNIFICANCE: CH is a precursor lesion for lymphoblastic leukemogenesis. ALL with MyM has distinct genetic and clinical characteristics, associated with adverse survival outcomes after chemotherapy. CH can precede ALL years before diagnosis, and ALL with MyM is enriched with activated T cells that respond to immunotherapies such as blinatumomab. See related commentary by Iacobucci, p. 142.

Real world predictors of response and 24-month survival in high-grade TP53-mutated myeloid neoplasms.

Current therapies for high-grade TP53-mutated myeloid neoplasms (≥10% blasts) do not offer a meaningful survival benefit except allogeneic stem cell transplantation in the minority who achieve a complete response to first line therapy (CR1). To identify reliable pre-therapy predictors of complete response to first-line therapy (CR1) and outcomes, we assembled a cohort of 242 individuals with TP53-mutated myeloid neoplasms and ≥10% blasts with well-annotated clinical, molecular and pathology data. Key outcomes examined were CR1 & 24-month survival (OS24). In this elderly cohort (median age 68.2 years) with 74.0% receiving frontline non-intensive regimens (hypomethylating agents +/- venetoclax), the overall cohort CR1 rate was 25.6% (50/195). We additionally identified several pre-therapy factors predictive of inferior CR1 including male gender (P = 0.026), ≥2 autosomal monosomies (P < 0.001), -17/17p (P = 0.011), multi-hit TP53 allelic state (P < 0.001) and CUX1 co-alterations (P = 0.010). In univariable analysis of the entire cohort, inferior OS24 was predicated by ≥2 monosomies (P = 0.004), TP53 VAF > 25% (P = 0.002), TP53 splice junction mutations (P = 0.007) and antecedent treated myeloid neoplasm (P = 0.001). In addition, mutations/deletions in CUX1, U2AF1, EZH2, TET2, CBL, or KRAS ('EPI6' signature) predicted inferior OS24 (HR = 2.0 [1.5-2.8]; P < 0.0001). In a subgroup analysis of HMA +/-Ven treated individuals (N = 144), TP53 VAF and monosomies did not impact OS24. A risk score for HMA +/-Ven treated individuals incorporating three pre-therapy predictors including TP53 splice junction mutations, EPI6 and antecedent treated myeloid neoplasm stratified 3 prognostic distinct groups: intermediate, intermediate-poor, and poor with significantly different median (12.8, 6.0, 4.3 months) and 24-month (20.9%, 5.7%, 0.5%) survival (P < 0.0001). For the first time, in a seemingly monolithic high-risk cohort, our data identifies several baseline factors that predict response and 24-month survival.

Contrasting effects of Deadend1 (Dnd1) gain and loss of function mutations on allelic inheritance, testicular cancer, and intestinal polyposis.

BACKGROUND: Certain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter. RESULTS: We found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1⁺). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis. CONCLUSION: These results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.

Postmortem genetic testing in sudden unexplained death: A public health laboratory experience.

Sudden unexplained death in the young poses a diagnostically challenging situation for practicing autopsy pathologists, especially in the absence of anatomic and toxicological findings. Postmortem genetic testing may identify pathogenic variants in the deceased of such cases, including those associated with arrhythmogenic channelopathies and cardiomyopathies. The Wisconsin State Laboratory of Hygiene (WSLH) is a state-run public health laboratory which performs postmortem genetic testing at no cost to Wisconsin medical examiners and coroners. The current study examines sequencing data from 18 deceased patients (ages 2 months to 49 years, 5 females) submitted to WSLH, from 2016 to 2021. Panel-based analysis was performed on 10 cases, and whole exome sequencing was performed on the most recent 8 cases. Genetic variants were identified in 14 of 18 decedents (77.8%), including 7 with pathogenic or likely pathogenic variants (38.9%). Whole exome sequencing was more likely to yield a positive result, more variants per decedent, and a larger number of variants of uncertain significance. While panel-based testing may offer definitive pathogenic variants in some cases, less frequent variants may be excluded. Whole exome testing may identify rare variants missed by panels, but increased yield of variants of uncertain significance may be difficult to interpret. Postmortem genetic testing in young decedents of sudden unexplained death can provide invaluable information to autopsy pathologists to establish accurate cause and manner of death and to decedent's relatives to allow appropriate management. A public health laboratory model may be a financially advisable alternative to commercial laboratories for medical examiner's/coroner's offices.

MUC4 expression by immunohistochemistry is a specific marker for BCR-ABL1+ and BCR-ABL1-like B-lymphoblastic leukemia.

BCR-ABL1-like B-acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of high-risk B-ALL that benefits from targeted tyrosine kinase inhibitor (TKI) therapy. The incidence of this high-risk B-ALL is relatively low and screening with surrogate markers will be useful to identify patients for further genetic testing. Here we demonstrate that widely available MUC4 protein immunohistochemistry (IHC) is predictive of a BCR-ABL1-like genotype for a subset of patients. Overall, MUC4 expression was observed in 36% (9/25) BCR-ABL1-like, 43% (3/7) BCR-ABL1+ and 9% (2/22) B-ALL other cases (p=.019 for BCR-ABL1 like and BCR-ABL1+ versus B-ALL others). Furthermore, 83% (5/6) of patients with ABL class fusions showed MUC4 expression when compared to 25% (4/16, p=.006) patients with JAK class fusions. Overall, the study demonstrates that MUC4 expression is highly specific (90.9%) for BCR-ABL1+ and BCR-ABL1-like B-ALL with high sensitivity for cases with ABL class fusions.

CDKN2A loss-of-function predicts immunotherapy resistance in non-small cell lung cancer.

Immune checkpoint blockade (ICB) improves outcomes in non-small cell lung cancer (NSCLC) though most patients progress. There are limited data regarding molecular predictors of progression. In particular, there is controversy regarding the role of CDKN2A loss-of-function (LOF) in ICB resistance. We analyzed 139 consecutive patients with advanced NSCLC who underwent NGS prior to ICB initiation to explore the association of CDKN2A LOF with clinical outcomes. 73% were PD-L1 positive (≥ 1%). 48% exhibited high TMB (≥ 10 mutations/megabase). CDKN2A LOF was present in 26% of patients and was associated with inferior PFS (multivariate hazard ratio [MVA-HR] 1.66, 95% CI 1.02-2.63, p = 0.041) and OS (MVA-HR 2.08, 95% CI 1.21-3.49, p = 0.0087) when compared to wild-type (WT) patients. These findings held in patients with high TMB (median OS, LOF vs. WT 10.5 vs. 22.3 months; p = 0.069) and PD-L1 ≥ 50% (median OS, LOF vs. WT 11.1 vs. 24.2 months; p = 0.020), as well as in an independent dataset. CDKN2A LOF vs. WT tumors were twice as likely to experience disease progression following ICB (46% vs. 21%; p = 0.021). CDKN2A LOF negatively impacts clinical outcomes in advanced NSCLC treated with ICB, even in high PD-L1 and high TMB tumors. This novel finding should be prospectively validated and presents a potential therapeutic target.

Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Rapid functional genetics of the oligodendrocyte lineage using pluripotent stem cells.

Oligodendrocyte dysfunction underlies many neurological disorders, but rapid assessment of mutation-specific effects in these cells has been impractical. To enable functional genetics in oligodendrocytes, here we report a highly efficient method for generating oligodendrocytes and their progenitors from mouse embryonic and induced pluripotent stem cells, independent of mouse strain or mutational status. We demonstrate that this approach, when combined with genome engineering, provides a powerful platform for the expeditious study of genotype-phenotype relationships in oligodendrocytes.

Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease.

Pelizaeus-Merzbacher disease (PMD) is a fatal X-linked disorder caused by loss of myelinating oligodendrocytes and consequent hypomyelination. The underlying cellular and molecular dysfunctions are not fully defined, but therapeutic enhancement of oligodendrocyte survival could restore functional myelination in patients. Here we generated pure, scalable quantities of induced pluripotent stem cell-derived oligodendrocyte progenitor cells (OPCs) from a severe mouse model of PMD, Plp1jimpy. Temporal phenotypic and transcriptomic studies defined an early pathological window characterized by endoplasmic reticulum (ER) stress and cell death as OPCs exit their progenitor state. High-throughput phenotypic screening identified a compound, Ro 25-6981, which modulates the ER stress response and rescues mutant oligodendrocyte survival in jimpy, in vitro and in vivo, and in human PMD oligocortical spheroids. Surprisingly, increasing oligodendrocyte survival did not restore subsequent myelination, revealing a second pathological phase. Collectively, our work shows that PMD oligodendrocyte loss can be rescued pharmacologically and defines a need for multifactorial intervention to restore myelination.

Implementation and Clinical Utility of an Integrated Academic-Community Regional Molecular Tumor Board.

PURPOSE: Precision oncology develops and implements evidence-based personalized therapies that are based on specific genetic targets within each tumor. However, a major challenge that remains is the provision of a standardized, up-to-date, and evidenced-based precision medicine initiative across a geographic region. MATERIALS AND METHODS: We developed a statewide molecular tumor board that integrates academic and community oncology practices. The Precision Medicine Molecular Tumor Board (PMMTB) has three components: a biweekly Web-based teleconference tumor board meeting provided as a free clinical service, an observational research registry, and a monthly journal club to establish and revise evidence-based guidelines for off-label therapies. The PMMTB allows for flexible and rapid implementation of treatment, uniformity in practice, and the ability to track outcomes. RESULTS: We describe the implementation of the PMMTB and its first year of activity. Seventy-seven patient cases were presented, 48 were enrolled in a registry, and 38 had recommendations and clinical follow-up. The 38 subjects had diverse solid tumors (lung, 45%; GI, 21%; breast, 13%; other, 21%). Of these subjects, targeted therapy was recommended for 32 (84%). Clinical trials were identified for 24 subjects (63%), and nontrial targeted medicines for 16 (42%). Nine subjects (28%) received recommended therapy with a response rate of 17% (one of six) and a clinical benefit rate (partial response + stable disease) of 38% (three of eight). Although clinical trials often were identified, patients rarely enrolled. CONCLUSION: The PMMTB provides a model for a regional molecular tumor board with clinical utility. This work highlights the need for outcome registries and improved access to clinical trials to pragmatically implement precision oncology.

Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells.

Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to induced oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.