Sequential tumor molecular profiling identifies likely germline variants.

PURPOSE: To identify likely germline DNA variants from sequential tumor profiling data from hematopoietic malignancies (HMs). METHODS: The coefficient of variance was calculated from variant allele frequency of next-generation sequencing assays. Variants' likelihood of being germline was ranked on a 1 to 5 scale. Outcomes were examined in patients with such variants. RESULTS: In a pilot set of 33 genes, 89% of grade 1, 77% of grade 2, 62% of grade 3, 52% of grade 4, and 21% of grade 5 variants were confirmed to be germline. Among those, 22% were pathogenic or likely pathogenic in genes recognized as conferring hereditary HM risk, including BRCA1/2, CHEK2, CSF3R, and DDX41. To determine if this approach identified genes with known autosomal dominant inheritance, we analyzed sequential data from 1336 genes in 1135 HM patients. Among unique variants, 16% occurred in hereditary HM genes, and 15% were deleterious. Patients with grade 1/2 alleles had decreased survival 2 years after initial molecular testing (78% versus 88%, P = .0037) and increased all-cause mortality compared with those without (hazard ratio 2.02, 95% CI 1.18-3.46, P = .019). CONCLUSION: Variant germline status may be predicted using sequential tumor profiling and patients with likely germline variants experience inferior outcomes compared with those without.

Feasibility and limitations of cultured skin fibroblasts for germline genetic testing in hematologic disorders.

To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed next-generation sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22-29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (-11.9%; p < 0.01), larger biopsy size (-10.6%; p < 0.01), or lymphoid malignancy (-8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow-up studies for optimal interpretation.

Therapy-related myeloid neoplasms with normal karyotype show distinct genomic and clinical characteristics compared to their counterparts with abnormal karyotype.

Therapy-related myeloid neoplasms (t-MNs) are a complication of treatment with cytotoxic chemotherapy and/or radiation therapy. The majority of t-MNs show chromosomal abnormalities associated with myelodysplastic syndrome (MDS) or KMT2A rearrangements and are characterized by poor clinical outcomes. A small but substantial subset of patients have normal karyotype (NK) and their clinical characteristics and mutational profiles are not well studied. We retrospectively studied patients diagnosed with t-MN at three institutions and compared the mutational profile and survival data between t-MNs with NK and t-MNs with abnormal karyotype (AK). A total of 204 patients with t-MN were identified including 158 with AK and 46 with NK. NK t-MNs, compared to AK, were enriched for mutations in TET2 (p < 0.0001), NPM1 (p < 0.0001), ASXL1 (p = 0.0003), SRSF2 (p < 0.0001), RUNX1 (p = 0.0336) and STAG2 (p = 0.0099) and showed a significantly lower frequency of TP53 mutations (p < 0.0001). Overall survival (OS) was significantly lower in AK t-MNs as compared to NK cases (p = 0.0094). In our study, NK t-MNs showed a significantly better OS, a higher prevalence of MN-associated mutations and a lower frequency of TP53 mutations compared to their AK counterparts. The distinct clinical and mutational profile of NK t-MNs merits a separate classification.

EWSR1-PATZ1 fusion renal cell carcinoma: a recurrent gene fusion characterizing thyroid-like follicular renal cell carcinoma.

Thyroid-like follicular renal cell carcinoma is an uncommon kidney tumor with no distinct molecular alteration described to date. This cohort of eight women with mean and median ages of 45 and 46 years, respectively (range 19-65 years), had unencapsulated, well-circumscribed tumors composed of tightly packed anastomosing follicle-like cysts filled with eosinophilic colloid-like material and lined by cuboidal cells with high nuclear to cytoplasmic ratios, oval to elongated nuclei with perpendicular arrangement toward the lumens, and prominent nuclear overlapping. The stroma between these was minimal with the exception of two tumors. Calcifications and necrosis were absent. Immunohistochemically, the tumors were positive for KRT19 (7/7), PAX8 (5/5), cyclin D1 (6/6), KRT7 (5/7), and AMACR (1/5; focal, weak), and were negative for WT1, TTF1 (transcription termination factor-1), and thyroglobulin. In three of three tumors tested molecularly, EWSR1-PATZ1 fusion was identified by RNA sequencing and confirmed by RT-PCR and Sanger sequencing. Over a follow-up period of 1-7 years, no evidence of recurrence or metastasis has been detected. The EWSR1-PATZ1 fusion has been recognized as a recurrent alteration in a subset of round to spindle cell sarcomas with EWSR1-non-ETS fusions (EWSR1-PATZ1 sarcoma) and in several central nervous system tumors. The finding of an EWSR1-PATZ1 fusion in all three of the thyroid-like follicular renal cell carcinomas for which sufficient tissue was available for genomic profiling provides the first distinct molecular abnormality in thyroid-like follicular renal cell carcinomas, supporting its designation as a distinct diagnostic entity.

Evaluation of SWI/SNF Protein Expression by Immunohistochemistry in Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinomas (OCCC) are known to harbor ARID1A mutations, and several recent studies have described immunohistochemical loss of SMARCA2, SMARCA4, and SMARCB1 in a subset of tumors. We performed ARID1A, SMARCA2, SMARCA4, and SMARCB1 immunohistochemistry on 105 OCCCs to identify possible associations with clinicopathologic features and assess their prognostic value in these tumors. ARID1A, SMARCA4, and SMARCB1 were considered retained if any tumor cell nucleus stained while for SMARCA2, >5% of tumor nuclei were required to be positive. Patients had a mean age of 56 yr and tumors averaged 13 cm in size. Most patients (63%) had stage I tumors with 47% being alive and well, 41% dead from disease, 10% dead from other causes, and 3% alive with disease at last follow-up (mean 72 mo). Tumors showed an admixture of architectural patterns, but papillary was most frequent (49%). Stromal hyalinization was detected in 83% of OCCCs and a background precursor in 78%. High-grade atypia and/or oxyphilic cells were noted in 45% and 29% of tumors, respectively. All OCCCs expressed SMARCA4 and SMARCB1, but the absence of ARID1A was noted in 30% of tumors and SMARCA2 in 8%. ARID1A-retained OCCCs were associated with a dominant tubulocystic or solid pattern, but no other clinicopathologic features reached statistical significance. No switch/sucrose non-fermentable protein expression was predictive of prognosis. Additional studies with known mutational status of these proteins are warranted to better assess their prognostic utility and develop a standardized immunohistochemical scoring system.

Aggressive morphologic variants of mantle cell lymphoma characterized with high genomic instability showing frequent chromothripsis, CDKN2A/B loss, and TP53 mutations: A multi-institutional study.

Aggressive morphologic variants of mantle cell lymphoma (MCL), including blastoid and pleomorphic (B/P-MCL), are rare and associated with poor clinical outcomes. The genomic landscape of these variants remains incompletely explored. In this multi-institutional study, we describe recurrent mutations and novel genomic copy number alterations (CNAs) in B/P-MCL, using next generation sequencing and SNP-array. Chromothripsis, a recently described phenomenon of massive chromosomal rearrangements, was identified in eight of 13 (62%) B/P MCL cases, and a high degree of genomic complexity with frequent copy number gains and losses was also seen. In contrast, a comparative cohort of nine cases of conventional MCL (C-MCL) showed no chromothripsis and less complexity. Twelve of 13 (92%) B/P-MCL cases showed loss of CDKN2A/B (6 biallelic and 6 monoallelic losses); while only one C-MCL showed monoallelic CDKN2A/B loss. In B/P-MCL, TP53 was the most commonly mutated gene, with mutations present in eight cases (62%), six of which showed concurrent loss of chromosome 17p. Of the eight cases with chromothripsis, six (85%) harbored TP53 mutations. Other recurrent mutations in B/P-MCL included ATM (7, 53%), CCND1 (5, 38%), NOTCH1 (2, 18%), NOTCH2, and BIRC3 (each in 3, 23%). Here, we describe high genomic instability associated with chromothripsis and a high frequency of CDKN2A/B and TP53 alterations in the aggressive variants of MCL. The nonrandom chromothripsis events observed in B/P-MCL may be an indicator of clinically aggressive MCL. In addition, frequent CDKN2A deletion and high genomic instability may provide potential targets for alternative treatment.

Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia.

Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 WHO classification, and is associated with a favorable prognosis. While previous studies have evaluated NPM1 in a binary fashion, we recently demonstrated a significant independent negative prognostic effect of high NPM1 mutant allele burden (VAF) at diagnosis in a cohort of de novo AML patients. Although the importance of minimal residual disease (MRD) monitoring in NPM1-mutated AML has been well characterized, the potential relationship between diagnostic allele burden and MRD is unknown. We retrospectively evaluated for MRD at first remission (CR1). We used either next-generation sequencing (NGS) [n = 71], and/or immunohistochemistry (IHC) for mutant NPM1 (NPM1c) [n = 60], in a subset of patients from our recently examined cohort. We identified a statistically significant positive correlation between the VAF at diagnosis, and at CR1 (Spearman r = 0.4, P = .006), and enrichment for MRD in high diagnostic VAF patients (P = .05), as previously defined. IHC-positivity also correlated significantly with a higher median diagnostic NPM1 VAF (0.42 vs 0.39, P = .02), and with the VAF at CR1 (Spearman r = 0.7, P = .003). In multivariable analyses, both high diagnostic VAF (P = .003) and MRD (P = .02) were independent predictors of shorter event-free survival (EFS). Our findings suggest a relationship between the NPM1 mutant allele burden at diagnosis, and the presence of MRD at first remission. Our findings support IHC as a potentially useful adjunctive tool for disease monitoring.

High prevalence of MiTF staining in undifferentiated pleomorphic sarcoma: caution in the use of melanocytic markers in sarcoma.

AIMS: The diagnosis of undifferentiated pleomorphic sarcoma (UPS) may be challenging, as other lesions with undifferentiated spindle cell morphology must be excluded, including melanoma. Microphthalmia-associated transcription factor (MiTF) stains naevi and epithelioid melanomas, as well as some mesenchymal neoplasms. The aim of this study was to evaluate the prevalence of MiTF and melanocytic markers in UPS and a subset of atypical fibroxanthoma (AFX). METHODS AND RESULTS: MiTF, SOX10, Melan-A, HMB45 and S100 immunostaining was performed on resection specimens from 19 UPSs and five AFXs. Next-generation sequencing of 50 genes was performed in UPSs to exclude dedifferentiated melanoma. In 17 of 19 UPSs (89%), tumour cells showed nuclear positivity for MiTF that was not eliminated by casein block. Three showed focal nuclear staining for HMB45, which was eliminated by casein block. One showed focal nuclear vacuole staining for S100 with red but not brown chromogen. None expressed SOX10 or Melan-A. Mutational analysis of 15 UPSs with adequate DNA showed no mutations within hotspot regions of BRAF, KIT, or NRAS. Four of five AFXs (80%) stained with MiTF; other markers were negative. CONCLUSION: There is a high prevalence of nuclear MiTF expression in UPSs (89%) and AFXs (80%). Rare UPSs showed non-specific nuclear HMB45 or S100 staining. These findings argue against using MiTF in isolation to differentiate between UPS or AFX and melanoma, and caution in interpreting focal staining for a single additional melanocytic marker. Casein block may eliminate non-specific staining. MiTF should be used to support a diagnosis of melanoma only if multiple melanocytic markers are positive.

Identifying Inherited and Acquired Genetic Factors Involved in Poor Stem Cell Mobilization and Donor-Derived Malignancy.

Analysis of the clinical characteristics of hematopoietic stem cell transplant (HSCT) donors has proven beneficial for identifying cases of heritable hematopoietic disorders. This study examines poor peripheral blood hematopoietic stem cell mobilization after granulocyte colony-stimulating factor administration among 328 donors as a potential marker for suspected familial predisposition to myeloid malignancies. Here, we present data comparing the clinical characteristics of poor-mobilizing versus nonpoor-mobilizing donors and the results of panel-based sequencing of hematopoietic genes in poor-mobilizing donors. From this analysis, we identified a novel case of a donor-derived myelodysplastic syndrome in an HSCT recipient that is consistent with clonal evolution of TET2-mutated clonal hematopoiesis of indeterminate potential (CHIP) within the donor. This study demonstrates the potential risk of using hematopoietic stem cells from a donor with CHIP and raises the question of whether there should be increased screening measures to identify such donors.

Polyglucosan bodies in intramuscular nerve branches are a poor predictor of GBE1 mutation and adult polyglucosan body disease.

INTRODUCTION: Adult polyglucosan body disease (APBD) is associated with formation of polyglucosan bodies in peripheral nerve branches. Some muscle biopsies show these inclusions in intramuscular nerve branches. It has not been established whether the presence of multiple polyglucosan bodies in intramuscular peripheral nerve branches could or should suggest testing for APBD. METHODS: Fifteen muscle biopsies from adults between the ages of 36 and 84 years, all showing polyglucosan bodies in intramuscular peripheral nerve twigs, were tested by sequencing of the GBE1 gene. RESULTS: In 4 patients, testing identified heterozygous missense mutations not previously described. No homozygous or compound heterozygous mutations were identified. CONCLUSIONS: The presence of polyglucosan bodies in intramuscular nerve twigs by itself, even if they are multiple, is not an indication of APBD. Further testing may only be indicated in patients with clinical disease manifestations.

Nfatc1 orchestrates aging in hair follicle stem cells.

Hair production is fueled by stem cells (SCs), which transition between cyclical bouts of rest and activity. Here, we explore why hair growth wanes with age. We show that aged hair follicle SCs (HFSCs) in mice exhibit enhanced resting and abbreviated growth phases and are delayed in response to tissue-regenerating cues. Aged HFSCs are poor at initiating proliferation and show diminished self-renewing capacity upon extensive use. Only modestly restored by parabiosis, these features are rooted in elevated cell-intrinsic sensitivity and local elevation in bone morphogenic protein (BMP) signaling. Transcriptional profiling presents differences consistent with defects in aged HFSC activation. Notably, BMP-/calcium-regulated, nuclear factor of activated T-cell c1 (NFATc1) in HFSCs becomes recalcitrant to its normal down-regulating cues, and NFATc1 ChIP-sequencing analyses reveal a marked enrichment of NFATc1 target genes within the age-related signature. Moreover, aged HFSCs display more youthful levels of hair regeneration when BMP and/or NFATc1 are inhibited. These results provide unique insights into how skin SCs age.

Clinical Utility and Benefits of Comprehensive Genomic Profiling in Cancer.

BACKGROUND: Comprehensive genomic profiling (CGP) with next-generation sequencing detects genetic alterations of hundreds of genes simultaneously and multiple molecular biomarkers with one test. In the personalized medicine era, CGP is increasingly used for cancer diagnosis, treatment selection, and prognosis prediction. CONTENT: In this review, we summarize the benefits of CGP, clinical utility of CGP, and challenges of setting up CGP in the clinical laboratories. Besides the genetic alterations identified in the cancer-related genes, other biomarkers such as tumor mutational burden, microsatellite instability, and homologous recombination deficiency are critical for initiating targeted therapy. Compared with conventional tests, CGP uses less specimen and shortens the turnaround time if multiple biomarkers need to be tested. RNA fusion assay and liquid biopsy are helpful additions to DNA-based CGP by detecting fusions/splicing variants and complementing tissue-based CGP findings, respectively. SUMMARY: Many previous hurdles for implementing CGP in the clinical laboratories have been gradually alleviated such as the decrease in sequencing cost, availability of both open-source and commercial bioinformatics tools, and improved reimbursement. These changes have helped to make CGP available to a greater population of cancer patients for improving characterization of their tumors and expanding their eligibility for clinical trials. Additionally, sequencing results of the hundreds of genes on CGP panels could be further analyzed to better understand the biology of various cancers and identify new biomarkers.

A novel telomere biology disease-associated gastritis identified through a whole exome sequencing-driven approach.

A whole exome sequencing (WES)-driven approach to uncover the etiology of unexplained inflammatory gastritides has been underutilized by surgical pathologists. Here, we discovered the pathobiology of an unusual chronic atrophic gastritis in two unrelated patients using this approach. The gastric biopsies were notable for an unusual pattern of gastritis with persistent dense inflammation, loss of both parietal and neuroendocrine cells in the oxyntic mucosa, and sparing of the antral mucosa. The patients were found to harbor pathogenic variants in telomeropathic genes (POT1 and DCLRE1B). Clonality testing for one of the patients showed evidence of evolving clonality of TCR-gene rearrangement. Both patients showed significantly decreased numbers of stem/progenitor cells by immunohistochemistry, which appears to be responsible for the development of mucosal atrophy. No such cases of unusual chronic atrophic gastritis in the setting of telomeropathy have been previously reported. The loss of stem/progenitor cells suggests that stem/progenitor cell exhaustion in the setting of telomere dysfunction is the likely mechanism for development of this unusual chronic atrophic gastritis. The results underscore the need for close monitoring of these gastric lesions, with special regard to their neoplastic potential. This combined WES-driven approach has promise to identify the cause and mechanism of other uncharacterized gastrointestinal inflammatory disorders.

Whole exome sequencing of placental chorangioma.

INTRODUCTION: Placental chorangioma is a benign non-trophoblastic vascular proliferation of the placental chorion favored to represent hamartoma-like or hyperplastic capillary lesions. As the exact pathophysiology has not been established, we investigated the molecular characteristics of placental chorangiomas using exploratory whole exome sequencing. METHODS: Three cases were retrospectively selected and whole exome sequencing was performed on macrodissected lesions. DNA extraction, DNA quantification, library preparation and sequencing were performed with IDT xGen™ Exome Hybridization Panel v2 for library capture. Sequencing data was analyzed with an in-house bioinformatics pipeline for single-nucleotide variants and insertions/deletions. RESULTS: All neonates were delivered at term and had birth weights ranging from 11th-35th percentile for gestational age. All mothers presented with hypertensive disorder during pregnancy. Chorangiomas ranged from 0.7 cm to 5.1 cm and were well-circumscribed near the fetal surface. Case 1 showed a background of chorangiosis and acute subchorionitis, while case 2 had foci of chronic lymphocytic villitis. Whole exome sequencing did not reveal any significant pathologic variants. DISCUSSIONS: The absence of molecular alteration in placental chorangioma is likely indicative of the reactive/non-neoplastic nature of this lesion. The presence of compromised blood flow in the form of hypertensive disorders in our cases may be one of its underlying pathophysiologic mechanisms.

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.

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.

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.