Citations

ABSTRACT Imaging mass spectrometry has made significant advancements in recent years owing to its ability to study the spatial localization and abundance of proteins, peptides, lipids, glycans, metabolites, and drugs. In this study, we employed an imaging mass spectrometry‐based workflow called MALDI‐IHC, which uses antibodies conjugated with photocleavable mass‐tags, to investigate the detection of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) along with pan‐cytokeratin in formalin‐fixed paraffin‐embedded (FFPE) sections from cases with breast cancer. The expression of ER, PR, and HER2 was consistent with the clinical diagnosis based on conventional immunohistochemistry, lacking signal in triple‐negative cases and concordant signals in the cases that were classified as ER+/PR+/HER2+, ER+/PR+/HER2−, and ER−/PR−/HER2+ groups, respectively. In addition, the potential utility of a seconday antibody‐based MALDI‐IHC approach for HER2 expression was evaluated. In agreement with immunohistochemistry results, HER2 expression co‐localized within tumor regions in a HER2+ breast cancer case, while no expression was observed in a HER2− case. Collectively, these results highlight the potential of imaging mass spectrometry‐based MALDI‐IHC workflow for multiplexed detection of proteins in clinically relevant tissue sections.

ABSTRACT Background Isolated chromosome 5/5q losses (–5/5q) without TP53 mutations are associated with favorable outcomes in myeloid neoplasms (MN) with <5% blasts. However, the clinical implication of concurrent −5/5q and TP53 aberrations in MN with ≥5% blasts is poorly understood. Methods Patients with TP53 ‐mutated MN carrying ≥5% blasts assessing the prognostic impact of ‐5/5q on 24‐month overall survival (OS24) were examined. Results Of 587 patients, 515 (88%) exhibited −5/5q overwhelmingly in the context of a complex karyotype (98.3% vs. 61.1% complex karyotype without −5/5q; p < .0001) and multihit TP53 allelic state (88.3% vs. 56.9%; p < .0001). Proportions of patients with blasts ≥20% were comparable between groups with and without −5/5q; p = 0.26. Notably, patients with −5/5q exhibited significantly fewer coalterations; p < .0001. Looking at outcomes, presence of −5/5q was associated with shorter median 24‐month overall survival (7.8 months vs. 11.2 months; p Log‐rank = .012), an effect restricted to subgroups with blasts <20% ( p = .039; N = 163), absent −7/7q ( p = .007; N = 225), or WHO5‐defined single hit allelic state ( p = 0.030; N = 91). Importantly, −5/5q retained independent adverse prognostic significance regardless of TP53 allelic state in a multivariable model. Furthermore, among the subset of 75 (13%) patients undergoing allogeneic stem cell transplantation, −5/5q predicted significantly shorter median 5‐year posttransplant survival (16.2 months vs. median not reached; p Log‐rank = .009). Conclusions These findings emphasize the independent prognostic relevance of chromosome 5/5q losses underscoring the clinical relevance of cytogenetic testing for −5/5q even in this high‐risk cohort.

Key Points Increased protein expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase was observed in old adults compared with young adults. In old adults, overexpressed proteins were enriched in mitochondrial processes while under expressed proteins enriched in RNA metabolism. Validated differentially expressed proteins were not significantly correlated with kidney pathology after adjusting for age. Background Kidney aging is characterized by a loss of glomeruli, predominantly in the superficial cortex, with a resultant decline in GFR and an increased risk of various kidney-related diseases. The early molecular alterations in glomeruli associated with the aging process are not well studied. Methods We combined laser capture microdissection and mass spectrometry–based unbiased proteomic analysis of nonsclerosed, nonischemic glomeruli in the superficial cortex from young and old adults who underwent a radical nephrectomy for a tumor to understand the age-related molecular changes in glomeruli. Twenty-four young and 30 old adults were used for the discovery dataset, and the significant differentially expressed proteins were further validated using an independent set comprising six young and eight old adults. Results Kidneys from old adults had lower eGFR, less kidney parenchyma on computed tomography imaging, and more glomerulosclerosis and arteriosclerosis on histology. Quantitative proteomic analysis of nonsclerosed, nonischemic glomeruli identified increased expression of tissue inhibitor of metalloproteinase-3, glypican 6, synuclein γ , apolipoprotein A-4, and Ecto-5′-nucleotidase in old adults that were further validated in an independent set. Pathway analysis indicated that proteins with increased expression in old adults were enriched in mitochondrial translational processes, aerobic respiration, and TCA cycle, whereas proteins with decreased expression in old adults were enriched in mRNA splicing, mRNA processing, and nonsense mediated decay. Furthermore, validated differentially expressed proteins did not show any significant correlation with the kidney pathology independent of age group. Conclusions Overall, this study identified proteins that are specifically associated with the aging process in otherwise normal-appearing glomeruli.

Importance Sleep disordered breathing (SDB) is a well-established contributor to cardiovascular morbidity, mediated by intermittent hypoxemia, autonomic dysregulation, and endothelial dysfunction. Patients with hypertrophic cardiomyopathy (HCM) may be especially at risk for SDB, but the clinical impact of SDB in this population remains unclear. Objective To define the prevalence and subtypes of SDB in HCM and examine their association with echocardiographic parameters and cardiac biomarker expression. Design, Setting, and Participants A prospective cohort study was conducted between April 18, 2018, and January 15, 2024, at a single tertiary referral center specializing in HCM care. Adults with HCM (left ventricular wall thickness ≥15 mm or pathogenic variants) were recruited from an institutional registry. Patients diagnosed with SDB or current pregnancy were excluded. Patients underwent polysomnography, with comparative assessment of echocardiographic, electrocardiographic, and biomarker indices. Observers were blinded to polysomnographic results. Data analysis was performed from April 11, 2024, to July 25, 2024. Exposures SDB classified via polysomnography using apnea-hypopnea index thresholds, with subtypes including obstructive sleep apnea and central sleep apnea and with event severity, hypoxemia, and sleep architecture disruption quantified. Main Outcomes and Measures Echocardiographic indices, cardiac biomarker expression, functional status, apnea-hypopnea index, and overnight hypoxemia. Results Among 154 patients (median [IQR] age, 60 [48-68] years; 102 [66.2%] male), 91 (59.1%) were diagnosed with SDB. Those with SDB, compared with those without SDB, had higher left ventricular mass index (median [IQR], 128 [107-161] vs 109 [96-134] g/m 2 ; P = .03), E/e′ ratio (median [IQR], 12.5 [10.0-15.0] vs 10.0 [8.3-14.5]; P = .04), and baseline troponin-T level (median [IQR], 0.013 [0.009-0.022] vs 0.011 [0.007-0.015] ng/mL [to convert to micrograms per liter, multiply by 1]; P = .04) and greater overnight troponin-T level increases (change in median [IQR], 0.0021 [−0.0029 to 0.0062] vs 0.0002 [−0.0022 to 0.0026] ng/mL; P = .02). New York Heart Association class II or III symptoms were more common in those with SDB (48 [52.7%] vs 17 [27.0%]; P = .005). Hypertension and diabetes were more prevalent among patients with SDB than without SDB (hypertension: 67 [73.6%] vs 36 [57.1%]; P = .03; diabetes: 14 [15.4%] vs 3 [4.8%]; P = .04), whereas rates of atrial fibrillation and prior myectomy did not differ significantly between groups. Conclusions and Relevance This study suggests that undiagnosed SDB is highly prevalent in patients with HCM and that SDB is associated with adverse myocardial remodeling, greater diastolic dysfunction, and elevated troponin-T levels, indicating subclinical myocardial injury. SDB may contribute to HCM pathophysiology and symptom burden, supporting the rationale for randomized clinical trials to determine the impact of treating SDB on symptoms and clinical outcomes in patients with HCM.

We investigated the prognostic impact of blast counts, TP53 allelic state determinants (number of hits, del(17p), variant allele frequency, complex karyotype),and a novel karyotypic clonal cell fraction (≤50% clonal cells vs >50% clonal cells (termed ‘CK50’)) in 495 individuals with TP53- mutated ( TP53 MUT ) myeloid neoplasms. Outcome examined was 24-month survival (OS24). The cohort (median age 71) included 29% (144/495) myelodysplastic syndromes (MDS)/MDS-acute myeloid leukaemia (AML) (1%–19% blasts) and 71% (351/495) AML (≥20% blasts), with 18% (81/460) having low CK50. Overall, 83% received front-line hypomethylating agents. Higher blast counts (<20% vs ≥20%) were marginally associated with CK50 (p=0.08). In the OS24 analysis, blast count showed a marginal association with OS24 (HR 1.3 (95% CI 1.0 to 1.6); p=0.07), while CK50 predicted significantly inferior outcomes (HR=1.7 (95% CI 1.2 to 2.3); p=0.002). In a multivariable model including all TP53 allelic state determinants, only CK50 and complex karyotype remained relevant for predicting adverse outcomes.

Context.— Morphologic evaluation of peripheral blood smears provides valuable information to diagnose and manage a variety of hematologic disorders. Objective.— To measure the competency of the technical staff in the morphologic evaluation of peripheral blood smears and provide performance trends. Design.— Participating technologists accessed 10 whole slide–imaged peripheral blood smears through a web-based imaging tool in 2 separate studies. Participants performed a 100-cell differential and morphologic evaluation for each slide image. Grading criteria, determined by 3 hematopathologists, were weighted according to their clinical significance (score range, 0–100 for each case). Each institution and participant answered a questionnaire to assess the impact of current practices and educational programs on competency scores. Results.— A total of 776 technologists from 92 institutions participated in study 1 and 1495 technologists from 179 institutions participated in study 2. Median performance scores for institutions were 78.9 and 87.6 for studies 1 and 2, respectively, encompassing a range of hematologic disorders. Based on results of the questionnaire for study 1, higher performance scores were seen when institutions required a specific number of continuing education credits per year through an agency ( P = .005). In study 2, institutions with remediation procedures following a failed competency demonstrated higher performance scores ( P = .03). Conclusions.— Medical technologist competency of peripheral blood smears improves with level of experience and is positively impacted through attending educational programs. Whole slide images offer a convenient means of assessing technical competence and provide data to allow institutions to appropriately focus their procedures and educational efforts.

Introduction: MPAL is a rare and heterogenous subtype of acute leukemia characterized by a single blast population with phenotypic expression of both myeloid and lymphoid lineages (biphenotypic) or multiple co-existing lineages of blasts that satisfy criteria for both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). MPAL presents both a diagnostic and therapeutic challenge given its rarity and resistance to traditional therapies. Data suggest ALL-directed or hybrid therapies are most effective at inducing durable remissions as a bridge to allogeneic stem cell transplantation (Allo-SCT; Lazzarotto 2023, Rasekh 2021); however, many are single-institution with limited sample sizes. Methods: A retrospective analysis at 7 US academic centers was performed. Adult patients (pts) with a confirmed diagnosis of MPAL between 2003-2023 were included. Comparisons of categorical and continuous variables utilized the Fisher exact test and Wilcoxon rank-sum or Kruskal-Wallis test as appropriate. Time-to-event outcomes were calculated using the Kaplan-Meier method and compared using log-rank test. Results: A total of 79 pts with MPAL were included in this study. Median age at diagnosis was 61 years (range, 18-89); 52% were ≥ 60 years. Majority of pts were male (60%), non-Hispanic white (64%), and had an ECOG ≤ 1 (83%). Of the 50 pts with baseline CSF analysis, 5 (10%) had CNS disease. Phenotypically, the predominant blast population was biphenotypic in 68%, myeloid in 17%, and lymphoid in 15% of cases. KMT2A-rearrangement and BCR::ABL1 translocation constituted 4% and 14% of cases, respectively, and 30% of pts had complex cytogenetics. Most common mutations at baseline included RUNX1 (33%), DNMT3A (30%), FLT3-ITD (26%), TET2 (19%), WT1 (19%), NRAS (18%), TP53 (17%), and ASXL1 (14%) mutations. Of the 79 pts, 61 (77%) received intensive chemotherapy (IC), 12 (15%) received HMA-based regimens, 4 (5%) received other non-intensive regimens, and 2 (3%) transitioned to hospice prior to therapy. Among those receiving IC, 37 (61%) received ALL-directed therapies, 16 (26%) AML-directed, and 8 (13%) received hybrid regimens which included FLAG-VIPR (6/8), 7+3+vincristine (1/8), and cladrabine/low-dose cytarabine/vincristine/dexamethasone (1/8). Therapies were phenotype-discordant in 4/61 pts receiving IC; 3 myeloblast-predominant MPAL pts received ALL-directed therapy and one lymphoid-predominant pt received AML-directed therapy. Among 54 pts with predominant biphenotypic blasts, 52% received ALL-directed regimens. Of the 11 pts with BCR::ABL1 translocation, 8 (73%) received dasatinib, 2 (18%) ponatinib, and 1 (9%) received both. Only 30% of pts with FLT3-ITD mutated MPAL (5/17) received a FLT3 inhibitor. Overall, 56% of pts achieved complete remission (CR) or CR with incomplete count recovery (CRi), with a median duration of response of 16.7 months (IQR: 8.1-46.1). The rates of CR/CRi were 39%, 56%, and 75% for myeloid, biphenotypic, and lymphoid MPAL, respectively. All pts with CNS disease (5/5) cleared after induction. Thirty-seven percent of pts proceeded with Allo-SCT in first remission (23% of myeloid pts, 58% lymphoid, 35% biphenotypic). Thirty-two percent of responders (CR/CRi; 14/44) relapsed with a median time to relapse of 9.4 months (range, 0.5-42). Median overall survival (OS) for the entire cohort was 51 months (95% CI: 15-89). The 2-year OS rate was higher in pts with lymphoid-predominant phenotype (83%) compared to biphenotypic (55%) or myeloid-predominant phenotype (38%; p=0.16). Median OS was significantly longer in transplant versus non-SCT recipients (121 vs 14 months) with a 1-year OS rate of 97% vs. 59% (p&lt;0.0001), respectively. Among pts who achieved CR/CRi, median OS significantly longer in transplant versus non-SCT recipients (121 vs 55 months, p=0.031), with a 2-year OS rate of 91% vs 75%. Conclusions In this large multi-center study, there were differences in survival based on predominant blast phenotype, with improved OS in lymphoid-predominant MPAL. Although most pts with biphenotypic-predominant MPAL received ALL-directed regimens, OS was worse in this subtype compared to lymphoid-predominant MPAL. There was significantly improved OS among Allo-SCT recipients underscoring the importance of Allo-SCT in this subtype of acute leukemia. Additional studies are needed to understand this rare and heterogenous disease.

Introduction: TP53 mutations are found in 18% of adult patients (pts) with acute lymphoblastic leukemias (ALL), and portends poor prognosis (Saygin, Blood Cancer Discov 2024). TP53 mutations are often associated with low hypodiploidy and Li Fraumeni syndrome (germline) in children. However, we and others have shown that TP53-mutant (TP53-m) ALL arises from pre-existing TP53-m clonal hematopoiesis in adults as opposed to germline origin. The impact of co-occurring mutations, TP53 allelic frequency (AF) and different types of treatment on outcomes of TP53-m ALL are not fully characterized. We set out to investigate the clinical and molecular predictors of outcomes in adults with TP53-mutant ALL. Methods: We retrospectively studied 107 patients with TP53-m ALL treated at seven different academic centers in North America. Genetic profiling of pre-treatment bone marrow samples was performed with a comprehensive panel covering 147 myeloid and lymphoid genes. We also performed RNA-sequencing to identify fusions that define ALL subtypes. Single nucleotide polymorphism (SNP) array was used to define low hypodiploidy. Results: The median age at diagnosis was 59 years (range, 10-80), 42% were female, 70% White. 89% of pts had B-ALL while 11% had T-lineage ALL. 24% of pts were classified to have therapy-related ALL based on their history of exposure to genotoxic therapy for another cancer. 45% of pts had hypodiploid karyotype, 79% of whom were low-hypodiploid. 7% had hyperdiploid karyotype and 5% had BCR::ABL1. 11 pts had two different TP53 variants. 90% of TP53 variants involved the DNA binding domain. Most frequently observed co-occurring mutations involved CDKN2A (20%), NOTCH1 (12%) and RB1 (10%) genes. 71% of pts were treated with standard cytotoxic chemotherapy, while the remaining pts received newer antibody-based therapies (i.e., inotuzumab, blinatumomab) or venetoclax in first-line setting. 11% of pts received allogeneic hematopoietic cell transplant (HCT) in first complete remission (CR1) while 3% received HCT after relapse (CR2 or CR3). When patients with vs without low hypodiploidy were compared, we did not observe a significant difference in overall survival (OS) as outcomes were poor in both groups (median, 17 vs 19 months, p= 0.89). However, pts who had TP53 AF &gt;0.5 had significantly worse OS when compared to pts with TP53 VAF &lt;0.5 (13 vs 25 months, p= 0.03). Conclusions: TP53-m ALL is a very high-risk disease subtype and its dismal outcome is independent from presence of co-existing low hypodiploidy. Loss of heterozygosity, characterized by TP53 AF &gt;0.5 is associated with even worse survival. New therapies are urgently needed to improve outcomes in this subset.

SummaryTo assess the relevance of co‐occurring somatic mutations in TP53‐mutated myeloid neoplasms with ≥10% blasts, we pooled 325 individuals from 10 centres. We focused on comparing three published somatic co‐alteration signatures comprising (1) nine MDS‐related genes (‘ICC‐MDSR’), (2) ICC‐MDSR + additional secondary mutations‐related genes (‘Tazi signature’) and (3) EPI6 (comprising six genes). Outcomes examined were 24‐month overall survival (OS24) and front‐line complete response (CR1). The median age was 69 years with 77% receiving front‐line hypomethylating agents (HMA). All three signatures ICC‐MDSR (p = 0.009), Tazi signature (p = 0.001) and EPI6 (p = 0.025) predicted inferior CR1. In the low‐intensity (HMA) subgroup, only Tazi signature (p = 0.026) predicted inferior CR1. In OS24 analysis of the HMA‐treated subgroup (N = 200), only Tazi signature was adverse (hazard ratio, HR = 1.6 [1.1–2.2]; p = 0.011). However, a forward stepwise multivariable age‐adjusted Cox model including all three signatures picked EPI6 as the sole significant adverse predictor in the entire cohort (p = 0.0001) as well as within the HMA‐treated subgroup (p = 0.0071). These data confirm the value of testing co‐occurring somatic alterations even within a high‐grade TP53‐mutated myeloid neoplasm cohort.

Background: Sleep disordered breathing (SDB) is frequently observed in patients with cardiovascular disease, and is associated with intermittent hypoxia, sympathetic activation, oxidative stress, and endothelial dysfunction, all of which may facilitate the development and progression of cardiovascular disease. Objectives: We sought to determine the prevalence, subtypes, and clinical implications of comorbid SDB in patients with hypertrophic cardiomyopathy (HCM). Methods: In this prospective observational study, patients with HCM without prior diagnosis of SDB were recruited to undergo inpatient polysomnography between May 1 st 2018 and October 31 st 2022. Electrocardiographic and echocardiographic parameters, cardiac biomarkers, and symptoms were comparatively assessed in patients with and without comorbid SDB. Patients were assessed for the development of major adverse cardiovascular events (MACE) during follow-up (median 29, range 3-57 months). Results Among 154 participants, 91 (59.1%) were diagnosed with SDB of whom 38 (41.8%) had mild, 22 (24.2%) moderate, 18 (19.8%) severe obstructive sleep apnea, and 13 (14.3%) had central sleep apnea. SDB was associated with greater left ventricular mass (median [IQR]) (128 [107, 161] vs. 109 [96, 134] g/m 2 , p =0.034) and E/e’ ratio (12.5 [10, 15] vs. 10.0 [8.3, 14.5], p =0.039). Patients with HCM over the age of 50 years, with body mass index ≥ 30kg/m 2 , who described their sleep as “poor” were at particularly high risk of having comorbid SDB, as were those who were diabetic, hypertensive, or described greater exertional limitation (NYHA Class ≥II 53% vs. 27%, p =0.005). Importantly, subjects with SDB had elevated baseline Troponin T levels (14 ng/L vs 11 ng/L, p =0.030), and further substantial overnight troponin increases following untreated apneic sleep (2.1ng/L vs 0.2ng/L, p =0.009). Finally, patients with comorbid SDB experienced a significantly higher proportion of incident adverse cardiovascular events (MACE hazard ratio 5.3, 95% CI, 1.6, 17.2, p =0.006) than those without SDB. Conclusions: We report a particularly high burden of previously undiagnosed SDB in patients with HCM, particularly in older, heavier, male patients. Undiagnosed SDB in HCM is not benign and is associated with symptom progression, comorbidity, and a poorer prognosis, particularly in the case of central sleep apnea. These important new findings provide rationale for heightened vigilance and routine screening for SDB in patients with HCM.