The human leukocyte antigen-G (HLA-G) gene encodes a tolerogenic protein known to promote tumor immune-escape. We investigated HLA-G polymorphisms and soluble molecules (sHLA-G) in 68 chronic myeloid leukemia (CML) patients. Patients with G*01:01:01 or G*01:01:02 allele had higher value of sHLA-G compared to G*01:01:03 (109.2 ± 39.5 vs 39.9 ± 8.8 units/ml; p = 0.03), and showed lower event free survival (EFS) (62.3% vs 90.0%; p = 0.02). The G*01:01:03 allele was associated with higher rates and earlier achievement of deep molecular response (MR)4.5 (100% vs 65%, median of 8 vs 58 months, p = 0.001). HLA-G alleles with higher secretion of sHLA-G seem associated with lower EFS, possibly because of an inhibitory effect on the immune system. Conversely, lower levels of sHLA-G promoted achievement of MR4.5, suggesting increased cooperation with immune system.

Supplemental Tables 1-4. Supplemental table S1: Primers used in RT-PCR; Supplemental table S2: PCR specificity and efficiency determined using plasmid standards; Supplemental table S3: Endpoint values of univariate analysis for event-free and overall survival for GLI1 expression in cohort B (n=290); Supplemental table S4: Gene expression in endothelial cell-enriched AML bone marrow.

Background: Relapsed/refractory (R/R) acute myeloid leukemia (AML) is a largely unmet medical condition with limited treatment options and dismal prognosis. In newly diagnosed AML patients (pts), a modified risk stratification based on the mutational status of four genes (FLT3-ITD, KRAS, NRAS and TP53) indicated distinct benefit from treatment with azacitidine and venetoclax (Döhner et al. ASH 2022). However, the role of venetoclax (VEN) in R/R AML is not well defined yet and predictors of response are unclear. Aims: To identify predictors of response and survival of off-label VEN+non-intensive therapy in pts with R/R AML reported to the venetoclax registry (NCT03662724). Methods: We retrospectively analyzed clinical characteristics and outcomes of R/R AML pts treated with VEN-based non-intensive treatment-regimens between February 2017 and February 2023. Cytogenetic and molecular studies were performed at the time of refractory or relapsed AML by conventional karyotyping and next-generation sequencing. Mutations associated with myeloid leukemias were detected using a custom myeloid sequencing panel, which included 48 genes. VEN was combined with either azacitidine, decitabine or LDAC. The overall response rate (ORR) was defined by complete remission (CR) + CR with incomplete hematologic recovery (CRi) + morphologic leukemia-free state (MLFS). Data cut-off was February 21, 2023. Results: One-hundred-sixty-three pts with either relapsed (n=67, 41%) or refractory (n=81, 50%; missing 9%) AML and a median age of 70 years (range, 24-86) were included in our analysis. Prior treatment lines included intensive chemotherapy (61%), HMA treatment (58%) and allogeneic transplantation (33%). ELN2017 risk was intermediate and adverse in 31% and 55% of pts in this high-risk cohort. Forty-five pts (28%) had complex cytogenetics and seventeen pts (10%) had extramedullary disease manifestation. In total, 29 genes were found mutated in ≥5 pts. The overall response rate (ORR) was 44% (n=77), including 38% of pts achieving CR/CRi and 6% MLFS. After a median follow-up of 25.9 months (mo), median overall survival (OS) and median event-free survival (EFS) was 8.4 mo (95% CI, 5.7-11.04) and 4.8 mo (95% CI, 3.6-6), respectively. Clinical predictors of survival included age above the median age (≥70 years vs. <70 years 6.4 vs. 10 mo, p=0.07), HMA pretreatment (pretreatment vs. no pretreatment 6.0 vs. 10.2 mo, p=0.02), extramedullary disease manifestation (present vs. absent 5.0 vs. 9.3 mo, p=0.01). A modified risk model was constructed based on the mutational status of nine genes that were associated with survival. Pts were classified into three risk groups with significantly different OS based on mutated STAG2, BCOR or SF3B1 (favorable risk; median OS 17.5 mo), mutated TP53, any FLT3, CBL, PTPN11 or NF1 mutation (adverse risk; median OS 4.6 mo), and all other pts (intermediate risk; median OS 9.6 mo; p<0.001) (Figure 1). This risk score was also prognostic for EFS and ORR. In multivariate analysis the genetic R/R AML VEN risk model (p<0.001), extramedullary disease (p=0.02), and HMA pretreatment (p=0.01) remained significant predictors of inferior survival. Summary/Conclusion: This real-world analysis comprising a large cohort of R/R AML pts treated with VEN-based non-intensive therapies identifies the presence of extramedullary disease and HMA pretreatment as clinical predictors of inferior survival. Further, our data suggest a novel prognostic risk classification based on the mutational status of nine genes with significant OS, EFS and ORR distinction. The genetic R/R AML VEN risk score requires independent validation. Figure 1Keywords: Real world data, Molecular markers, Venetoclax, Acute myeloid leukemia

Supplementary Figure from Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia

The mortality rate of diabetic patients on dialysis is higher than that of non-diabetic patients. Asymmetric dimethylarginine and inflammation are strong predictors of death in hemodialysis. This study aimed to evaluate asymmetric dimethylarginine and C-reactive protein interaction in predicting mortality in hemodialysis according to the presence or absence of diabetes.Asymmetric dimethylarginine and C-reactive protein were measured in 202 patients in maintenance hemodialysis assembled from 2011 to 2012 and followed for four years. Effect modification of C-reactive protein on the relationship between asymmetric dimethylarginine and all-cause mortality was investigated dividing the population into four categories according to the median of asymmetric dimethylarginine and C-reactive protein.Asymmetric dimethylarginine and C-reactive protein levels were similar between diabetics and non-diabetics. Asymmetric dimethylarginine – median IQR μM – (1.95 1.75–2.54 versus 1.03 0.81–1.55 P = 0.000) differed in non-diabetics with or without evolution to death (HR 2379 CI 1.36–3.68 P = 0.000) and was similar in diabetics without or with evolution to death. Among non-diabetics, the category with higher asymmetric dimethylarginine and C-reactive protein levels exhibited the highest mortality (69.0% P = 0.000). No differences in mortality were seen in diabetics. A joint effect was found between asymmetric dimethylarginine and C-reactive protein, explaining all-cause mortality (HR 15.21 CI 3.50–66.12 P = 0.000).Asymmetric dimethylarginine is an independent predictor of all-cause mortality in non-diabetic patients in hemodialysis. Other risk factors may overlap asymmetric dimethylarginine in people with diabetes. Inflammation dramatically increases the risk of death associated with high plasma asymmetric dimethylarginine in hemodialysis.La tasa de mortalidad de los pacientes diabéticos em diálisis se ha referido que es superior a la de los no diabéticos. La dimetilarginina asimétrica y la inflamación son potentes predictores de muerte en hemodiálisis. Este estudio tuvo como objetivo evaluar la interacción de dimetilarginina asimétrica y proteína C reactiva en la predicción de mortalidad en hemodiálisis según la presencia o ausencia de diabetes.Se midieron dimetilarginina asimétrica y proteína C reactiva en 202 pacientes en hemodiálisis de mantenimiento reclutados entre 2011 a 2012 y seguidos durante cuatro años. Se investigó la modificación del efecto de la proteína C reactiva en la relación entre dimetilarginina asimétrica y la mortalidad por todas las causas dividiendo la población en cuatro categorías según la mediana de dimetilarginina asimétrica y proteína C reactiva.Los niveles de dimetilarginina asimética y proteína C reactiva fueron similares entre diabéticos y no diabéticos. Dimetilarginina asimétrica - mediana IQR μM - (1,95 1,75 - 2,54 versus 1,03 0,81 - 1,55 P = 0,000) difirió en los no diabéticos con o sin evolución a la muerte (OR 2379 IC 1,36 - 3,68 P = 0,000) y fue similar en los diabéticos sin o con evolución a muerte. Entre los no diabéticos, la categoría con niveles más altos de dimetilarginina asimétrica y proteína C reactiva presentó la mayor mortalidad (69,0% P = 0,000). No se observaron diferencias en la mortalidad en los diabéticos. Se encontró un efecto conjunto entre la dimetilarginina asimétrica y la proteína C reactiva, lo que explica la mortalidad por todas las causas (OR 15,21 IC 3,50-66,12 P = 0,000).La dimetilarginina asimétrica es un predictor independiente de mortalidad por todas las causas en pacientes no diabéticos en hemodiálisis. Otros factores de riesgo pueden superponerse a la dimetilarginina asimétrica en personas con diabetes. La inflamación aumenta drásticamente el riesgo de muerte asociado con niveles plasmáticos elevados de dimetilarginina asimétrica en pacientes en hemodiálisis.

The precise diagnostic tests and subsequent prognostic stratification for patients with myelodysplastic syndrome (MDS) are often cumbersome, yet they are the basis of successful therapy. Diverse treatment options are available for these patients; however, the decisions in real-life are often not grounded on the available evidence. Although the International Prognostic Scoring System and revised International Prognostic Scoring System are still driving the medical approach to MDS patients, additional variables must be considered when therapeutic intervention is needed. A rational scheme for first-line therapy is described that allows for the possibility of selecting the optimal individual therapy for MDS patients.

The aim of this randomized clinical trial was to evaluate the impact of all- trans retinoic acid (ATRA) in combination with non-intensive chemotherapy in older unfit patients (> 60 years) with newly diagnosed NPM1 -mutated acute myeloid leukemia. Patients were randomized (1:1) to low-dose chemotherapy with or without open-label ATRA 45 mg/m 2 , days 8–28; the dose of ATRA was reduced to 45 mg/m 2 , days 8–10 and 15 mg/m 2 , days 11–28 after 75 patients due to toxicity. Up to 6 cycles of cytarabine 20 mg/day s.c., bid, days 1–7 and etoposide 100 mg/day, p.o. or i.v., days 1–3 with (ATRA) or without ATRA (CONTROL) were intended. The primary endpoint was overall survival (OS). Between May 2011 and September 2016, 144 patients (median age, 77 years; range, 64–92 years) were randomized (72, CONTROL; 72, ATRA). Baseline characteristics were balanced between the two study arms. The median number of treatment cycles was 2 in ATRA and 2.5 in CONTROL. OS was significantly shorter in the ATRA compared to the CONTROL arm ( p = 0.023; median OS: 5 months versus 9.2 months, 2-years OS rate: 7% versus 10%, respectively). Rates of CR/CRi were not different between treatment arms; infections were more common in ATRA beyond treatment cycle one. The addition of ATRA to low-dose cytarabine plus etoposide in an older, unfit patient population was not beneficial, but rather led to an inferior outcome. The clinical trial is registered at clinicaltrialsregister.eu (EudraCT Number: 2010-023409-37, first posted 14/12/2010).

Background: Donor lymphocyte infusions (DLIs) can be employed for acute myeloid leukemia (AML) patients as therapeutic or prophylactic treatment after allogeneic hematopoietic cell transplantation (alloHCT). Application of DLI after alloHCT may induce graft-versus-host disease (GVHD). Therefore, biomarkers that predict the effect of DLIs may be useful to guide DLI use. Molecular measurable residual disease (MRD) is prognostic for the response to standard chemotherapy as well as for targeted therapies. Aims: To evaluate MRD as a prognostic marker for DLIs after alloHCT. Methods: Patients with AML or myelodysplastic syndrome (MDS) aged ≥18 years undergoing DLI at Hannover Medical School between 1998 and 2018 with available PB and/or BM DNA samples were included. Patients were excluded if they had no detectable mutations at diagnosis, had a second alloHCT in the observed time or if MDS patients had <10% bone marrow blasts at diagnosis. DNA libraries were prepared using the TruSight Myeloid Panel or Nextera Flex for enrichment (Illumina) and sequenced on a MiSeq device. Error-corrected sequencing of patient specific mutations was performed 30 days (Follow Up 30 days, FU30) and 90 days (FU90) after the first DLI. All mutations detected at diagnosis were used for MRD monitoring. Marker positivity was defined as variant allele frequency above the limit of detection as not all patients were in complete remission (CR), in CR with incomplete hematological recovery (CRi) or in morphologic leukemia free state (MLFS). Results: The median time from alloHCT to DLI was 11.3 months (range 3.1 to 74.4 months). Of 78 patients, 24 patients were marker negative and 54 patients were marker positive before DLI (of the marker positive patients 23 were in CR/CRi/MLFS). As expected, marker negative patients before DLI had a significantly better overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) than marker positive patients. From the 54 marker positive patients before DLI, 27 patients were in CR/CRi at FU90, 22 patients did not reach CR/CRi and five patients died before FU90. OS, EFS and RFS were significantly improved in patients that were in CR/CRi at FU90. CR/CRi status at FU90 correlated strongly with remission status before DLI (p<0.001). Next, the prognostic effect of MRD in CR/CRi patients at FU90 (n=27) was analyzed. 18 patients reached MRD negativity at FU90 and nine patients stayed MRD positive. Baseline characteristics such as patient sex, cytogenetic risk group, age at diagnosis, the median time between alloHCT and DLI or use of prophylactic vs. therapeutic DLIs were similar between MRD negative and positive patients. Treatment intensity until FU90 was similar between the two groups. Remission status before DLI was not associated with MRD negativity at FU90 (p=0.782). There was no significant difference in OS (p=0.593), EFS (p=0.229) and RFS (p=0.226) between FU90 MRD negative and positive patients. Exclusion of DTA (DNMT3A, TET2, ASXL1) mutations as MRD markers affected the number of patients in CR/CRi at FU90 (20 patients MRD negative and five patients MRD positive), while patient characteristics and outcome did not differ between the two groups. Summary/Conclusion: Patients that achieve CR/CRi until FU90 after DLIs have a significantly better outcome than the remaining patients. However, the MRD status in patients in CR/CRi at FU90 had no prognostic effect in the patient cohort investigated here.

Background: Most of myelofibrosis (MF) cases harbor somatic mutations in the driver genes JAK2, CALR, or MPL. Other somatic nondriver mutations have been increasingly detected with the use of high-throughput sequencing. The role of TP53 in prognosis of MF has long been unclear, but recent first studies suggested worse survival for patients harboring this mutation. Aims: While allogeneic stem cell transplantation still remains the only curative treatment option, outcome of patients with TP53 has not been evaluated yet. Methods: Here, we analysed in 417 patients with PMF or post ET/PV MF incidence and impact on otcome of TP53. We detected 46 patients with detectable TP53 mutation using next-generation sequencing, and compared characteristics and outcome after allograft with 371 non-TP53 patients with primary or secondary MF. Samples were collected at time of transplantation. Primary end points were overall survival (OS) and nonrelapse mortality. Secondary objectives were relapse, relapse-free survival, and graft-versus-host disease (GVHD). Results: Median follow-up from transplantation was 6.1 years for the TP53 group and 7.0 years for the non-TP53 group, respectively (P=0.50). Baseline characteristics were generally balanced, whereas more patients in the TP53 group had Karnofsky performance score <90 (57% versus 40%; P=0.04). Median OS was 2.1 years (0.1-5.4 years) for the TP53 group versus 15.4 years (11.5-19.3 years) for the non-TP53 group (P=0.002), and the 6-year OS was 40% (24-55%) for the TP53 group versus 65% (60-70%) for the non-TP53 group. 6-year nonrelapse mortality was 35% (21-49%) for the TP53 group versus 27% (22-32%) for the non-TP53 group (P=0.16) and 6-year incidence of relapse was 26% (13-39%) for the TP53 group versus 18% (14-21%) for the non-TP53 group (P=0.09). 6-year relapse-free survival was 35% (20-50%) versus 53% (48-58%; P=0.01). Acute GVHD occurred in 63% versus 57% (P=0.43). Of patients with relapse, 92% in the TP53 group versus 63% in the non-TP53 group died (P=0.05). Risk for death appeared to be associated with variant allele frequency (P=0.06). Summary/Conclusion: MF patients with TP53 appeared to have higher nonrelapse mortality and relapse incidence but significantly worse relapse-free and overall survival, mainly because of worse survival after relapse.

Background: Mutations of CEBPA (CEBPAmut) are present in ~5-10% of newly diagnosed adult acute myeloid leukemia (AML), and approximately half of the patients (pts) exhibit biallelic mutations (CEBPAbi). CEBPAbi defines a distinct entity within the 2016 WHO classification and is categorized as favorable in the 2017 risk stratification by the European LeukemiaNet. CEBPAmut can be divided into basic leucine zipper domain (bZIP) or transcription activation domains (TAD) mutations, respectively. Recent studies have demonstrated CEBPAbZIP mutations, in particular in-frame mutations (CEBPAbZIP-inf), to be associated with favorable outcome, regardless of mono- or biallelic status. Aims: To evaluate the prognostic impact of CEBPAbZIP, in particular CEBPAbZIP-inf mutations in AML. Methods: Investigating a cohort of 454 intensively treated CEBPA mutated AML pts entered into the AMLSG BiO Registry study (NCT01252485). Results: Of the 454 pts, 223 had CEBPAbi and 231 monoallelic CEBPA mutations (CEBPAsm) affecting bZIP in 78 pts or TAD in 153 pts. Genotypes differed significantly with regard to clinical and genetic features: CEBPAbi pts were younger than CEBPAsmbZIP and CEBPAsmTAD (median age in yrs: 52 vs 59 vs 60; P<.001), had a higher rate of de novo AML (97% vs 81% vs 84%; P<.001), lower platelet counts (median G/l 36 vs 57 vs 52; P<.001), higher peripheral blood (PB) blast counts (median 71% vs 60% vs 44%; P<.001), and showed an inverse correlation with FLT3 internal tandem duplication (FLT3-ITD) and NPM1 mutation (NPM1mut) (6% vs 21% vs 35% and 0% vs 26% vs 50%; P<.001 each). Outcome analysis revealed a significant improved overall (OS) and event-free survival (EFS) for CEBPAbi with no difference between CEBPAsmbZIP and CEBPAsmTAD (5-year OS: 62% vs 42% vs 51%; P<.001, 5-year EFS: 47% vs 27% vs 43%; P<.022). Subgroup specific analysis within CEBPAsmbZIP revealed an improved outcome for CEBPAbZIP-inf pts (n=46) (5-year OS: 52% vs 29%; P=.001, and 5-year EFS: 34% vs 18%; P=.018). To further address the impact of CEBPAbZIP-inf, pts were categorized as CEBPAbZIP-inf (n=250), irrespective of the allelic status, vs all others (CEBPAother) (n=204). CEBPAbZIP-inf pts were younger (median age in yrs: 52 vs 62; P<.001), had a higher rate of de novo AML (97% vs 81%; P<.001), lower platelet counts (median G/l 36 vs 53; P<.001), higher white blood cell (WBC) (median G/l 25.4 vs 16.1; P=.029), and higher PB blast counts (median 72% vs 46%; P<.001); FLT3-ITD and NPM1mut were less common in CEBPAbZIP-inf pts (9% vs 30% and 3% vs 44%; P<.001 each). CEBPAbZIP-inf exhibited a significant improved OS (5-year OS: 60% vs 48%; P<.001) and EFS (5-year EFS: 47% vs 37%; P=.007); in multivariate Cox models for OS and EFS, including allogeneic hematopoietic cell transplantation (HCT) in first complete remission as time-dependent covariate, age (HR: 1.50; P<.001), WBC (HR: 1.40; P=.018), and adverse cytogenetics (HR: 2.33; P=.010) were unfavorable factors for OS, whereas NPM1mut (HR: 0.63; P=.040), HCT (HR: 0.46; P=.010), and CEBPAbZIP-inf (HR: 0.59; P=.007) revealed as favorable. For EFS, age (HR: 1.19; P=.002), WBC (HR: 1.30; P=.028), and FLT3-ITD (HR: 1.83; P=.003) were unfavorable, whereas NPM1mut (HR: 0.65; P=.028) and HCT (HR: 0.33; P<.001) were favorable. Summary/Conclusion: In this cohort of 454 CEBPA mutated adult AML pts, CEBPAbZIP-inf was associated with specific clinical and genetic characteristics. Furthermore, CEBPAbZIP-inf pts had a significantly superior outcome irrespective of the allelic status. This study confirms recent findings suggesting a prognostic role of this mutation type. FGR and AC contributed equally

Background: Acute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and erythroid/myeloid (EML) phenotypes based on the degree of erythroid hyperplasia. Despite an intensive mutational analysis, the mechanism of erythroid hyperplasia in AEL is still poorly understood and so are feasible therapeutic targets. Aims: To understand the mechanism of erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL. Methods: We analyzed a total of 124 adult AEL cases, where whole genome/exome sequencing of 35 cases were followed by targeted-capture sequencing in all cases. RNA sequencing was also performed in 23 cases. The mutational profile of AEL cases was compared to that of 409 cases with non-erythroid AML (non-AEL). Patient-derived xenograft (PDX) mouse models of AEL with JAK2 and/or EPOR amplification were established from 6 AEL patients with these abnormalities. These models were tested for their response to JAK1/2 inhibitor. Results: In accordance with a recent report, AEL cases were classified into 4 genomic groups (A-D), which are characterized by biallelic TP53 mutations and complex karyotype (Group-A), mutated NPM1 (Group-B) and STAG2 (Group-C), and other mutations in histone modifiers and transcription factors (Group-D). In particular, all but one PEL cases belonged to Group-A. Also found in non-AEL cases, these group-defining lesions in AEL were uniquely associated with focal gains/amplifications of EPOR, JAK2, and/or ERG/ETS2 loci (Group-A), PTPN11 mutations (Group-B), and KMT2A-PTD (Group-C), which might be responsible for the AEL phenotype. Highly enriched in PEL cases (7/13), EPOR/JAK2 focal gains/amplifications were implicated in their extreme erythroid hyperplasia and associated with particularly poor prognosis, even compared to other Group-A cases, who had shorter survival than those in other groups. As expected, JAK2/EPOR-amplified cases showed upregulated STAT5 signaling compared to non-AEL cases, which however, was also observed in other AEL cases, suggesting that upregulated STAT5 signaling is a hallmark of AEL. Based on these findings, we tested the effect of JAK2 inhibition on cell growth of EPOR/JAK2-amplified AEL cells in in vitro culture and xenograft model. Of interest, ruxolitinib-mediated JAK2 inhibition resulted in significantly suppressed in vitro cell growth of EPOR/JAK2-amplified AEL cells and prolonged overall survival in 4 PDX models with phospho-STAT5 downregulation, although other 2 models were resistant to JAK2 inhibition with persistent STAT5 activation. Summary/Conclusion: AEL is a heterogenous subgroups of AML characterized in common by upregulated JAK/STAT5 signaling. Gains/amplifications of JAK2/EPOR are frequent in TP53-mutated cases, particularly those with the PEL phenotype, and could be exploited as potential therapeutic targets using JAK2 inhibitors.