Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study.

BACKGROUND: Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours. METHODS: Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort. FINDINGS: MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups. INTERPRETATION: Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs. FUNDING: US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

Differential outcomes and immune checkpoint inhibitor response among endometrial cancer patients with MLH1 hypermethylation versus MLH1 "Lynch-like" mismatch repair gene mutation.

OBJECTIVES: To identify differential survival outcomes and immune checkpoint inhibitor (ICI) response in MLH1 hypermethylated versus MLH1 mutated ("Lynch-like") endometrial tumors and determine whether their molecular profiles can elucidate the differential outcomes. METHODS: 1673 mismatch repair deficient endometrial tumors were analyzed by next-generation sequencing and whole transcriptome sequencing (Caris Life Sciences, Phoenix, AZ). PD-L1, ER, and PR were tested by immunohistochemistry and immune cell infiltrates were calculated using MCP-counter. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons. Overall survival (OS) was depicted using Kaplan-Meier survival curves. RESULTS: The endometrial cancer cohort comprised 89.2% patients with MLH1 hypermethylated tumors and 10.8% with MLH1 mutated tumors, with median ages of 67 and 60 years, respectively (p < 0.01). Patients with MLH1 hypermethylated tumors had significantly worse OS and trended toward worse OS following ICI treatment than patients with MLH1 mutated tumors. The immune microenvironment of MLH1 hypermethylated relative to MLH1 mutated was characterized by decreased PD-L1 positivity, immune checkpoint gene expression, immune cell infiltration, T cell inflamed scores, and interferon gamma (IFNγ) scores. MLH1 hypermethylation was also associated with decreased mutation rates in TP53 and DNA damage repair genes, but increased rates of JAK1, FGFR2, CCND1, and PTEN mutations, as well as increased ER and PR positivity. CONCLUSIONS: Endometrial cancer patients with MLH1 hypermethylation display significantly decreased survival and discrepant immunotherapy responses compared to patients with MLH1 mutated tumors, which was associated with differential mutational profiles, a more immune cold phenotype, and increased ER/PR expression in MLH1 hypermethylated tumors. Providers may consider early transition from single agent ICI to a multi-agent regimen or hormonal therapy for patients with MLH1 hypermethylated tumors.

HER2+ endometrioid endometrial cancer possesses distinct molecular and immunologic features associated with a more active immune microenvironment and worse prognosis.

OBJECTIVE: HER2 status is not routinely evaluated in endometrioid endometrial cancer (E-EMCA), though it is frequently overexpressed or amplified in high grade E-EMCA and uterine serous carcinoma. Defining characteristics and survival outcomes of HER2+ E-EMCA could reveal subsets of patients who may benefit from targeted therapies. METHODS: 2927 E-EMCA tumors from the Caris Life Sciences database were analyzed by next-generation sequencing and whole exome sequencing, whole transcriptome sequencing, and immunohistochemistry for molecular and genomic features in a CLIA/CAP-certified laboratory (Caris Life Sciences, Phoenix, AZ). HER2 status was determined by transcriptomic cutoff extrapolated from uterine serous carcinoma. The relationship between HER2 status and patient outcomes was determined by Kaplan-Meier analysis. RESULTS: HER2 positivity was detected in 5.47% of E-EMCA. Differences in molecular alterations based on HER2 status were most apparent in microsatellite stable (MSS) tumors, which displayed increased TP53 mutations and loss of heterozygosity (LOH) and decreased PTEN and CTNNB1 mutations. HER2+ tumors had increased immune checkpoint gene expression and immune cell infiltration, particularly among MSS tumors. All HER2+ tumors displayed increased MAPK pathway activation scores (MPAS) and patients with HER2+ tumors experienced worse overall survival. CONCLUSIONS: HER2 positivity in E-EMCA corresponds with a unique molecular landscape, particularly in MSS tumors. HER2+ tumors are also associated with increased MAPK pathway activation and exhibit features of a more active immune microenvironment. These findings suggest a potential benefit of HER2 and MAPK targeted therapies as well as immunotherapies in this patient population.

The Genomic Landscape of Vulvar Squamous Cell Carcinoma.

Vulvar squamous cell cancer (VSC) accounts for 90% of vulvar cancers. Next-generation sequencing studies of VSC imply human papillomavirus (HPV) and p53 status play separate roles in carcinogenesis and prognosis. We sought to describe the genomic landscape and analyze the immunologic profiles of VSC with respect to HPV and p53 status. A total of 443 VSC tumors underwent tumor profiling. Next-generation sequencing was performed on genomic DNA isolated from formalin-fixed paraffin-embedded tumor samples. PD-L1, microsatellite instability were tested by fragment analysis, IHC, and next-generation sequencing. Tumor mutational burden-high was defined as >10 mutations per MB. HPV 16/18 positive (HPV+) status was determined using whole exome sequencing on 105 samples. Three cohorts were identified from 105 samples with known HPV: HPV+, HPV-/p53wt, and HPV-/p53mt. Where HPV and p53 status were examined, TP53 mutations were exclusive of HPV+ tumors. In all, 37% of samples were HPV+. Among the 66 HPV- tumors, 52 (78.8%) were HPV-/p53mt and 14 (21.2%) were HPV-/p53wt. The HPV-/p53wt cohort had a higher rate of mutations in the PI3KCA gene (42.9% HPV-/p53wt vs 26.3% HPV+ vs. 5.8% HPV-/p53mt, q =0.028) and alterations in the PI3K/AkT/mTOR pathway (57.1% HPV-/p53wt vs. 34.2% HPV+ vs. 7.7% HPV-/p53mt, q =0.0386) than the other 2 cohorts. Ninety-eight VSC tumors with HPV16/18 information underwent transcriptomic analysis and immune deconvolution method. No differences were observed in immune profiles. The HPV-/p53wt VSC tumors had significantly higher rates of mutations in the PI3KCA gene and alterations in the PI3K/AkT/mTOR pathway, a potential target that merits further investigation in this subgroup.

Molecular characterization of ESR1 variants in breast cancer.

PURPOSE: Estrogen receptor 1 (ESR1) mutations and fusions typically arise in patients with hormone receptor-positive breast cancer after aromatase inhibitor therapy, whereby ESR1 is constitutively activated in a ligand-independent manner. These variants can impact treatment response. Herein, we characterize ESR1 variants among molecularly profiled advanced breast cancers. METHODS: DNA next-generation sequencing (592-gene panel) data from 9860 breast cancer samples were retrospectively reviewed. Gene fusions were detected using the ArcherDx fusion assay or whole transcriptome sequencing (n = 344 and n = 4305, respectively). Statistical analyses included Chi-square and Fisher's exact tests. RESULTS: An ESR1 ligand-binding domain (LBD) mutation was detected in 8.6% of tumors evaluated and a pathogenic ESR1 fusion was detected in 1.6%. Most ESR1 LBD mutations/fusions were from estrogen receptor (ER)-positive samples (20.1% and 4.9%, respectively). The most common ESR1 LBD mutations included D538G (3.3%), Y537S (2.3%), and E380Q (1.1%) mutations. Among biopsy sites, ESR1 LBD mutations were most observed in liver metastases. Pathogenic ESR1 fusions were identified in 76 samples (1.6%) with 40 unique fusion partners. Evaluating co-alterations, ESR1 variant (mutation/fusion) samples more frequently expressed androgen receptor (78.0% vs 58.6, P < 0.0001) and less frequently immune checkpoint proteins than ESR1 wild-type (PD-1 20.0% vs 53.4, P < 0.05; immune cell PD-L1 10.0% vs 30.2, P < 0.0001). CONCLUSION: We have described one of the largest series of ESR1 fusions reported. ESR1 LBD mutations were commonly identified in ER-positive disease. Limited data exists regarding the clinical impact of ESR1 fusions, which could be an area for future therapeutic exploration.

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Distinct molecular profile of IRF4-rearranged large B-cell lymphoma.

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma-related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

Association of clinical trial enrollment and survival using contemporary therapy for pediatric acute lymphoblastic leukemia.

While early studies reported superior survival for cancer patients enrolled on clinical trials, recent findings are inconclusive. We investigated the association between enrollment on contemporary trials and event-free survival (EFS) in pediatric B-cell acute lymphoblastic leukemia (B-ALL). In a retrospective cohort of 274 children (1-21 years) treated for B-ALL from 2008 to 2015, 55.5% enrolled with no disparity in enrollment by age, sex, or ethnicity. Three-year EFS was similar for enrolled and not enrolled patients (90.1% [95% CI, 82.5-94.5] versus 86.5% [95% CI, 77.7-92.0]). Clinical trial enrollment did not affect pediatric B-ALL survival, albeit in a limited-size cohort treated at a single academic institution.

Predictors of hepatotoxicity and pancreatitis in children and adolescents with acute lymphoblastic leukemia treated according to contemporary regimens.

BACKGROUND: Hepatotoxicity and pancreatitis are common treatment-related toxicities (TRTs) during contemporary treatment regimens for acute lymphoblastic leukemia (ALL). Limited detailed data from Children's Oncology Group (COG) regimens has been previously reported to enable identification of patient and treatment risk factors for these toxicities and their impact on outcomes. PROCEDURE: We analyzed a retrospective pediatric ALL cohort treated at a single institution according to COG regimens from 2008 to 2015. The primary endpoint was cumulative incidence of study-defined "severe" hepatotoxicity (Common Terminology Criteria for Adverse Events [CTCAE] Grade ≥ 4 transaminitis or Grade ≥ 3 hyperbilirubinemia) and clinically significant pancreatitis (any grade). Pancreatitis was additionally classified using the Ponte di Legno (PdL) toxicity criteria. Secondary endpoints were chemotherapy interruptions, early disease response (end of induction [EOI] minimal residual disease [MRD]), and event-free survival (EFS). RESULTS: We identified 262 patients, of whom 71 (27%) and 28 (11%) developed hepatotoxicity and pancreatitis, respectively. Three cases of pancreatitis did not fulfill PdL criteria despite otherwise consistent presentations. Both TRTs occurred throughout therapy, but approximately 25% of hepatotoxicity (18/71) and pancreatitis (8/28) occurred during induction alone. Both obesity and age (≥10 years) were identified as predictors of hepatotoxicity (subdistribution hazard ratio [SHR] obesity = 1.75, 95% confidence interval [95% CI] 1.04-2.96; SHR age ≥10 = 1.9, 95% CI 1.19-3.10) and pancreatitis (SHR obesity = 2.18, 95% CI 1.01-4.67; SHR age ≥ 10 = 2.76, 95% CI 1.19-6.39, P = 0.018). Dose interruptions were common but neither toxicity influenced EOI MRD nor EFS. CONCLUSIONS: Obese and/or older children are particularly at risk for hepatotoxicity and pancreatitis, and may benefit from toxicity surveillance and chemoprotective strategies to prevent or mitigate associated morbidity.

Myeloid lineage switch following chimeric antigen receptor T-cell therapy in a patient with TCF3-ZNF384 fusion-positive B-lymphoblastic leukemia.

A pediatric patient diagnosed initially with B-lymphoblastic leukemia (B-ALL) relapsed with lineage switch to acute myeloid leukemia (AML) after chimeric antigen receptor T-cell (CAR-T) therapy and hematopoietic stem cell transplant. A TCF3-ZNF384 fusion was identified at diagnosis, persisted through B-ALL relapse, and was also present in the AML relapse cell population. ZNF384-rearrangements define a molecular subtype of B-ALL characterized by a pro-B-cell immunophenotype; furthermore, ZNF384-rearrangements are prevalent in mixed-phenotype acute leukemias. Lineage switch following CAR-T therapy has been described in patients with KMT2A (mixed lineage leukemia) rearrangements, but not previously in any patient with ZNF384 fusion.

Outcomes of adults and children with primary mediastinal B-cell lymphoma treated with dose-adjusted EPOCH-R.

Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3-91·5] and overall survival was 95·4% (95% CI 91·8-99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1-3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P < 0·001). Our data support the use of DA-EPOCH-R for the treatment of PMBCL in children and adults. Patients with a positive end-of-therapy FDG-PET scan have an inferior outcome.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Babesia microti, an intraerythrocytic parasite, is tickborne in nature. In contrast to transmission by blood transfusion, which has been well documented, transmission associated with solid organ transplantation has not been reported. We describe parasitologically confirmed cases of babesiosis diagnosed ≈8 weeks posttransplantation in 2 recipients of renal allografts from an organ donor who was multiply transfused on the day he died from traumatic injuries. The organ donor and recipients had no identified risk factors for tickborne infection. Antibodies against B. microti parasites were not detected by serologic testing of archived pretransplant specimens. However, 1 of the organ donor's blood donors was seropositive when tested postdonation and had risk factors for tick exposure. The organ donor probably served as a conduit of Babesia parasites from the seropositive blood donor to both kidney recipients. Babesiosis should be included in the differential diagnosis of unexplained fever and hemolytic anemia after blood transfusion or organ transplantation.

Genomic Profiling of Rare Undifferentiated Sarcomatoid Subtypes of Pancreatic Carcinomas: In Search of Therapeutic Targets.

PURPOSE: The highly aggressive undifferentiated sarcomatoid carcinoma (USC) subtype of pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized because of its rarity. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy, but the prevalence of established predictive biomarkers of response is largely unknown. The objective of this study was to leverage comprehensive genomic profiling of USC PDAC tumors to determine the prevalence of biomarkers associated with potential response to targeted therapies. METHODS: USC tumors (n = 20) underwent central pathology review by a board-certified gastrointestinal pathologist to confirm the diagnosis. These samples were compared with non-USC PDAC tumors (N = 5,562). Retrospective analysis of DNA and RNA next-generation sequencing data was performed. RESULTS: USC PDACs were more frequently PD-L1+ by immunohistochemistry than non-USC PDAC (63% v 16%, respectively, P < .001). Furthermore, USC PDAC had an increase in neutrophils (8.99% v 5.55%, P = .005) and dendritic cells (1.08% v 0.00%, q = 0.022) and an increased expression of PDCD1LG2 (4.6% v 1.3%, q = 0.001), PDCD1 (2.0% v 0.8%, q = 0.060), and HAVCR2 (45.9% v 21.7%, q = 0.107) than non-USC PDAC. Similar to non-USC PDAC, KRAS was the most commonly mutated gene (86% v 90%, respectively, P = 1). CONCLUSION: To our knowledge, this work represents the largest molecular analysis of USC tumors to date and showed an increased expression of immune checkpoint genes in USC tumors. These findings provide evidence for further investigation into immune checkpoint inhibitors in USC tumors.

Differential landscape of immune evasion in oncogenic RAS-driven primary and metastatic colorectal cancers.

Oncogenic drivers such as KRAS extensively modulate the tumor inflammatory microenvironment (TIME) of colorectal cancer (CRC). The influence of KRAS on modulating immune cell composition remains unclear. The objective of this study was to identify signatures of infiltrative immune cells and distinctive patterns that differ between RAS wild-type (WT) and oncogenic mutant (MT) CRC that explain immune evasion in MT tumors. A total of 7,801 CRC specimens were analyzed using next-generation DNA sequencing, whole-exome sequencing, and/or whole transcriptome sequencing. Deficiency of mismatch repair (dMMR)/microsatellite instability (MSI) and tumor mutation burden (TMB) were also assessed. KRAS mutations were present in 48% of CRC, similarly distributed in patients younger than vs. 50 years and older. In microsatellite stable (MSS) KRAS MT tumors, composition of the TIME included higher neutrophil infiltration and lower infiltration of B cells. MSI-H/dMMR was significantly more prevalent in RAS WT (9.1%) than in KRAS MT (2.9%) CRC. In MSS CRC, TMB-high cases were significantly higher in RAS MT (3.1%) than in RAS WT (2.1%) tumors. KRAS and NRAS mutations are associated with increased neutrophil infiltration, with codon-specific differences. These results demonstrate significant differences in the TIME of RAS mutant CRC that match previous reports of immunoevasive characteristics of such tumors.

Immunohistochemical evaluation of MYC expression in mantle cell lymphoma.

AIM: To assess the validity and potential clinical utility of evaluating MYC expression by immunohistochemistry (IHC) in mantle cell lymphoma (MCL). METHODS AND RESULTS: MYC IHC was scored on a tissue microarray containing 62 MCLs and 29 controls by two pathologists. Inter-observer correlation was high (intra-class correlation of 0.98). MYC IHC scores correlated with MYC expression (Spearman's rank correlation 0.69, P < 0.0001) and weakly with Ki67 proliferation index (Spearman's rank correlation 0.30, P = 0.03). Six blastic MCLs did not have higher mean MYC IHC scores or MYC mRNA expression than non-blastic MCLs. None of 57 cases assessed, including all of the blastic cases, showed MYC rearrangement by fluorescence in-situ hybridization. Multivariate analysis with backward selection from potential predictors including age, lactate dehydrogenase, leukocyte count, MIPI score, ECOG performance status, blastic morphology and Ki67 index showed that MYC IHC score is an independent predictor of progression-free survival (hazard ratio 2.34, 95% CI 1.42-3.88, P = 0.0009) and overall survival (hazard ratio 1.90, 95% CI 1.05-3.43, P = 0.034). CONCLUSIONS: We show that a new monoclonal anti-MYC antibody can enable accurate and reproducible visual assessment of MYC expression that is independently predictive of clinical outcomes in MCL.

Laboratory testing for cobalamin deficiency in megaloblastic anemia.

Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed.

Opposing Roles of SPOP Mutations in Human Prostate and Endometrial Cancers.

PURPOSE: Recurrent gene mutations in speckle-type POZ protein (SPOP), the substrate-binding component of E3 ubiquitin ligase, are associated with tumor progression in prostate and endometrial cancers. Here, we characterized SPOP mutations in these cancers and explored their association with molecular and immune signatures and patient outcomes. METHODS: There were 7,398 prostate cancer and 19,188 endometrial cancer samples analyzed for clinical and molecular profiles at Caris Life Sciences. Overall survival (OS) was analyzed using Kaplan-Meier survival curves. Statistical significance was determined using chi-square and Mann-Whitney U tests, with P values adjusted for multiple comparisons. RESULTS: SPOP mutations were identified in 9.2% of prostate and 4.3% of endometrial cancers. Mutations clustered in the SPOP meprin and TRAF-C homology domain, with no significant overlap between cancer types. SPOP mutation was associated with differential comutation profiles and opposing tumor immune microenvironment signatures for each cancer, with greater immune infiltration in SPOP-mutated endometrial cancer. SPOP-mutated prostate and endometrial cancers displayed altered epigenetic gene expression, including opposite regulation of BRD2 transcripts. In SPOP-mutant prostate cancer, higher expression of androgen receptor-regulated transcripts and improved OS after treatment with hormonal agents were observed. In endometrial cancer, hormone receptor expression was significantly lower in SPOP-mutated tumors and differences in OS were highly dependent on the particular hotspot mutation and histologic subtype. CONCLUSION: These data indicate that SPOP mutations drive opposing molecular and immune landscapes in prostate and endometrial cancers-suggesting a loss-of-function mechanism in prostate cancer and gain-of-function mechanism in endometrial cancer-and provide a rationale for tailored therapeutic approaches.

Multi-omic characterization reveals a distinct molecular landscape in young-onset pancreatic cancer.

Purpose: Using a real-world database with matched genomic-transcriptomic molecular data, we sought to characterize the distinct molecular correlates underlying clinical differences between young-onset pancreatic cancer (YOPC; <50-yrs.) and average-onset pancreatic cancer (AOPC; ≥70-yrs.) patients. Methods: We analyzed matched whole-transcriptome and DNA sequencing data from 2430 patient samples (YOPC, n=292; AOPC, n=2138) from the Caris Life Sciences database (Phoenix, AZ). Immune deconvolution was performed using the quanTIseq pipeline. Overall survival (OS) data was obtained from insurance claims (n=4928); Kaplan-Meier estimates were calculated for age-and molecularly-defined cohorts. Significance was determined as FDR-corrected P -values ( Q )<0.05. Results: YOPC patients had higher proportions of mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H), BRCA2 -mutant, and PALB2 -mutant tumors compared with AOPC patients, but fewer SMAD4-, RNF43-, CDKN2A- , and SF3B1- mutant tumors. Notably, YOPC patients demonstrated significantly lower incidence of KRAS mutations compared with AOPC patients (81.3% vs. 90.9%; Q =0.004). In the KRAS- wildtype subset (n=227), YOPC tumors demonstrated fewer TP53 mutations and were more likely driven by NRG1 and MET fusions, while BRAF fusions were exclusively observed in AOPC patients. Immune deconvolution revealed significant enrichment of natural killer (NK) cells, CD8 + T cells, monocytes, and M2 macrophages in YOPC patients relative to AOPC patients, which corresponded with lower rates of HLA-DPA1 homozygosity. There was an association with improved OS in YOPC patients compared with AOPC patients with KRAS -wildtype tumors (median 16.2 [YOPC- KRAS WT ] vs. 10.6 [AOPC- KRAS WT ] months; P =0.008) but not KRAS -mutant tumors ( P =0.084). Conclusion: In this large, real-world multi-omic characterization of age-stratified molecular differences in PDAC, YOPC is associated with a distinct molecular landscape that has prognostic and therapeutic implications.