Unmutated Group Research Articles

Impact of spliceosome mutation on outcomes of myelodysplastic syndrome and chronic myelomonocytic leukemia patients undergoing allogeneic hematopoietic cell transplantation

IntroductionAllogeneic Hematopoietic cell transplantation (allo-HCT) remains the only curative therapy for myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). The impact of spliceosome mutations on allo-HCT outcome is unclear and further understanding is needed to assess the implications of this class of mutations on risk of relapse, overall survival (OS) and non-relapse mortality (NRM) in order to make decision regarding timing of allo-HCT. We examined the allo-HCT outcomes of MDS/CMML patients based on their spliceosome mutation profile to understand the impact of these mutations on transplant outcomes. ObjectiveTo compare outcomes of MDS/CMML patients with and without spliceosome mutations undergoing allo-HCT. MethodsThis is a single institution, retrospective study of MDS/CMML patients who underwent allo-HCT with myeloablative or reduced intensity conditioning (RIC) regimen at City of Hope from January 2016 to December 2021. Among them, patients who underwent molecular mutation profiling by NGS (Next Generation Sequencing) for a set of genes known to be mutated in myeloid neoplasms are included in this analysis. We compared OS, relapse free survival, NRM and acute/chronic graft versus host disease (GVHD) incidence between the spliceosome-mutated and unmutated groups. ResultsWe identified 258 consecutive MDS/CMML patients who underwent allo-HCT. Of these, 126 (48.8 %) patients had molecular profiling done among whom 57 (45.2 %) patients carried a spliceosome mutation. 84.9 % of patients had MDS and 55.6 % underwent a matched unrelated donor transplant. The median age for the whole cohort was 66 years (range 12–77).78.6 % and 73.7 % received RIC in the spliceosome and non-spliceosome groups, respectively. The 2-year OS for the whole cohort was 66.5 % (95 %CI 0.55–0.75) with a day 100 NRM of 7.1 % and 2-year cumulative incidence of relapse of 20 %. Grade II-IV acute GVHD at day 100 was 36.3 % (95 % CI 0.27–0.44) and any chronic GVHD at 2-years was 48.4 % (95 % CI 0.37–0.58). Patients who carried a spliceosome mutation had a significantly better 2-year survival of 83.8 % vs 55.9 % in the non-spliceosome group (P=0.002) and a better PFS of 73.7 % vs 50.0 % (P=0.007). There was no difference in the cumulative incidence of relapse at 2-years 15.9 % vs 18.5 % (P=0.59) between two groups but the spliceosome group had a significantly lower NRM at 2-years 10.4 % vs 31.5 % (P=0.009). There was no difference in incidence of acute or chronic GVHD between the two groups. ConclusionsAmong patients with MDS or CMML who underwent allo-HCT, our study shows better OS for patients who have spliceosome mutations due to lower NRM compared to those carrying non- spliceosome mutations. This favorable outcome of the spliceosome-mutated patients could have implications for timing of allo-HCT, particularly for patients in the intermediate MDS prognostic risk groups.

Chronic Lymphocytic Leukemia (CLL) with Borderline Immunoglobulin Heavy Chain Mutational Status, a Rare Subgroup of CLL with Variable Disease Course.

Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; p = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; p = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.

Clinical characteristics and outcome of PIK3CA mutated hormone receptor positive (HR+), human epidermal growth factor 2 negative (HER2-), advanced breast cancer (BC) treated with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i): Real world data.

e13078 Background: BC is the most common malignant disease and leading cancer-related cause of death in women. Despite significant achievements in the treatment, advanced BC (aBC) is still an incurable disease. HR+/HER2- subtype accounts for 70% of all BC. One of the most common mutations in BC is the PIK3CA gene mutation which can induce endocrine and chemo- resistance. In early BC, patients (pts) with PIK3CA mutations mostly have better, but in aBC worse prognosis. Standard first-line treatment for HR+/HER2- aBC is a combination of CDK4/6i and endocrine therapy. PIK3CA status is most often assessed in this group of pts and combination of alpelisib - PIK3CA inhibitor and fulvestrant is second-line treatment option in PIK3CA mutated pts. Methods: Medical records of HR+/HER2- aBC pts who started CDK4/6i therapy between 01.08.2018. and 01.10.2020. were analyzed with prior University Hospital Center Zagreb Ethics Committee approval. Demographics, tumor characteristics, clinical presentation and therapy information were collected. Molecular analysis of PIK3CA mutations was performed on tumor tissue samples. Time to treatment failure (TTF) was analyzed using the Kaplan-Meier method and the data cut-off was 31.12.2022. Results: Of the 103 pts with determined PIK3CA status, 45 (43.7%) had a detectable mutation. Median follow-up was 35 months. CDK4/6i were first line therapy in 31 (68.9%) pts with and 29 (50%) pts without PIK3CA mutation. Endocrine resistance, de novo metastatic disease and bone marrow infiltration were more common in PIK3CA mutated subgroup. The incidence of visceral metastases was similar in both groups, whereas liver metastases were more common in the unmutated group. Disease progression was observed in 69 (67%) pts, 28 (40.6%) of whom were PIK3CA mutated. Median TTF in mutated subgroup was 24 months (95% CI 21-33) and 25 months in unmutated subgroup (95% CI 19-36). During the follow-up period, 35 (34%) pts died, 14 (40%) of whom were PIK3CA mutated. Odds ratios in pts with a PIK3CA mutation. Conclusions: There was no relevant difference in TTF between two groups. However, PIK3CA mutated subgroup had more aggressive clinical features - more often presented with bone marrow infiltration and de novo aBC, statistically relevant OR 5.158 and 2.323 respectively, which is related to lower quality of life and highlights the importance of treatment improvement for this subgroup of pts. [Table: see text]

Metabolic Biomarkers Affecting Cell Proliferation and Prognosis in Polycythemia Vera

Background Polycythemia vera (PV) is a malignant clonal hematological disease of hematopoietic stem cells characterized by proliferation of peripheral blood cells, and JAK2 mutation is one of the main causes of PV peripheral blood cell proliferation. Abnormal cell metabolism is a new feature of malignant proliferation of tumor cells, but the role of metabolism in the pathogenesis and prognosis of PV remains unclear. Methods Metabolic profiling of peripheral blood sera from 32 patients with PV and 20 HCs was performed using liquid chromatography-mass spectrometry (LC-MS). Partial least-squares with latent structure discriminant analysis (PLS-DA) was used for data analysis. Results To assess whether there were significant changes in the metabolic profiles of PV patients and healthy controls, we performed a multifactorial analysis of the metabolic profiles of the two groups. Figure 1 shows the score plots of the PLS-DA in positive and negative modes. Both data from positive and negative modes suggested that the serum metabolites of PV patients were significantly changed. Subsequently, we identified endogenous metabolites between PV patients and HCs. These potential biomarkers were eventually identified as fatty acids, acylcarnitine, sphingolipids, amino acids, etc. To clarify the abnormal metabolic pathways in PV, we performed enrichment analysis of the metabolic pathways of the biomarkers. The results showed there were 9 important metabolic pathways associated with PV, namely: D-glutamine and D-glutamate metabolism, sphingolipid metabolism, pyruvate metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, tryptophan metabolism, lysine degradation, arginine biosynthesis and glycolysis/gluconeogenesis (Figure 2). Therefore, fatty acid metabolism, glucose metabolism, sphingolipid metabolism and amino acid metabolism were significantly altered in PV. To explore the effect of JAK2-associated metabolic abnormalities on cell proliferation in PV patients, we screened for endogenous metabolites associated with JAK2, and found 7 metabolites that differed significantly between the JAK2 mutated and unmutated groups. The levels of Cer(d18:2/22:6-2OH(7S, 17S)), SM(d18:0/PGF1α), CerP(d18:1/16:0), glutamic acid, lactic acid, melibiose and xylose were significantly higher in the JAK2-mutated group than in the JAK2-unmutated group. Among them, Cer(d18:2/22:6-2OH(7S, 17S)) (r=0.817; P<0.001) and SM(d18:0/PGF1α) (r=0.463; P=0.021) levels were positively correlated with JAK2 mutational burden. Then we evaluated the relationship between JAK2 mutation and blood cell count in PV patients. PLT and HCT levels were significantly higher in JAK2 mutated PV patients than in JAK2 unmutated PV patients (P=0.006; P=0.044) and found a significant positive correlation between JAK2 mutational burden and WBC and PLT levels (r=0.62, P=0.001; r=0.55, P=0.007). Finally, we found that Cer(d18:2/22:6-2OH(7S, 17S)) levels were positively correlated with WBC (r=0.650, P<0.001), and SM(d18:0/PGF1α) levels were positively correlated with WBC and PLT (r=0.444, P=0.011 and r=0.623; P<0.001, respectively). Therefore, we speculate that Cer(d18:2/22:6-2OH(7S, 17S)) and SM(d18:0/PGF1α) are closely associated with JAK2 mutations and may contribute to the proliferation of peripheral blood cells in PV patients. According to the prognostic point system, PV patients can be divided into low-risk group (n=11) and intermediate/high-risk group (n=21). Compared to the low-risk group, the levels of 8 endogenous metabolites in the intermediate/high-risk group were significantly elevated. We next used binary logistic regression to analyze the combined biomarkers and then determine the ROC curve. A set of potential biomarkers containing four endogenous metabolites with an AUC > 0.77 was found to provide an AUC of 0.952, with a sensitivity of 90.905% and specificity of 90.909% at the optimal cutoff point. Therefore, elevated levels of Cer(d18:2/22:6-2OH(7S, 17S)), SM(d18:0/PGF1α), CerP(d18:1/16:0) and octadec-13-enoylcarnitine could be used as potential metabolic biomarkers of poor prognosis in patients with PV. Conclusion Metabonomics provides an important tool for the study of the pathogenesis of PV and the relationship between JAK2 gene mutation. Furthermore, the potential biomarkers of this study may provide a reference for the prognosis of PV. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE

Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021). Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations. Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM-rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1mut. On average, SF3B1mut patients harbored 2.85 mutations (including SF3B1). The most frequent additional mutations of SF3B1mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1, the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1mut; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1mut AML seems to be dominated by the sub-entity. Image:Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1mut AML, it might be unidentifiable in others. Alternatively, SF3B1mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1mut AML.

Prognostic Role of DNA Damage Response Genes Mutations and their Association With the Sensitivity of Olaparib in Prostate Cancer Patients

ObjectiveEvidence shows that gene mutation is a significant proportion of genetic factors associated with prostate cancer. The DNA damage response (DDR) is a signal cascade network that aims to maintain genomic integrity in cells. This comprehensive study was performed to determine the link between different DNA damage response gene mutations and prostate cancer.Materials and methodsA systematic literature search was performed using PubMed, Web of Science, and Embase. Papers published up to February 1, 2022 were retrieved. The DDR gene mutations associated with prostate cancer were identified by referring to relevant research and review articles. Data of prostate cancer patients from multiple PCa cohorts were obtained from cBioPortal. The OR or HR and 95% CIs were calculated using both fixed-effects models (FEMs) and random-effects models (REMs).ResultsSeventy-four studies were included in this research, and the frequency of 13 DDR genes was examined. Through the analysis of 33 articles that focused on the risk estimates of DDR genes between normal people and PCa patients, DDR genes were found to be more common in prostate cancer patients (OR = 3.6293 95% CI [2.4992; 5.2705]). Also, patients in the mutated group had a worse OS and DFS outcome than those in the unmutated group (P < .05). Of the 13 DDR genes, the frequency of 9 DDR genes in prostate cancer was less than 1%, and despite differences in race, BRCA2 was the potential gene with the highest frequency (REM Frequency = .0400, 95% CI .0324 - .0541). The findings suggest that mutations in genes such as ATR, BLM, and MLH1 in PCa patients may increase the sensitivity of Olaparib, a PARP inhibitor.ConclusionThese results demonstrate that mutation in any DDR pathway results in a poor prognosis for PCa patients. Furthermore, mutations in ATR, BLM, and MLH1 or the expression of POLR2L, PMS1, FANCE, and other genes significantly influence Olaparib sensitivity, which may be underlying therapeutic targets in the future.

BRAF V600E mutation and correlation with clinicopathologic features in radioactive iodine refractory papillary thyroid carcinoma

Objective: To define the rate of BRAF V600E mutation in radioactive iodine (RAI) refractory papillary thyroid carcinoma (PTC). The correlation BRAF V600E mutation with some clinicopathologic features in RAI refractory PTC. Subject and method: 92 patients with RAI refractory PTC were operated recurrent lesions at 108 Military Central Hospital from 9/2009 to 3/2018. Recurrent lesions were evaluated histopathological characteristics, immunohistochemical staining with BRAF V600E (Ventana, Roche), BRAF V600E gene test by real-time PCR. Evaluating the status of mutated BRAF V600E and correlate with some clinicopathologic features. Result: Average age: 47.1 ± 15.6 (12 - 81); the rate of BRAF V600E mutation accounted for 80.4%; the average age of unmutated BRAF V600E RAI refractory PTC group (40.2 ± 17.2) was lower than the group of BRAF V600E mutation (48.5 ± 14.9) (p=0.042); BRAF V600E mutation in early-stage disease (I - II) had a higher rate than ones in advanced disease (II-IV); similarly, this gene mutation occurred more frequent in the group received more than 600mCi cumulative dose of RAI compared with the group received less than 600mCi cumulative dose. There was no correlation of BRAF V600E mutation with age, tumor size, lymph node, distant metastasis. BRAF V600E mutation in tall cell variant of PTC was seen more frequently than other variants and it was also seen in cases of extracapsular lymph node invasion. There was no relationship between the existence of mutated BRAF V600E with vessel invasion and calcification. Conclusion: The rate of BRAF V600E mutation in RAI refractory PTC is high, accounting for 80.4%. It correlates with some clinicopathologic characteristics such as disease stage, cumulative dose of radioactive iodine, tall cell variant of PTC; no correlation of BRAF V600E mutation with age, tumor size, lymph node and distant metastasis, extracapsular lymph node invasion

Comorbidity Evaluation Identifies Smoking Dose Density to be Associated with TP53 Mutation in Myeloid Malignancies

Background: Inflammation and aging are factors associated with mutagenicity. In acute Myelogenous Leukemia (AML), detectable mutations (mut) are a powerful tool for identification of patients (pt) at a higher risk for relapse. One frequently observed mutation is TP53, which is particularly unresponsive to treatment. Known risk factors for TP53 acquisition include prior chemotherapy or radiation exposure. It is possible that TP53 mut initiates at low allele frequency and expands in a “permissible marrow microenvironment”. Cell extrinsic inflammation, collectively called extracellular stressor, is a theory of mutation acquisition which is gaining momentum given its recently recognized role in clonal initiation, expansion, and dominance. Smoking is one such possible extracellular stressor that dramatically increases risk of human cancer. Lung tissue directly exposed to smoking develops higher frequency of mut; however, other tissue not directly exposed (e.g. bladder, pancreas) exhibit similar mutation frequencies. This suggests that active smoking metabolites systemically induce inflammation and/or mutagenicity. Indeed, TP53 and ASXL1 mut have been associated with clonal hematopoiesis (CH). In this study, we seek to create “proof of clinical concept” for association between smoking “dose” and TP53 myeloid malignancy development when controlled by an age-adjusted effect. Methods: After IRB approval, AML and Myelodysplastic Syndrome (MDS) AML patients with available Next Generation Sequencing (NGS) were isolated from MDS/AML databases. “Smoking dose” was estimated in patients exhibiting or not exhibiting TP53 mut by standard pack-year calculation. Demographics, clinical, and laboratory categorical and continuous variables were analyzed by chi-square and t-test using SAS software. Logistic regression analysis allowed for identification of variables with independent predictive value for TP53 acquisition. Results: Our cohort represented 52 patients (pt) [AML N=47/52 (90.3%) and MDS N=5/52 (9.6%)]. 23/52 (44.2%) pt harbored TP53-mut and 29/52 pt (55.8%) were part of a genomic control group harboring ASXL1, RUNX1, FLT3, NPM1, and KRAS mut. Median age for pt with and without TP53 mut was 71 y (49-81) vs 60 y (22-88), respectively, p= 0.04 [Fig. 1B]. In TP53 mutated vs TP53 unmutated group, 14/23 (60.8%) vs 13/20 (65%) pt were male. 9/23 (39.1%) and 7/20 (35%) of pt with and without TP53 mut had available smoking history. Pack-year smoking was 53.6 [20-100] and 23.2 [10-40], in pt wit and without TP53 mut, respectively, p=0.02 [Fig. 1A]. When adjusted by age and prior chemotherapy exposure, smoking dose density was the only potential factor with relevance for TP53 acquisition (p=0.05). Conclusions: In myeloid malignancies, our data suggests that TP53 acquisition is associated with a dose dependent smoking history. This smoking dose dependency seems to be independent from aging and prior chemotherapy exposure in AML. Previous studies have demonstrated strong associations between smoking and inflammation. It is possible that higher dosages of smoking induce marrow microenvironment inflammatory changes permissible for TP53 initiation. These microenvironmental changes may be associated with mutational signatures similar to those found in other smoking-associated malignancies, including lung adenocarcinoma. Our report adds to the body of evidence for mechanistic link between magnitude of smoking exposure and marrow environmental stress to promote selective mutagenicity, though larger study is needed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Expression of WTAP Gene in Acute Myeloid Leukemia and Its Clinical Significance

To investigate the expression of WTAP gene in acute myeloid leukemia (AML) and its clinical significance. 74 acute myeloid leukemia patients with non-M3 type and 19 normal donors were selected, and real-time quantitative polymerase chain reaction was used to detect the mRNA expression level of WTAP gene in their bone marrow cells. The relationship between the mRNA expression level of WTAP gene and the clinical characteristics was analyzed. The relative mRNA expression of WTAP gene in the non-M3 AML group was significantly higher than that in the healthy control group, and the difference showed statistically significant (P<0.01). There showed no statistically significant difference in WTAP gene expression among each subtypes (all P>0.05) according to the classification of FAB. The mRNA expression level of WTAP gene in FLT3-ITD mutated AML patients was higher than that in FLT3-ITD unmutated group (P=0.016), and the mRNA expression level of WTAP gene in AML patients with CEBPα mutation was lower than that in CEBPα unmutated group (P=0.016). The expression level of WTAP mRNA was positively correlated with WT1 expression (r=0.6866, P<0.01). There was no relationship between WTAP mRNA expression level and other clinical parameters, such as age, gender, white blood cell count, hemoglobin level, platelet count, bone marrow original proportion of immature cells, chromosome karyotype, and NPM1, DNMT3A, ASXL1, NRAS, TET2 genes mutation status (P>0.05). The expression level of WTAP mRNA showed no obvious effect on the complete remission of patients after first treatment. The different expression level of WTAP gene at initial diagnosis showed also no effect on the overall survival time of patients. The expression level of WTAP gene is increasing in new diagnosed non-M3 acute myeloid leukemia. There is a positive correlation between the expression level of WTAP gene and the expression level of WT1 fusion gene. WTAP mRNA always shows higher expression in patients with FLT3-ITD mutation than that in patients without FLT3-ITD mutation, and shows lower expression in patients with CEBPα mutation than that in unmutated group.

Effect of enhancer of zeste homolog 2 mutations on the prognosis of patients with myelodysplastic syndrome: A meta-analysis.

Background:Gene mutations with important prognostic roles have been identified in patients with myelodysplastic syndrome (MDS). Overall, it is not yet fully clear whether enhancer of zeste homolog 2 (EZH2) is affected and contributes to the disease in MDS patients. Thus, we performed a meta-analysis to investigate the effects of EZH2 mutations on the prognosis of patients with MDS.Methods:We searched English-language databases (PubMed, Embase, and Cochrane Library) for studies published on the effects of EZH2 mutations in MDS patients. The study had to include at least 1 of the following indices as therapeutic evaluation data: overall survival (OS), transformation time to leukemia, and International Prognostic Scoring System risk. Revman, version 5.2 software was used for all statistical processing. We calculated the risk ratio and the 95% confidence interval (CI) of continuous variables, and determined the hazard ratio and 95% CI of time-to-event data.Results:We included 5 studies with a total enrolment of 994 patients. There was a significant adverse effect on OS in the EZH2-mutation group compared to the unmutated group (hazard ratio = 2.47, 95% CI: 1.37–4.47, P < .00001), while the heterogeneity was relatively high (I2 = 68%). There was no significant correlation between EZH2 mutations and IPSS risk (low/int-1 vs int-2/high) (odds ratio: 0.69, 95% CI: 0.14–3.39, P = .65), with significant heterogeneity (I2 = 78%). The analysis did not show significant publication bias in the studies.Conclusion:This meta-analysis indicated an adverse effect of EZH2 mutations with regard to OS in patients with MDS. However, larger cohort trials are still needed to better understand the prognostic impacts of EZH2 mutations on MDS patients.

Final Results from the Multicenter, Open-Label, Phase II GIBB Study of Obinutuzumab+Bendamustine in Previously Untreated Patients with Chronic Lymphocytic Leukemia

Final Results from the Multicenter, Open-Label, Phase II GIBB Study of Obinutuzumab+Bendamustine in Previously Untreated Patients with Chronic Lymphocytic Leukemia

Maintenance of Long-Term Undetectable Minimal Residual Disease after Combination of Ibrutinib with Abbreviated Chemotherapy in the Icll-07 Filo Trial

Maintenance of Long-Term Undetectable Minimal Residual Disease after Combination of Ibrutinib with Abbreviated Chemotherapy in the Icll-07 Filo Trial

Effect of RNA splicing machinery gene mutations on prognosis of patients with MDS: A meta-analysis.

Background:Gene mutations with important prognostic role have been identified in patients with myelodysplastic syndrome (MDS). We performed a meta-analysis to investigate the effects of RNA splicing machinery gene mutations on prognosis of MDS patients.Methods:We searched English database including PubMed, Embase, Cochrane Library for literatures published within recent 10 years on the effect of RNA splicing machinery genes in MDS. Revman version 5.2 software was used for all the statistical processing. We calculated risk ratio and 95% confidence interval (CI) of continuous variables, and find hazard ratio (HR) and 95% CI of time-to-event data.Results:We included 19 studies enrolling 4320 patients. There is a significant superior overall survival (OS) in splicing factor 3b, subunit 1 (SF3B1)-mutation group compared to unmutated group (HR = 0.58, 95% CI: 0.5–0.67, P < .00001); OS decreased significantly in serine/arginine-rich splicing factor 2/ U2 auxiliary factor protein 1 (SRSF2/U2AF1) mutation group compared to unmutated group, (HR = 1.62, 95% CI: 1.34–1.97, P < .00001 and HR = 1.61, 95% CI: 1.35–1.9, P < .00001, respectively). In terms of leukemia-free survival (LFS), the group with SF3B1 mutation had better outcome than unmutated group, HR = 0.63 (95% CI: 0.53–0.75, P < .00001). Other RNA splicing gene mutation group showed significant poor LFS than unmutated groups, (HR = 1.89, 95% CI: 1.6–2.23, P < .00001; HR = 2.77, 95% CI: 2.24–3.44, P < .00001; HR = 1.48, 95% CI: 1.08–2.03, P < .00001; for SRSF2, U2AF1, and zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 [ZRSR2], respectively). As for subgroup of low- or intermediate-1-IPSS risk MDS, SRSF2, and U2AF1 mutations were related to poor OS. (HR = 1.83, 95% CI: 1.43–2.35, P < .00001; HR = 2.11, 95% CI: 1.59–2.79, P < .00001 for SRSF2 and U2AF1, respectively). SRSF2 and U2AF1 mutations were strongly associated with male patients. SF3B1 mutation was strongly associated with disease staging.Conclusion:This meta-analysis indicates a positive effect of SF3B1 and an adverse prognostic effect of SRSF2, U2AF1, and ZRSR2 mutations in patients with MDS. Mutations of RNA splicing genes have important effects on the prognosis of MDS.

Identification and Characterization of Potential Candidates for Treatment Targeting Apoptosis Pathways in Patients with Hematological Neoplasms

Background: Resistance to apoptosis is one of the hallmarks in hematological neoplasms, most typically via dysregulation of the intrinsic mitochondrial pathway. The most important antiapoptotic/pro-survival proteins in this pathway are BCL2, BCL2L1 and MCL1. Molecules targeting each of these proteins are in various stages of preclinical and clinical development. The BCL2 inhibitor venetoclax is the first FDA approved drug in this setting and was shown to be highly effective in CLL and some B-cell lymphoma subtypes. Kumar et al. (Blood 2017) reported that in multiple myeloma response to venetoclax correlated with higher BCL2, BCL2:BCL2L1 and BCL2:MCL1 mRNA expression ratios.Aim: To determine the gene expression levels of BCL2, BCL2L1 and MCL1 across various hematological neoplasms in order to identify best candidates for pro-survival protein inhibitors.Patients and Methods: 2239 patients with hematological neoplasms (Table1) were evaluated by whole transcriptome sequencing (WTS) and 64 cases without hematological neoplasms were analysed as controls. Total RNA was sequenced on the NovaSeq 6000 with a median of 50 million reads per sample. The obtained estimated gene counts were normalized and the resulting log2 counts per million (L2CPM) were used as a proxy of gene expression.Results: The median BCL2 expression (exp) was significantly higher in lymphoid malignancies (lyM) compared to controls and myeloid malignancies (myM) (L2CPM: 5.8 vs 3.4 vs 3.4, p<0.0001) with the highest exp noticed in CLL, FL and MM (Table 1). On the other hand median BCL2L1 exp was significantly higher in myM and lower in lyM as compared to controls (6.7 vs 5.2 vs 5.8, for both p<0.0001). Median MCL1 exp was comparable to controls in myM, while it was significantly lower in lyM (9.4 vs 9.5 vs 8.5, p<0.0001). Comparing the different entities 8 patterns based on significant differences in exp of BCL2, BCL2L1 and MCL1 compared to controls emerged: 1) increased BCL2, lower BCL2L1, lower MCL1 (CLL, MCL, DLBCL, HGBL, PPBL, T-ALL and AML), 2) increased BCL2, lower BCL2L1, increased MCL1 (MM), 3) increased BCL2, lower MCL1 (B-ALL, Burkitt, FL, LPL, MZL), 4) increased BCL2, increased BCL2L1, lower MCL1 (ET, HCL), 5) increased BCL2L1 (MDS/MPN-U, MPN-U, PMF, PV), 6) lower BCL2, increased BCL2L1, increased MCL1 (CNL), 7) lower BCL2, increased BCL2L1, lower MCL1 (MDS, MDS/MPN RS-T), 8) no significant changes compared to controls in the expression of any of these genes (aCML, CML, CMML). Entities with pattern 1 are most likely to respond, followed by those with patterns 2, 3 and 4. Within most entities the BCL2 exp varied only within small ranges (Figure1).AML was the only myM with a significantly increased median BCL2 exp. In more detail, 21% of AML patients showed BCL2 exp above control values. The mean BCL2 exp was significantly higher in the following subgroups: PML-RARA rearranged (rear) (5.5, p<0.001), KMT2A rear (5.0, p=0.009), IDH1 mutated (mut) (5.1, p=0.025), and GATA2mut (4.9, p=0.04), while it was significantly lower in the following subgroups: DNMT3Amut (4.2; p=0.049), SF3B1mut (3.59; p=0.03), TET2mut (4.1, p=0.016), RAD21mut (3.7, p=0.05), RUNX1-RUNX1T1 rear (3.6, p<0.001), and complex karyotype (3.8, p=0.003). In B-ALL BCL2 exp was found to be higher in ALL with KMT2A rear, high hyperdiploidy and low hypodiploidy while it was lower in BCR-ABL1+ ALL. In T-ALL no association between BCL2 exp and phenotype or genotype was observed.In MDS BCL2 exp was lower compared to controls (3.1 vs 3.4, p=0.004), while BCL2L1 exp was higher (7.3 vs 5.8, p<0.0001). The BCL2 exp was lowest in the very low cytogenetic risk group according to IPSS-R, increased with risk category and was highest in the very poor cytogenetic risk group (2.9, 3.1, 3.1, 3.7, 3.9). SF3B1mut and TET2mut were associated with a significantly lower BCL2 exp as compared to the respective unmutated group (2.7 vs 3.3; 2.9 vs 3.2; for both p<0.0001).Conclusions: 1) The pattern of BCL2, BCL2L1 and MCL1 expression differs significantly between entities. 2) More than 85% of patients with CLL, FL and MM show an increased BCL2 expression, while in myeloid malignancies BCL2 expression is low and only 21% of AML patients demonstrate an increased BCL2 expression. 3) Prospective studies are mandatory to evaluate the predictive value of BCL2, BCL2L1 and MCL1 expression for response to therapies targeting pro-survival molecules in order to identify patients with the highest likelihood of response. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

BRAFV600E mutation in hairy cell leukemia: A single-center experience.

BRAFV600E mutation has been reported as a unique genetic lesion of hairy cell leukemia (HCL), a subset of which lacks this lesion and shows adverse outcomes. To determine the prevalence of BRAFV600E in HCL from our center and derive clinicopathological correlation, if any. A 9-year retrospective analysis of 46 consecutive cases of HCL diagnosed on morphology and immunophenotyping was done. Stained smears were used as samples for amplification refractory mutation system polymerase-chain reaction using fluorescent primers for mutation detection. BRAFV600E mutation was detected in 41/46 patients (89.1%) while absent in control samples of chronic lymphocytic leukemia. Cases mimicking HCL-variant clinically or immunophenotypically too showed the presence of this mutation. HCL with mutated BRAF presented at a younger age. No statistical difference in blood counts, tumor load, and immunophenotype patterns existed among BRAF mutated and unmutated group. Nine patients (45%) with mutated BRAF had residual disease following treatment with cladribine. BRAFV600E mutation analysis has a definitive role in the diagnosis of HCL.

The absolute percent deviation of IGHV mutation rather than a 98% cut-off predicts survival of chronic lymphocytic leukaemia patients treated with fludarabine, cyclophosphamide and rituximab.

The degree of somatic hypermutation, determined as percent deviation of immunoglobulin heavy chain gene variable region sequence from the germline (IGHV%), is an important prognostic factor in chronic lymphocytic leukaemia (CLL). Currently, a cut-off of 2% deviation or 98% sequence identity to germline in IGHV sequence is routinely used to dichotomize CLL patients into mutated and unmutated groups. Because dissimilar IGHV% cut-offs of 1-5% were identified in different studies, we wondered whether no cut-off should be applied and IGHV% treated as a continuous variable. We analysed the significance of IGHV% in 203 CLL patients enrolled on the original frontline fludarabine, cyclophosphamide and rituximab (FCR) trial with a median of 10years follow-up. Using the Cox Proportional Hazard model, IGHV% was identified as a continuous variable that is significantly associated with progression-free (PFS) and overall survival (OS) (P<0·001). Furthermore, we validated this finding in 323 patients treated with FCR off-protocol and in the total cohort (n=535). Multivariate analysis revealed a continuous trend. Higher IGHV% levels were incrementally associated with favorable PFS and OS in both FCR-treated cohorts (P<0·001, both cohorts). Taken together, our data suggest that IGHV% is a continuous variable in CLL patients treated with FCR.

Novel prognostic molecular factors: a quantum leap in the field of chronic lymphocytic leukemia

Cytogenetic lesions do not completely explain clinical heterogeneity of chronic lymphocytic leukemia (CLL). The 2016 revision of the World Health Organization classification 2008 indicated that molecular lesions of TP53, NOTCH1, SF3B1 and BIRC3 have potential clinical relevance and could be integrated into an updated risk profile. The negative clinical implications of TP53 disruptions are well constituted and patients with these mutations should be considered for novel, small molecule signal transduction inhibitors therapies. Mutations of NOTCH1, SF3B1 and BIRC3 are associated with poor prognosis. Patients with mutated SF3B1 or NOTCH1 genes present shorter time to first treatment compared to unmutated group. NOTCH1 mutations are related to a high risk of Richter's syndrome transformation, especially in case of TP53 disruptions' coexistence. Large studies on MYD88 mutations in CLL have not explained clearly their clinical importance.The aim of this paper is to provide a comprehensive review on novel molecular aberrations identified in CLL.

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Introduction:MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable.Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes.Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available.Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations.Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort.Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases.Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

VIII. Treatment of chronic lymphocytic leukaemia, where are we heading?

VIII. Treatment of chronic lymphocytic leukaemia, where are we heading?

The Prognostic Significance of TP53 Mutations for Overall Outcome in Lower-Risk MDS Patients

BACKGROUND AND AIMSRecent results have indicated that the TP53 mutations had a negative prognostic relevance for lower-risk myelodysplastic syndrome (MDS) patients but the relations between the presence of mutations and treatment has not been clarified yet. Therefore, we analyzed the prevalence of TP53 mutations in lower-risk MDS patients and determined the proportion of TP53 mutated bone marrow cells during the disease. We further observed mutual relation of treatment and mutated clones and defined impact of mutations on survival of patients.METHODSTP53 mutations were analyzed in DNA from bone marrow of 141 low risk or intermediate-1 risk MDS patients included into the cohort according to the International Prognostic Scoring System using primers designed within the IRON-II study research consortium (Roche Applied Science) on the GS Junior system. If mutations were detected, we searched for mutations at different timepoints of the disease and overall 210 samples were examined for mutations. Ten of the 16 patients with TP53 mutation received lenalidomide and/or 5-azacytidine. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden.RESULTSThe overall incidence of TP53 mutations was 11.3 % (16/141), three patients harbored two mutated clones. The mutation frequency in patients with and without del(5q) was 21% (13/62) and 3.8% (3/79), respectively. During the observation period, 37 patients (26.2%) died, the data obtained from remaining patients were censored either at the last date they were known to be alive (94 patients - 66.7%) or at the time of stem cell transplantation (HSCT) (10 patients - 7.1%). The median overall survival was 164.2 months in TP53 unmutated group and 84.5 in mutated group respectively calculated by Kaplan-Meier Survival Curves and the Log-Rank Test (P=0.18). Mutations were detectable in eight of the sixteen available patients (50%) at the time of diagnosis and remaining patients (50 %) displayed mutations in an average 35.1 months (range 12-69 months) from diagnosis.Three patients with mutational burden below 5% are alive more than 3 years without signs of disease progression and three patients with clone size above 40% died. The mutated clone disappeared after bone marrow transplantation in one patient. The proportion of TP53 mutated cells decreased radically after successful treatment with azacytidine in two patients. The clone size increased in four patients treated with lenalidomide and all of them died. Two patients with mutated clone sizes below 20% are still under observation.CONCLUSIONSWe confirmed the higher frequency of TP53 mutations in lower- risk MDS with del(5q) in a large cohort of patients. Patients with the TP53 mutations had shorter survival compared to those without mutation. The mutations were detected in a half of the patients at the time of diagnosis and approximately three years after diagnosis in remaining patients. The presence of TP53 mutation as an adverse prognostic factor should be routinely investigated in lower risk MDS patients at the time of the diagnosis as well as repeatedly in the course of the disease.Supported by grant (NT/13899, NT/14377, NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. DisclosuresNo relevant conflicts of interest to declare.