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Background: CDCP1 has been associated with reverse remodeling in human dilated cardiomyopathy (DCM) mediating its effect by reducing cardiac fibrosis which has been demonstrated in-vitro using human cardiac fibroblasts (CF) and in-vivo histologically in mice. However, the role of and the molecular mechanisms by which CDCP1 attenuates cardiac fibrosis in-vivo is unknown. Methods: To characterize the transcriptomic profiles of CDCP1 in cardiac fibrosis, Cdcp1 KO FVB/NJ mice were generated, and implanted with osmotic minipumps containing angiotensin II and phenylephrine (Ang II/PE) or saline at age of 10 weeks. There were 4 experimental groups (6 mice each), Saline_WT, Saline_KO, AngII/PE_WT, and AngII/PE_KO. After 4 weeks of AngII/PE induction, mice were euthanized, RNA was extracted from their heart tissue followed by RNA-seq to explore transcriptomic profiles. Fibrosis was histologically determined using picosirius (PSR) staining and was quantified by percentage of fibrosis area. Results: Histological analysis demonstrated that Cdcp1 KO attenuated severe cardiac fibrosis by 32.7% determined by quantification of percentage of fibrosis area. When comparing the transcriptome-wide gene expression in WT mice hearts from Saline to AngII/PE induction, a total of 316 differentially expressed genes (DEGs) were identified ( Fig. 1A ). Expression of Nppb (encodes ProBNP), a marker for heart failure, was significantly upregulated. The most significantly upregulated DEG after AngII/PE induction was Crlf1 ( Fig. 1A ), a gene predominantly expressed in CF, consistent with previous findings. We next compared transcriptomic profiles of AngII/PE induction WT and Cdcp1 KO mice hearts, to explore the role of CDCP1 in cardiac fibrosis. A total of 163 DEGs were identified ( Fig. 1B ). Notably, Cdcp1 -mediated DEGs enriched GO Pathways associated with extracellular matrix organization (Biological Process) and collagen-containing extracellular matrix (Cellular Component) ( Figs. 1C, 1D ), pivotal processes in cardiac fibrosis. In addition, Cdcp1 KO resulted in several “top” DEGs (including Rnase1, Ccl7, Ccl12 Il21r , and Il6 ) which have known function in inflammation, suggesting that CDCP1 might attenuate cardiac fibrosis through immune regulation . Conclusion: Our findings underscore the pivotal role of CDCP1 in modulating cardiac fibrosis in-vivo, potentially through immune regulation. Targeting CDCP1 may offer promising therapeutic avenues for mitigating myocardial fibrosis.

Abstract Despite the growing evidence supporting the existence of CNS involvement in acute and chronic graft-versus-host disease (CNS-GvHD), the characteristics and course of the disease are still largely unknown. In this multicentre retrospective study, we analysed the clinical, biological, radiological and histopathological characteristics, as well as the clinical course of 66 patients diagnosed with possible CNS-GvHD (pCNS-GvHD), selected by predetermined diagnostic criteria. Results were then contrasted depending on whether pCNS-GvHD onset occurred before or after Day 100 following allogeneic haematopoietic stem cell transplantation (allo-HSCT). The median time between allo-HSCT and pCNS-GvHD onset was 149 days (interquartile range25–75 48–321), and pCNS-GvHD onset occurred before Day 100 following transplantation in 44% of patients. The most frequent findings at presentation were cognitive impairment (41%), paresis (21%), altered consciousness (20%), sensory impairment (18%) and headache (15%). Clinical presentation did not significantly differ between patients with pCNS-GvHD occurring before or after Day 100 following transplantation. Brain MRI found abnormalities compatible with the clinical picture in 57% of patients, while CT detected abnormalities in only 7%. Seven patients had documented spinal cord MRI abnormalities, all of them with pCNS-GvHD occurring after Day 100 following transplantation. In the CSF, the white blood cell count was increased in 56% of the population (median 18 cells/μl). Histopathological analyses were performed on 12 specimens and were suggestive of pCNS-GvHD in 10. All compatible specimens showed parenchymal and perivascular infiltration by CD3+ and CD163+ cells. Immunosuppressive therapy was prescribed in 97% of patients, achieving complete clinical response in 27%, partial improvement in 47% and stable disease in 6%. Response to immunosuppressive therapy did not differ significantly between patients with pCNS-GvHD occurring before or after Day 100 following transplantation. Clinical relapse was observed in 31% of patients who initially responded to treatment. One-year overall survival following pCNS-GvHD onset was 41%. Onset before Day 100 following haematopoietic stem cell transplantation [hazard ratio with 95% confidence interval: 2.1 (1.0–4.5); P = 0.041] and altered consciousness at initial presentation [3.0 (1.3–6.7); P = 0.0077] were associated with a reduced 1-year overall survival probability. Among surviving patients, 61% had neurological sequelae. This study supports that immune-mediated CNS manifestations may occur following allo-HSCT. These can be associated with both acute and chronic GvHD and carry a grim prognosis. The clinical presentation as well as the radiological and biological findings appear variable.

Abstract Background Few antiviral therapies have been studied in patients with coronavirus disease 2019 (COVID-19) and kidney impairment. Herein, the efficacy, safety, and pharmacokinetics of remdesivir, its metabolites, and sulfobutylether-β-cyclodextrin excipient were evaluated in hospitalized patients with COVID-19 and severe kidney impairment. Methods In REDPINE, a phase 3, randomized, double-blind, placebo-controlled study, participants aged ≥12 years hospitalized for COVID-19 pneumonia with acute kidney injury, chronic kidney disease, or kidney failure were randomized 2:1 to receive intravenous remdesivir (200 mg on day 1; 100 mg daily up to day 5) or placebo (enrollment from March 2021 to March 2022). The primary efficacy end point was the composite of the all-cause mortality rate or invasive mechanical ventilation rate through day 29. Safety was evaluated through day 60. Results Although enrollment concluded early, 243 participants were enrolled and treated (remdesivir, n = 163; placebo, n = 80). At baseline, 90 participants (37.0%) had acute kidney injury (remdesivir, n = 60; placebo, n = 30), 64 (26.3%) had chronic kidney disease (remdesivir, n = 44; placebo, n = 20), and 89 (36.6%) had kidney failure (remdesivir, n = 59; placebo, n = 30); and 31 (12.8%) were vaccinated against COVID-19. Composite all-cause mortality or invasive mechanical ventilation rates through day 29 were 29.4% and 32.5% in the remdesivir and placebo group, respectively (P = .61). Treatment-emergent adverse events were reported in 80.4% for remdesivir versus 77.5% for placebo, and serious adverse events in 50.3% versus 50.0%, respectively. Pharmacokinetic plasma exposure to remdesivir was not affected by kidney function. Conclusions Although the study was underpowered, no significant difference in efficacy was observed between treatment groups. REDPINE demonstrated that remdesivir is safe in patients with COVID-19 and severe kidney impairment. Clinical Trials Registration EudraCT 2020-005416-22; Clinical Trials.gov NCT04745351.

Abstract Use of surrogates as primary end points is commonplace in hematology/oncology clinical trials. As opposed to prognostic markers, surrogates are end points that can be measured early and yet can still capture the full effect of treatment, because it would be captured by the true outcome (eg, overall survival). We discuss the level of evidence of the most commonly used end points in hematology and share recommendations on how to apply and evaluate surrogate end points in research and clinical practice. Based on the statistical literature, this clinician-friendly review intends to build a bridge between clinicians and surrogacy specialists.

Abstract Among 281 patients with essential thrombocythemia and calreticulin (CALR) mutation, we found a variant allele frequency of ≥60% to be associated with significantly shortened myelofibrosis-free survival, mostly apparent with CALR type-1 and CALR type-indeterminate mutations.

ImportancePostoperative radiation therapy for close surgical margins in low- to intermediate-grade salivary carcinomas lacks multi-institutional supportive evidence.ObjectiveTo evaluate the oncologic outcomes for low- and intermediate-grade salivary carcinomas with close and positive margins.Design, Setting, and ParticipantsThe American Head and Neck Society Salivary Gland Section conducted a retrospective cohort study from 2010 to 2019 at 41 centers. Margins were classified as R0 (negative), R1 (microscopically positive), or R2 (macroscopically positive). R0 margins were subclassified into clear (>1 mm) or close (≤1 mm). Data analysis was performed from June to October 2023.Main Outcomes and MeasuresMain outcomes were risk factors for local recurrence.ResultsA total of 865 patients (median [IQR] age at surgery, 56 [43-66] years; 553 female individuals [64%] and 312 male individuals [36%]) were included. Of these, 801 (93%) had parotid carcinoma and 64 (7%) had submandibular gland carcinoma, and 748 (86%) had low-grade tumors and 117 (14%) had intermediate-grade tumors, with the following surgical margins: R0 in 673 (78%), R1 in 168 (19%), and R2 in 24 (3%). Close margins were found in 395 of 499 patients with R0 margins (79%), for whom margin distances were measured. A total of 305 patients (35%) underwent postoperative radiation therapy. Of all 865 patients, 35 (4%) had local recurrence with a median (IQR) follow-up of 35.3 (13.9-59.1) months. In patients with close margins as the sole risk factor for recurrence, the local recurrence rates were similar between those who underwent postoperative radiation therapy (0 of 46) or observation (4 of 165 [2%]). Patients with clear margins (n = 104) had no recurrences. The local recurrence rate in patients with R1 or R2 margins was better in those irradiated (2 of 128 [2%]) compared to observed (13 of 64 [20%]) (hazard ratio [HR], 0.05; 95% CI, 0.01-0.24). Multivariable analysis for local recurrence found the following independent factors: age at diagnosis (HR for a 10-year increase in age, 1.33; 95% CI, 1.06-1.67), R1 vs R0 (HR, 5.21; 95% CI, 2.58-10.54), lymphovascular invasion (HR, 4.47; 95% CI, 1.43-13.99), and postoperative radiation therapy (HR, 0.10; 95% CI, 0.04-0.29). The 3-year local recurrence-free survivals for the study population were 96% vs 97% in the close margin group.Conclusions and RelevanceIn this cohort study of patients with low- and intermediate-grade major salivary gland carcinoma, postoperative radiation therapy for positive margins was associated with decreased risk of local recurrence. In isolation from other risk factors for local recurrence, select patients with close surgical margins (≤1 mm) may safely be considered for observation.

MB and VA equal contributors AB and STO co-corresponding authors This study was initiated following evaluation of a 32 year-old woman who presented with a history of thrombocytopenia identified in childhood who subsequently developed features of myelofibrosis (MF). A bone marrow biopsy demonstrated hypercellularity in conjunction with megakaryocyte hyperplasia and marked reticulin fibrosis. Molecular testing for JAK2, CALR, and MPL mutations was negative. Given the unusual association between congenital thrombocytopenia and MF in this patient, exome sequencing was performed, revealing a heterozygous R304W mutation in the coiled coil (CC) domain of STIM1. Activating mutations in the CC and EF hand domains of STIM1 have been associated with Stormorken syndrome, a rare congenital platelet disorder associated with abnormal store operated calcium entry (SOCE). We subsequently identified a second patient with MF associated with a STIM1 activating mutation. This individual was found to have severe thrombocytopenia at birth, and exome sequencing revealed a heterozygous S88G mutation in the EF hand of STIM1. A bone marrow biopsy obtained at 6 months of age revealed atypical megakaryocytes and grade 1-2 MF. A repeat biopsy at age 2 showed persistence of stable MF. The unusual finding of MF in these two patients suggested the possibility of altered calcium signaling as a shared mechanism driving congenital platelet disorders such as Stormorken syndrome and myeloproliferative neoplasms (MPNs) including MF. In support of this notion, we identified elevated STIM1 expression in MF vs normal megakaryocyte progenitors, as well as in platelets from patients with essential thrombocythemia (ET) vs healthy controls. Additionally, we found that STIM1 expression was significantly elevated in CD34+ hematopoietic stem/progenitor cells (HSPCs) from both ET and MF patients vs healthy controls. Notably, STIM1 expression was increased in both JAK2 and CALR-mutant MF patients. Collectively, these findings provide evidence of aberrant STIM1 expression in MPN patient cells. To determine the functional role of STIM1 in MPN disease development, colony assays and patient-derived xenograft (PDX) experiments were performed with MF patient CD34+ cells subjected to CRISPR ablation of STIM1. Strikingly discordant results were observed with JAK2 vs CALR-mutant patient samples. Abrogation of STIM1 in CALR-mutant CD34+ cells led to decreased colony formation, and NSGS mice engrafted in parallel with STIM1-targeted cells exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. These findings suggest an important role for STIM1 in CALR-mutant MPN disease phenotypes. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to increased colony formation and exacerbated disease phenotypes in vivo as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Similar results were obtained in experiments utilizing pharmacologic inhibitors of SOCE activity. Taken together, these findings indicate that the consequences of aberrant STIM1 activity may be context-dependent relating to specific MPN driver mutations. To expand these observations, we identified a separate cohort of 9 family members in Italy with Stormorken syndrome and confirmed STIM1 EF hand mutations. In ex vivo megakaryocytic differentiation assays, cells from affected individuals exhibited a defect in proplatelet formation. These observations were corroborated by initial analyses of a newly generated Stim1 R304W conditional knock-in mouse which recapitulated the characteristic thrombocytopenia found in patients with Stormorken syndrome. In summary, this study represents the first demonstration of MF development in patients with Stormorken syndrome, thereby uncovering a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying Stormorken syndrome and MPNs. Our findings suggest distinct mechanisms relating to the interaction between JAK2 vs CALR mutation and altered STIM1 activity. Further studies of these relationships may have important ramifications for potential therapeutic approaches targeting these pathways.

Background. A mutation in calreticulin ( CALRm) is found in 30-35% of patients (pts) with Essential Thrombocythemia (ET). There are 2 main CALRm mutation types, Type 1/Type 1 like (T1) a 52bp deletion, and Type 2/Type 2 like (T2) a 5bp insertion. Other atypical mutations (T3) occur in a minority of pts. Recent study ( Guglielmelli P et al, BCJ 2023) in pts with myelofibrosis (MF) showed that higher CALR variant allele frequency (VAF) was associated with anemia at diagnosis and during FU, and to features of more advanced disease (CD34 + cell counts, ASXL1 mutation (mut), >2 mutated myeloid genes, and shorter leukocytosis-free survival. Aim. To evaluate whether the CALRm VAF in pts with ET was associated with predefined major clinical outcomes: evolution to MF, transformation to acute leukemia (AML), thrombosis, major bleeding, and survival (OS). Patients and Methods. Diagnosis of ET was strictly according to 2023 WHO and ICC to avoid mis-inclusion of prefibrotic MF. CALRm VAF was determined by capillary gel electrophoresis as the ratio (%) of areas under the curve of CALRm/ CALRm+ CALRwt. A panel of 45 myeloid neoplasm-associated genes was sequenced by NGS. Results. A total of 281 CALRm ET pts were identified from CRIMM (Florence, I), Quebec MPN Research Group centers' (Canada) and Mayo Clinic (Rochester, US) databases; 152 (54%), 101 (36%) and 28 (10%) were T1, T2 and T3 CALRm, respectively. Overall FU was 8.6y (0.3-39.4), median survival was not reached. Rate of death at 10y, 20y and 30y was 8%, 15% and 25%. MF transformation occurred in 50 pts (18%), AML in 2 (0.7%), 25 pts (8.9%) died. A major thrombotic event before or at ET diagnosis occurred in 17 (6.0 %) pts (14 arterial and 3 venous), whereas 28 pts (10.0%) had >1 major thrombosis during follow-up (53.6% arterial, 46.4% venous). Major bleeding occurred in 19 pts (6.8%; 16 at or before diagnosis). Ascertainment of correlation of CALRm VAF with predefined outcomes by continuous variable analysis (ROC curve) highlighted a significant correlation of VAF >60% with MF-free survival (MFS), with HR of 2.85 (95%CI, 1.4-5.6; P= .002. Fig. 1A). On the contrary, there was no impact of CALRm VAF as continuous variable on AML, thrombosis, bleeding and OS. We therefore used a threshold of >60% to compare main clinical and hematologic characteristics at diagnosis and outcomes during FU in pts categorized as CALR-high and CALR-low. Of all pts, 21 (7.5%) harbored a VAF >60%; of these, 52%, 19% and 29% were T1, T2, T3, respectively, compared to 54%, 37%, 8% of CALR-low (P= .008). We found no difference in age, gender, constitutional symptoms, splenomegaly, arterial and venous thrombosis at diagnosis and FU, IPSET revised risk stratification, and bleeding events. Hemoglobin was lower (12.8 g/dL (8.4-14.0) versus 13.6 (10.1-16.0); P=.02), while leukocytes and platelets did not differ. The proportion of pts with additional mutations in myeloid genes was significantly greater in CALR-high pts compared to CALR-low (66.7% vs 30.2%; P=0.01), as it was the proportion of pts with >2 mut myeloid genes (25% vs 11.6%, P=0.04). During FU, 52% of CALR-high pts (n=11) transformed to MF compared to 15% (n=39) of CALR-low (P<.0001); AML and death not differ. Anemia (<10g/dL) during the FU developed in 69% of CALR-high pts vs 18%, leukocytosis (>15x10 9/L) in 46% vs 8% and splenomegaly (greater than 5 cm from LCM) in 50% vs 12% (P<.0001 for all). The HR for anemia-free survival was 2.87 (95%CI, 1.3-6.3), for leukocytosis HR 3.71 (95%CI, 1.3-10.5) and splenomegaly HR 3.40 (95%CI, 1.2-9.2), all P<.01. MFS was significantly shorter in T1 (HR 2.0; P=0.04) or T3 (HR 2.7; P=0.03) using T2 as reference. Finally, we compared C ALR-mut, JAK2V617F mut and triple-negative (TN) ET pts. ROC analysis indicated a JAK2VAF >35% ( JAK2-high) as the best cutoff for shorter MFS. As in Fig. 1B, the MFS survival of CALR-high and JAK2-high was superimposable, and significantly shorter that in CALR/JAK2-low and TN pts. Conclusions. A CALR VAF >60% in pts with ET is associated with greater risk and shorter time to MF progression, development of anemia and splenomegaly. Similar findings in JAK2-high pts reinforce that that accumulation of mutated CALR and JAK2 alleles, evident since diagnosis, is detrimental for evolution to post-ET myelofibrosis. Longitudinal studies after diagnosis might provide further information about the kinetics of allele accumulation.

Multiple CD19-directed therapies are available for the treatment of relapsed and refractory (R/R) DLBCL, including CAR T cells, tafasitamab/lenalidomide (TL), and loncastuximab tesirine (lonca-T) (Sermer D., Blood Rev 2023). However, there are few data available on the efficacy of sequential CD19-directed therapies. We performed a retrospective study evaluating all patients with R/R LBCL treated consecutively with TL at 11 institutions from 8/2020 to 8/2022, identifying 178 patients. In this study, we specifically evaluate outcomes in patients who received anti-CD19 therapy prior to or after TL. The primary outcome of interest was progression-free survival (PFS) in TL recipients who previously received CD19-directed CAR T therapy, with overall response rate (ORR), complete response rate (CRR), and overall survival (OS) also evaluated. Outcomes of lonca-T and CAR T after TL therapy were evaluated. Data on CD19 expression was assessed on biopsies after TL; this was determined locally using flow cytometry or immunohistochemistry per institutional protocols. In subgroup analyses, ORR and CRR were compared using Fisher's exact test. PFS and OS were estimated using the Kaplan-Meier method, with differences tested using the log-rank test. Of 178 patients who received TL, 52 had received prior anti-CD19 CAR T therapy, at a median of 7.4 months before TL (Table 1). Median PFS was lower in prior CAR T recipients (1.6 mo, 95% CI 1.1-2.5) vs no prior CAR T (2.1 mo, 95% CI 1.8-3.2), with hazard ratio 1.46 (p = 0.04; Figure 1). Best ORR was lower in patients with prior CAR T exposure (15% vs 33%, p = 0.02) though CRR was not significantly different (15% vs 18%, p = 1.0). We specifically evaluated TL outcomes based on prior response to CAR T, given the previously identified association of refractory disease with poor TL outcomes (Qualls D et al., presented at ASH 2022). Of 19 patients who had progressive disease more than 6 months after CAR T, ORR and CRR to TL therapy were 31.6% (all responses were CRs); in 33 patients with refractory disease (less than CR or relapse within 6 months) after CAR T, ORR and CRR to TL were significantly lower, at 6.1% (p = 0.04). Median PFS after TL was shorter in CAR-refractory than CAR-relapsed disease, at 1.4 vs 3.5 months (HR 2.2, p = 0.02). Of 88 patients who received therapy after TL, 20 (23%) received lonca-T and 14 (16%) received CAR T. Data on survival and initiation of new treatment after CAR T or lonca-T was available, but response and progression data after CAR T or lonca-T therapy is pending. These data allowed an estimate of OS and event-free survival (EFS), defined as survival without initiation of a new treatment after CAR T or lonca-T. With a median 3.9 months' follow-up, median EFS after CAR T administration was 3.7 months (95% CI, 0.9 - 6.5), and median OS was 8.1 months (95% CI, 0.6 - 15.7). In patients who received lonca-T, with median 2.7 months' follow-up from initiation, median EFS was 2.8 months (95% CI, 0.9 - 4.7), and median OS was 3.5 months (95% CI, 1.2 - 5.7). When comparing CAR T and lonca-T to polatuzumab vedotin- or chemotherapy-based therapies after TL, there were no significant differences in OS or EFS. In patients who discontinued TL, 24 had a biopsy a median of 18 days after the last dose of tafasitamab. Of these, 13 (54%) were CD19-positive and 11 (46%) were CD19-negative. All samples obtained more than 21 days after tafasitamab (6/6) were CD19-positive, while 46% obtained within 21 days were CD19-positive. These time-dependent findings may reflect “epitope masking” by tafasitamab rather than CD19 loss, reflecting other recent reports (Fitzgerald K., Leuk Lym 2022; Duell J., Leuk Lym 2022). In patients receiving TL after CAR T, responses and PFS were lower than in patients without prior CAR T therapy; however, with disease that was not refractory to prior CAR T, results were similar to patients who had not had prior CAR T. This may reflect that refractory disease, regardless of prior therapy, is associated with shorter PFS, though the possibility of CD19-mediated mechanisms of resistance remains. Modest EFS and OS were seen in patients receiving CAR T therapy or lonca-T after already receiving TL, though this may also reflect the high-risk disease characteristics of this cohort. Overall, in a high-risk, real-world cohort of TL-treated patients, these data suggest that some patients may benefit from subsequent CD19-directed therapies, but better therapeutic options are needed.