Unfavorable Mutations Research Articles

Characteristics and outcomes of therapy-related myeloid neoplasms after peptide receptor radionuclide/chemoradionuclide therapy (PRRT/PRCRT) for metastatic neuroendocrine neoplasia: a single-institution series.

Peptide receptor radionuclide/chemoradionuclide therapy (PRRT/PRCRT) is an effective therapy for metastatic neuroendocrine neoplasia (NEN), but therapy-related myeloid neoplasms (t-MN) remain of concern. The study reviewed the clinicopathological features and outcomes of patients who developed t-MN. Retrospective analysis of all patients diagnosed with t-MN by 2016 WHO classification, from a cohort of 521 patients who received PRRT/PRCRT over a 12-year period. Molecular next-generation sequencing using an in-house 26-gene panel was performed. Twenty-five of 521 (4.8%) patients were diagnosed with t-MN, including six acute myeloid leukaemia (AML) and 19 myelodysplastic syndrome (MDS). The median time from first cycle PRRT/PRCRT to diagnosis of t-MN was 26months (range 4-91). Twenty-two of 25 (88%) patients had grade 1-2 pancreatic or small bowel NEN with moderate metastatic liver burden. Six patients (24%) had prior chemotherapy. Median number of PRRT cycles = 5 (22/25 (88%) with concomitant radiosensitising chemotherapy). All 25 patients achieved disease stabilisation (68%) or partial response (32%) on RECIST 1.1 at 3months post-PRRT. At t-MN diagnosis, all patients presented with thrombocytopenia (median nadir 33 × 109/L, range 3-75) and 17 (68%) remained NEN progression-free. Marrow genetic analysis revealed unfavourable karyotype in 16/25 (66%) patients with tumour protein 53 (TP53) mutation in nine (36%). Azacitidine therapy was utilised in ten eligible patients, while four received induction chemotherapy for AML. The median overall survival from first PRRT was 62months (19-94), but from t-MN diagnosis was only 13months (1-56), with death due primarily to haematological disease progression. The diagnosis of t-MN after PRRT/PRCRT is an infrequent but serious complication with poor overall survival. Most patients present with thrombocytopenia; unfavourable genetic mutations have a poor response to t-MN treatment. Prospective data are needed to explore potential pre-existing genetic factors and predictive biomarkers to minimise the risk of t-MN.

Protein kinase A inhibits tumor mutator APOBEC3B through phosphorylation

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.

PB1746 PRACINOSTAT PLUS AZACITIDINE IN OLDER PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (AML): A CASE REPORT FROM A PHASE 2 STUDY

Background:Older patients with newly diagnosed AML have limited treatment options and poor prognosis. They usually have poor tolerance and low response rates to intensive chemotherapy, poor candidacy for allogeneic stem cell transplant, and modest responses with the commonly used low‐intensity palliative regimens. A recent phase 2 study has shown that the pan‐histone deacetylase inhibitor pracinostat combined with azacitidine was well tolerated and active in older patients unfit for intensive chemotherapy, leading to a complete remission (CR) rate of 42% and median overall survival of 19.1 months (Garcia‐Manero G. Blood Advances, 2019).Aims:To describe novel and rare findings in an older patient with AML who received treatment with pracinostat and azacitidine.Methods:The patient is a 67‐year‐old man who presented with AML and severe symptomatic anemia, resulting in unstable angina and non–ST‐elevation myocardial infarction. Due to comorbidities that included advanced chronic obstructive pulmonary disease and symptomatic angina, the patient was deemed unfit for induction chemotherapy or allogeneic stem cell transplantation. The patient was enrolled in a phase 2 open‐label, single‐arm study (NCT01912274) assessing the safety and efficacy of pracinostat combined with azacitidine in older (≥65 years) patients with newly diagnosed de novo, secondary, or therapy‐related AML ineligible for induction chemotherapy. The patient received oral pracinostat 60 mg/day, 3 days/week, for 3 consecutive weeks plus azacitidine 75 mg/m2 daily for 7 days in a 28‐day cycle. Bone marrow (BM) biopsies were performed at screening, cycles 2, 4, and 6, and every 3 cycles thereafter until CR or as clinically indicated. A peripheral blood sample was also obtained from the patient at baseline, to determine the presence of any possible mutations.Results:The mutation analysis revealed that the patient presented with 5 mutations: CBL p.P433L variant allele frequency (VAF) 0.386243, fms‐like tyrosine kinase 3‐internal tandem duplication (FLT3‐ITD) VAF 0.2738, nucleophosmin (NPM1) p. L287fs 0.2167, RAD21 p. R172fs 0.2632, and Wilms tumor protein 1 (WT1) p.V379fs 0.3089. The patient has received over 48 cycles of pracinostat combined with azacitidine and remains on treatment to date (>49 months). After the first 2 treatment cycles, the patient achieved a BM partial remission, with CR being attained by cycle 6 and maintained for over 43 months. Due to treatment‐related neutropenia, the administration of cycles 5, 48, and 49 was delayed by 1, 2, and 1.5 weeks, respectively. Due to treatment‐related pneumonia, the administration of cycle 43 was delayed for approximately 7 weeks. He remained on therapy with no treatment‐related toxicities, and significant improvement in his performance status and quality of life.Summary/Conclusion:This case demonstrates the potential of pracinostat in combination with azacitidine to be a long‐term effective and safe regimen for older patients with newly diagnosed AML, achieving long‐lasting CR beyond what is expected with single‐agent azacitidine. These results bear increased relevance for patients with an unfavorable baseline mutation status and a consequent particularly poor prognosis.

Characteristics and Outcomes of Therapy-Related Myeloid Neoplasms after Peptide Receptor Radionuclide/Chemoradionuclide Therapy (PRRT/PRCRT) for Metastatic Neuroendocrine Neoplasia: A Single Institutional Series

Background: Peptide receptor radionuclide/chemoradionuclide therapy(PRRT/PRCRT) is an emerging therapy in patients with metastatic neuroendocrine neoplasia (NEN). However, therapy-related myeloid neoplasms (t-MN) remain a potential complication with substantial adverse outcomes. The study reviewed our PRRT/PRCRT patients who developed t-MN, with regard to their clinicopathological features and outcomes. Methods: Retrospective analysis of all patients diagnosed with t-MN, defined as per revised 2016 WHO classification, from this single centre cohort of 521 patients who received PRRT/PRCRT over a 12-year period. Molecular next generation sequencing using an in-house 26 gene panel were performed. Findings: Twenty-five (4.8%) patients were diagnosed with t-MN, including six acute myeloid leukaemia (AML) and 19 myelodysplastic syndrome (MDS). The median time from first cycle PRRT/PRCRT to diagnosis of t-MN was 26 months (range 4 - 91). Twenty-two of 25 (88%) patients had Grade 1-2 pancreatic or small bowel NEN with moderate metastatic liver burden. The majority, 22 of 25 (88%) received concomitant radiosensitising chemotherapy. All 25 patients achieved disease stabilisation (68%) or at least a partial response (32%) on RECIST 1.1 assessment at 3 months post-PRRT. At t-MN diagnosis, all patients presented with thrombocytopenia (median nadir 33 x 109/L, range 3 - 75) and 17 (68%) were NEN progression free. Marrow genetic analysis revealed unfavourable karyotype in 16/25 (66%) patients and tumour protein 53 (TP53) mutations in nine (36%). Azacitidine therapy was utilised in ten eligible patients, while four received induction chemotherapy for AML. The median overall survival from the time of t-MN diagnosis was 13 months (range 1 - 56), with cause of death due largely to haematological disease progression. Interpretation: The diagnosis of t-MN after PRRT/PRCRT is uncommon but a serious complication with poor overall survival. Most patients present with thrombocytopenia, unfavourable genetic mutations and poor response to t-MN treatment. Funding: No funding source involved. Declaration of Interest: RJH holds shares in Telix Pharmaceuticals on behalf of the Peter MacCallum Cancer Centre. MSH reports personal fees and non-financial support from Ipsen and Sanofi Genzyme, personal fees and other from Endocyte, outside the submitted work. All remaining authors declare no competing interests. Ethical Approval: The study was approved by the hospital’s medical ethics committee as a retrospective audit with approval of waiver for patient consent (HREC Project number 18/61R). All patients had provided written, informed consent for PRRT/PRCRT.

Prognostic Value of Genetic Mutations in Patients with Acute Myeloid Leukemias: Results of a Cooperative Study of Hematology Clinics of Saint Petersburg (Russia) and Charite Clinic (Germany)

Aim. To analyze the effect on prognosis of mutations that are typical of acute myeloid leukemia (AML) patients. Materials & Methods. The study included 620 AML patients surveyed at Hematology Clinics of Saint Petersburg (Russia) and Charite Clinic (Berlin, Germany). G-banding of chromosomes was employed for cytogenetic testing. Aberration screening in DNMT3A, IDH1/2 genes was based on real-time polymerase chain reaction (PCR) with subsequent analysis of melting and sequencing profiles. Mutations in FLT3, NPM1 genes were revealed by PCR. Results. Mutations were identified in 343 (55.3 %) out of 620 patients. Significantly more often mutations were discovered in patients with normal karyotype (NK) (p = 0.001). FLT3-ITD mutation was associated with reduced medians of overall survival (OS) and disease-free (DFS) survival: 11.3 vs. 15.8 months with FLT3-ITD- (p = 0.005) and 10.0 vs. 13.3 months with FLT3-ITD+ (p = 0.009), respectively. The relation of FLT3-ITD allele burden to OS duration was also assessed. In the ITD<sup>low</sup>/ITD- group the OS median was considerably longer than in the ITD<sup>high</sup> group (p = 0.028). In the group of patients with 1 mutation in NPM1 gene OS and DFS were much better in comparison with other patients (medians of 27.4 and 13.9 months, respectively, p = 0.040; 19.3 and 12.0 months, p = 0.049). Negative impact of mutations in DNM-T3A gene was noticed while assessing OS median: 12 (DN-MT3A+) and 15 months (DNMT3A-), respectively (p = 0.112). Mutations in IDH1 gene correlated with a better OS than in the group without mutations (p = 0.092). The rs11554137 polymorphism in IDH1 gene was associated with worse OS in the group of patients with NK (p = 0.186). In 144 patients various mutation combinations (from 2 to 5) were identified. It was demonstrated that mutations in FLT3 (FLT3-ITD), NPM1, DNMT3A, and IDH2 were identified significantly more often in combinations with other mutations (p = 0.001): NPM1+/ FLT3-ITD+ (20.8 %), NPM1+/FLT3-ITD+/DNMT3A+ (8.3 %), and FLT3-ITD+/DNMT3A+ (8.3 %). Patients with 1 mutation had a noticeably longer OS median compared with patients with 2 mutations (18.1 and 12.2 months; p = 0.003). In patients with NPM1+ according to their OS the most unfavorable additional mutation was FLT3-ITD (median 27.4 vs. 9.2 months; p = 0.019) and the combination of NPM1+/FLT3-ITD+/DNM-T3A+ (median 27.4 vs. 14.6 months; p = 0.141). OS of patients with DNMT3A+ showed a downward trend if FLT3-ITD additional mutation was identified (17.3 vs. 7.1 months; p = 0.074). Conclusion. Mutations in FLT3, DNMT3A, IDH1/2, NPM1 genes frequently occur in AML intermediate-risk patients, i.e. they determine the intermediate prognosis group in AML. The studied mutations considerably impact prognosis. It is important to take into consideration mutation type, its allele burden, and the presence of additional mutations. A patient with 2 mutations has a considerably worse OS compared with a patient with 1 mutation. The studied group of patients with the combination of NPM1+/FLT3-ITD+, NPM1+/ FLT3-ITD+/DNMT3A+, DNMT3A+/FLT3-ITD+ mutations has the poorest prognosis. Comprehensive analysis of genetic damages in AML patients allows to most accurately predict the course and prognosis of the disease and to plan targeted therapy.

Using Informatics Tools to Characterize Precision Medicine Treatments for Diffuse Large B-Cell Lymphoma (DLBCL)

▪Diffuse large B-cell lymphoma (DLBCL) is genetically and clinically heterogeneous. Despite immunohistochemical and genomic subtyping, standard firstline DLBCL treatment remains rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). As genetic profiling becomes more accessible and pervasive, studies identifying specific drug-gene interactions in DLBCL are needed to develop future patient-specific DLBCL treatment strategies that improve the delivery of effective and cost-effective, patient-centered care. Methods: We performed a systematic literature review using PubMed Medical Subject Heading and text word terms for DLBCL/genetics and exome sequencing and recorded DLBCL mutations, associated mutation frequencies, pathways, and implications for overall survival (OS) with a focus on recent publications from Reddy et al 2017, Chapuy et al 2018, and Schmitz et al 2018 and related articles. Data for mutation frequencies and OS were extracted from Reddy et al (2017; https://dlbcl.davelab.org/). Mutations were categorized as either favorable or unfavorable. Favorable mutations were associated with 3-year OS ≥ OS for patients equivalent or better than IPI score ≤1 in this dataset (≥90.1%), whereas unfavorable mutations were associated with 3-year OS ≤ OS for patients with an IPI score ≥2 from this dataset (≤64.0%). Potential mutation-specific treatments were identified from the initial literature review, using the Drug-Gene Interaction database (DGIdb, http://www.dgidb.org/, Cotto et al, 2017) and the Open Targets Platform (OTP, https://www.targetvalidation.org/, Koscielny et al, 2017). DLBCL therapeutic groups were defined for mutations/mutation groups that affected ≥5% of DLBCL patients and were associated with targeted therapies based on DGIdb and OTP. Specific drug-gene interactions were characterized using a series of PubMed searches. Results: The analysis identified 32 articles with 55 total therapeutic clusters. 3 mutations were associated with favorable outcomes with an expected 3-year OS of 90.1% (95% CI: 81.1%-95.0%], and 7 mutations were associated with unfavorable outcomes with an expected 3-year OS of 64.0% (95% CI: 56.3%-70.7%) (Figure). The intersection of mutations associated with unfavorable outcomes and mutations associated with targeted therapies yielded 16 therapeutic clusters and revealed a predicted population of 17.6% of DLBCL with poor risk disease and potentially targetable mutations (Table). Drug-gene interaction characterizations identified 267 pertinent studies that included: in vitro studies, mouse models, or clinical trials of the interactions' roles in any cancer. Of these, 7 studies (1 clinical trial, 6 in vitro studies, and 1 mouse model) directly investigated DLBCL specific effects for fostamatinib, ibrutinib, and crizotinib. Conclusions: Linking systematic review with informatics tools identified DLBCL populations, DLBCL specific gene-drug interactions, and potential therapies for trials for patients who relapse and frontline combinations with R-CHOP that can be used in precision medicine strategies for improving outcomes in this disease. DisclosuresFlowers:Genentech/Roche (unpaid): Consultancy; Pharmacyclics/ Janssen: Consultancy; Gilead: Research Funding; Millennium/Takeda: Research Funding; TG Therapeutics: Research Funding; Janssen Pharmaceutical: Research Funding; Karyopharm: Consultancy; Denovo Biopharma: Consultancy; Abbvie: Consultancy; Genentech/Roche: Research Funding; Gilead: Consultancy; Bayer: Consultancy; Celgene: Research Funding; Abbvie: Research Funding; Acerta: Consultancy; Beigene: Consultancy; Pharmacyclics: Research Funding.

Targeted Next-Generation Sequencing of TP53, BIRC3, NOTCH1, SF3B1, and ATM Identifies a Significant Proportion of Lower-Risk Chronic Lymphocytic Leukemia with Unfavorable Prognostic Indicators

IntroductionThe clinical course of chronic lymphocytic leukemia (CLL) is heterogeneous, ranging from indolent disease with essentially normal life expectancy to rapidly progressive disease with significantly decreased survival. Traditional risk stratification is dependent in large part on cytogenetic analysis by fluorescence in situ hybridization (FISH), with del(13q) as a sole abnormality associated with a favorable prognosis, trisomy 12 or normal cytogenetics associated with an intermediate prognosis, and del(11q) or del(17p) associated with an unfavorable prognosis.1 In the last few years, recurrent mutations in key genes including TP53, BIRC3, NOTCH1, SF3B1, and ATM have also been identified as unfavorable prognostic indicators in CLL.2-4 In a study by Rossi et al, integration of mutations and cytogenetic abnormalities was found to significantly improve the accuracy of survival prediction in CLL beyond that of FISH alone.2 The aim of this study is to evaluate the frequency of these unfavorable mutations in an unselected cohort of CLL patients seen in the community setting, and to determine the percentage of lower-risk patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.MethodsAn unselected cohort of 633 CLL patients seen in the community setting was included in this study. Demographic data included a mean age of 69 years, male to female ratio of 1.8:1, and mean WBC count of 48 g/dL. DNA sequences of key exons in TP53, BIRC3, NOTCH1, SF3B1, and ATM were determined from peripheral blood or bone marrow aspirates using a clinically validated next-generation sequencing (NGS) assay with a 5% lower limit of detection for single nucleotide variants. Results were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. FISH analysis utilized probes specific for 11q22.3 (ATM), chromosome 12, 13q14.3/13q34, and 17p13.1 (TP53). CLL diagnoses were confirmed by flow cytometry, with FISH for t(11;14)(IGH-CCND1) also performed to exclude mantle cell lymphoma. The percentage of CLL cells by flow cytometry ranged from 18-98%, with a mean of 69%.ResultsCytogenetic abnormalities were detected in 85% of CLL patients in this study, with del(13q) the most frequent (65%), followed by trisomy 12 (19%), del(11q) (17%), and del(17p) (10%). Mutations in any one of the 5 genes were detected in 37% of patients, with TP53 mutation the most frequent (12%), followed by mutations in NOTCH1 (11%), SF3B1 (10%), ATM (7%), and BIRC3 (3%). TP53 disruption as a whole was found in 14% of patients, consisting of del(17p) only (2%), TP53 mutation only (5%), and biallelic TP53 disruption (7%). ATM disruption as a whole was found in 21% of patients, consisting of del(11q) only (14%), ATM mutation only (4%), and biallelic ATM disruption (4%). Lower-risk patients, as defined by FISH showing either isolated del(13q), trisomy 12, or normal results, comprised 75% of patients, with 29% showing one or more unfavorable mutation: NOTCH1 (12%), SF3B1 (8%), TP53 (6%), ATM (4%), and BIRC3 (3%).ConclusionsMutations in TP53, BIRC3, NOTCH1, SF3B1, and ATM have previously been shown to be indicators of unfavorable prognosis in CLL. Using a clinically validated NGS assay, 37% of the CLL patients in our cohort were shown to have a mutation in any one of the 5 genes. In patients with lower-risk disease as defined by FISH, 29% showed one or more unfavorable mutation, including 6% with a TP53 mutation. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk CLL patients with a worse than expected prognosis who may potentially benefit from earlier and/or novel therapy.ReferencesDohner H, et al. N Engl J Med 2000;343:1910-1916Rossi D, et al. Blood 2013;121:1403-1412Austen B, et al. J Clin Oncol 2007;25:5448-5457Skowronska A, et al. J Clin Oncol 2012;30:4524-32 DisclosuresNo relevant conflicts of interest to declare.

Impact of Race on Outcomes in Intermediate Risk Acute Myeloid Leukemia

Introduction:A 2012 retrospective review of acute myeloid leukemia (AML) patient outcomes in the SEER national database showed higher mortality rates for African Americans and Hispanics despite lower rates of incidence and more favorable genetics at presentation compared to non-Hispanic whites (Patel. Cancer Causes & Control, 2011). While age is a known negative prognostic factor, it has been difficult to analyze the contribution of additional demographic factors predicting outcomes in AML. The purpose of this study was to investigate the impact of race on disease outcome in a diverse patient cohort of intermediate risk AML treated at two urban academic centers in Chicago.Methods:We retrospectively analyzed patients with intermediate risk AML cytogenetics treated with intensive chemotherapy with or without allogeneic stem cell transplant from 2003-2018 at Northwestern Memorial and the University of Illinois Hospital. Baseline clinical and molecular characteristics and treatment response were compared across racial groups. Categorical variables were compared using Pearson's chi square or the Fisher's exact test while overall survival (OS) was estimated by the Kaplan-Meier analysis and compared by the log-rank test. Risk factors for OS were assessed by the Cox regression model.Results:A total of 111 intermediate risk AML patients were included with the 3 major groups being Caucasian (non-Hispanic white), African American (AA) and Hispanic patients (Figure 1). Nearly half of each group was> 60 years of age at presentation. At the time of diagnosis, AA had a significantly higher body mass index (BMI) with a mean of 33.7 kg/m2 compared to 28.1 kg/m2 in the Caucasian group and 27.7 kg/m2 of Hispanic/others (p = 0.0047). There was no statistically significant difference in presenting labs including LDH, WBC count, blast percentage or molecular characteristics including FLT3-ITD or NPM1 mutation status (Figure 2).In log-rank analysis, Caucasians had significantly improved median OS compared to AA and Hispanics/others (31.6 vs. 16.7 vs.18.1 mos; p=0.026) (Figure 3). Multivariate analysis indicated that non-Caucasian patients had a 1.8 times higher risk of death compared to Caucasians (p = 0.059) even when adjusted for age and NPM1 and FLT3-ITD mutational status.We sought to examine whether this outcome disparity could be explained by chemotherapy resistance. The predominantly Hispanic group had a decreased response to induction chemotherapy compared to other groups (52% complete response (CR) vs. 70.6% of Caucasians and 68.8% of AA ; p=NS). In multivariate analysis, age (p = 0.046) and NPM1 status (p = 0.0006) predicted response to therapy while race did not. The favorable risk NPM1 mutation was enriched in Caucasians while the unfavorable FLT3 mutation was more commonly found in the primarily Hispanic group of patients (p = NS).Transplant emerged as a significant predictor of overall survival (p = 0.03). There was a difference approaching statistical significance in transplant utilization between the groups with only 36.8% of AA transplanted compared to 58.1% of non-African Americans (p = 0.09). Underrepresentation of minorities in the transplant registry was reflected in the significantly higher utilization of alternate donor sources in the AA and Hispanic patients. The proportion of patients receiving haploidentical and cord blood transplants was 5.7% of Caucasians vs. 57.1% of AA and 40% of Hispanic/others (p = 0.0006).Conclusion:In a cytogenetically homogenous cohort of AML patients we show that Caucasian patients had significantly superior overall survival compared to other racial groups. We found a statistically significant difference in BMI between racial groups, which may be reflective of comorbidities and chronic illnesses that affect clinical outcome. Our data also showed there was increased utilization of transplant in Caucasian patients likely related to donor availability and insurance coverage. We will next use Geographic Information Systems and payer source data to dissect the contribution of socioeconomic status and health care access to these disparities in disease outcome. Identifying health service gaps in these populations will enable healthcare providers to provide personalized and equitable care to an increasingly diverse patient population. [Display omitted] DisclosuresFrankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Khan:Teva: Speakers Bureau.

Distinct Gene Expression Patterns of Minimal Residual Disease (MRD) Cells in High-Risk AML Patients Identified By RNA-Sequencing

INTRODUCTIONEvolving techniques have made possible the direct detection, physical isolation, and study of AML minimal residual disease (MRD) after treatment. This could allow for better identification of therapeutic vulnerabilities in AML. Prior studies have focused on cells that initiate leukemia in mouse models, known as leukemia-initiating cells (LIC), generally with a foundational CD34+CD38- immunophenotype. LIC are typically derived from diagnostic samples of untreated patients. Such stem-like cells do not necessarily represent the residual fraction of AML after treatment. Relapse may originate from non-LIC, and the presence of phenotypically and molecularly defined MRD is now firmly established as a critical prognostic factor for patients. High-risk AML is characterized by relapse, despite morphologic complete remission with initial therapy in most cases. RNA-sequencing was performed on pre- and post-treatment AML subpopulations, including MRD, from high-risk patients, to determine differences in gene expression.METHODSMatched primary AML samples were collected from marrow and peripheral blood of patients with high-risk AML (including patients with unfavorable karyotype and/or TP53 mutation) at diagnosis and after treatment. Mononuclear cells were flow-sorted for bulk (CD45dim) and LIC (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells (MNC) and MRD, based on difference-from-normal/MRD immunophenotype specific for each patient as determined from established 20-marker clinical flow cytometry analysis. RNA was isolated using low-input methodology, and RNA-sequencing was performed using Illumina HiSeq 2000. Gene expression was assessed using GO-Elite, and differences between patients and subpopulations were assessed using rank product method.RESULTSGene expression in MRD was analyzed by RNA-sequencing in comparison to diagnostic samples in eight patients with high-risk AML. Four patients had unfavorable karyotype, including two with TP53 mutations. Patients had additional high-risk features, such as FLT3-ITD or RUNX1 mutations, or secondary/therapy-related AML. Treatment consisted of chemotherapy (6/8) or hypomethylating agents (2/8), with or without other targeted drugs. Residual leukemia was detected in post-treatment samples in all study patients. Significant differences in gene expression were detected between MRD and other sorted populations, including diagnostic bulk AML and LIC. Relevant MRD pathways included those with strong interactions with the microenvironment. Anti-apoptotic mechanisms, cytoskeletal, and cell adhesion related genes, WNT/beta-catenin signaling, and TGFbeta signaling ranked among the most relevant processes in AML MRD subpopulations (Figure 1A, GO-Elite interactome of highly expressed genes in AML MRD). To identify potentially critical and unique MRD-specific genes, rank product method was applied using 1) the most highly expressed genes in AML MRD, 2) the most differential expressed genes between MRD and bulk AML at diagnosis, and 3) the most differentially expressed genes between MRD and bulk MNC after treatment. Among the top 50 scoring genes using this approach (Figure 1B), 16 genes were among the top 5% of genes expressed in MRD among all patients and 20 genes have cell surface gene-products (shown in yellow). Several potential leukemia- and cancer-related genes of interest were identified (shown in bold).CONCLUSIONSKey differences exist between the gene expression profiles of post-treatment MRD from high-risk AML patients, in comparison to other populations and subpopulations of sorted cells before and after treatment. The highlighted differences suggest that MRD relies on specific intrinsic gene expression changes and microenvironmental interactions, and therefore may be targetable after elimination of bulk AML with initial therapy. Accessible surfacesome targets are among top hits. [Display omitted] DisclosuresKonopleva:cellectis: Research Funding; Immunogen: Research Funding; abbvie: Research Funding; Stemline Therapeutics: Research Funding. Andreeff:Astra Zeneca: Research Funding; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; Celgene: Consultancy; Reata: Equity Ownership; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

JCSE01.19 ALTER-0303 Study: Tumor Mutation Index (TMI) For Clinical Response to Anlotinib in Advanced NSCLC Patients at 3rd Line

Anlotinib is an effective multi-targeted receptor tyrosin kinase inhibitor (TKI) for refractory advanced Non-Small Cell Lung Cancer (NSCLC) therapy at 3rd line. ALTER-0303 clinical trial has been revealed that Anlotinib significantly prolongs progression free survival (PFS; Anlotinib: 5.37 months vs Placebo: 1.40 months) and overall survival (OS; Anlotinib: 9.63 months vs Placebo: 6.30 months) with the objective response rate (ORR) of 9.18% and the disease control rate (DCR) of 80.95%. Here, we sought to understand the gene mutation determinants for clinical response to Anlotinib via next generation sequencing (NGS) upon cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) at baseline. Totally 437 advanced NSCLC patients enrolled in ALTER-0303 study, and 294 patients received Anlotinib therapy. Of the 294 patients, 80 patients were analyzed in the present study. Capture-based targeted ultradeep sequencing was performed to obtain germline and somatic mutations in cfDNA and ctDNA. Response analyses upon discovery cohort (n = 62) and validation cohort (n = 80) were performed by use of germline and somatic (G+S) mutation burden, somatic mutation burden, nonsynonymous mutation burden, and unfavorable mutation score (UMS), respectively. Based on the above independent biomarkers and their subtype factors, tumor mutation index (TMI) was developed, and then used for response analysis. Our data indicated that the patients harbouring less mutations are better response to Anlotinib therapy (G+S muatation burden, cutoff = 4000, Median PFS: 210 days vs 127 days, p = 0.0056; somatic mutation burden, cutoff = 800, Median PFS: 210 days vs 130 days; p = 0.0052; nonsynonymous mutation burden, cutoff = 50, Median PFS: 209 days vs 130 days; p = 0.0155; UMS, cutoff = 1, Median PFS: 210 days vs 131 days; p = 0.0016). TMI is an effective biomarker for Anlotinib responsive stratification (Median PFS: 210 days vs 126 days; p= 0.0008; AUC = 0.76, 95% CI: 0.62 to 0.89) upon discovery cohort and validation cohort (Median PFS: 210 days vs 127 days; p = 0.0006). Lastly, integrative analysis of TMI and IDH1 mutation suggested a more promising result for Anlotinib responsive stratification upon validation cohort (Median PFS: 244 days vs 87 days; p < 0.0001; AUC = 0.90, 95% CI: 0.82 to 0.97). This study provide a biomarker of TMI to stratify Anlotinib underlying responders, that may improve clinical outcome for Anlotinib therapy on refractory advanced NSCLC patients at 3rd line. Clinical trial information: NCT02388919.

P3.13-09 ALTER-0303 Study: Tumor Mutation Index (TMI) For Clinical Response to Anlotinib in Advanced NSCLC Patients at 3rd Line

Anlotinib is an effective multi-targeted receptor tyrosin kinase inhibitor (TKI) for refractory advanced Non-Small Cell Lung Cancer (NSCLC) therapy at 3rd line. ALTER-0303 clinical trial has been revealed that Anlotinib significantly prolongs progression free survival (PFS; Anlotinib: 5.37 months vs Placebo: 1.40 months) and overall survival (OS; Anlotinib: 9.63 months vs Placebo: 6.30 months) with the objective response rate (ORR) of 9.18% and the disease control rate (DCR) of 80.95%. Here, we sought to understand the gene mutation determinants for clinical response to Anlotinib via next generation sequencing (NGS) upon cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) at baseline. Totally 437 advanced NSCLC patients enrolled in ALTER-0303 study, and 294 patients received Anlotinib therapy. Of the 294 patients, 80 patients were analyzed in the present study. Capture-based targeted ultradeep sequencing was performed to obtain germline and somatic mutations in cfDNA and ctDNA. Response analyses upon discovery cohort (n = 62) and validation cohort (n = 80) were performed by use of germline and somatic (G+S) mutation burden, somatic mutation burden, nonsynonymous mutation burden, and unfavorable mutation score (UMS), respectively. Based on the above independent biomarkers and their subtype factors, tumor mutation index (TMI) was developed, and then used for response analysis. Our data indicated that the patients harbouring less mutations are better response to Anlotinib therapy (G+S muatation burden, cutoff = 4000, Median PFS: 210 days vs 127 days, p = 0.0056; somatic mutation burden, cutoff = 800, Median PFS: 210 days vs 130 days; p = 0.0052; nonsynonymous mutation burden, cutoff = 50, Median PFS: 209 days vs 130 days; p = 0.0155; UMS, cutoff = 1, Median PFS: 210 days vs 131 days; p = 0.0016). TMI is an effective biomarker for Anlotinib responsive stratification (Median PFS: 210 days vs 126 days; p = 0.0008; AUC = 0.76, 95% CI: 0.62 to 0.89) upon discovery cohort and validation cohort (Median PFS: 210 days vs 127 days; p = 0.0006). Lastly, integrative analysis of TMI and IDH1 mutation suggested a more promising result for Anlotinib responsive stratification upon validation cohort (Median PFS: 244 days vs 87 days; p < 0.0001; AUC = 0.90, 95% CI: 0.82 to 0.97). This study provide a biomarker of TMI to stratify Anlotinib underlying responders, that may improve clinical outcome for Anlotinib therapy on refractory advanced NSCLC patients at 3rd line. Clinical trial information: NCT02388919.

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia.

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR.

Expression of CD274 (PD‐L1) is associated with unfavourable recurrent mutations in AML

Expression of <scp>CD</scp>274 (<scp>PD</scp>‐L1) is associated with unfavourable recurrent mutations in <scp>AML</scp>

Response and survival rates with frontline hypomethylating agent (HMAs) in favorable risk AML.

7039 Background: HMAs are an accepted frontline therapy for AML patients (pts) who are unfit for intensive induction therapy (IIT), particularly pts with unfavorable cytogenetics and/or p53 mutations. However, little is known about the response of favorable risk AML to HMAs. We previously reported that NPM1 mutated and/or CD34- AML status were predictors of response to HMAs. Here, we evaluated responses to frontline HMAs in AML. Methods: A total of 117 patients with de novo AML diagnosed between 7/2013 and 9/2016 were evaluated based on pt and disease related variables, overall response rate (ORR = CR + CR with incomplete count recovery + hematologic remission (ANC &gt; 1000/µL, Hgb &gt; 10g/dL, Plts &gt; 100,000/µL, &amp; no circulating blasts)), and overall survival (OS). Categorical variables were compared using Fisher’s exact test. Kaplan Meier methods estimated survival outcomes, and log rank tests compared survival between groups. Multivariable analyses were performed using Cox proportional hazards models. Results: 51 pts, considered unfit for IIT, received frontline HMAs. ORR and OS were highest in the ELN favorable risk AML pts (n = 13; ORR = 92%, p = .009; median OS = 17.5 months, p = .022). Among 41 NPM1 mutated pts, 15 received HMAs; and 26 received intensive induction. ORRs were 73% and 84%, respectively (p = .434). No difference was found in OS distributions between the HMA and IIT groups in univariate and multivariate (adjusted for age and FLT3 status) models (p = .329 and .241, respectively). Interestingly, ORR was 100% among 9 HMA-treated pts with NPM1 mutated, CD34-, FLT3/ITD-, cytogenetically normal AML. Conclusions: HMA therapy is highly effective frontline treatment in favorable risk AML pts considered unfit for IIT. Survival results with HMAs in NPM1 mutated AML are comparable to those of fitter pts treated with IIT. In selected favorable risk pts considered unfit for standard induction, HMAs can be a successful bridge to potentially curative therapy, including more intensive therapy or transplant. Cytogenetically normal AML with an isolated NPM1 mutation and CD34- status appears to be exceptionally responsive to frontline treatment with HMAs. Prospective validation of these findings is necessary.

Crispr/Cas9-Mediated ASXL1knockout in U937 Human Leukemic Cells Perturbs Myeloid Differentiation

Introduction.Mutations in the Additional Sex Combs Like1 (ASXL1) gene are frequent in myeloid malignances (myelodysplastic syndromes [MDS], myeloproliferative neoplasms, chronic myelomonocytic leukemia and acute myeloid leukemia [AML]), and they predict poor survival. As we recently published (Yoshizato T, et al., N Engl J Med 2015), mutations in ASXL1 as well as BCOR/BCORL1, PIGA, and DNMT3A are most prevalent in patients with aplastic anemia (AA). ASXL1 grouped with "unfavorable mutations" that conferred poor survival and increased risk of "clonal evolution" of AA to MDS and AML. As an epigenetic modifier, ASXL1mutations may be involved in myeloid malignant transformation (Davies C, et al., Br J Haematol 2013; Abdel-Wahab O, et al., Cancer Cell 2012) but precise mechanisms have not been delineated.Methods.The CRISPR/Cas9 system was employed to generate ASXL1-knockout clones from the U937 human leukemic cell line. Single cell clones were sorted by flow cytometry, and ASXL1 mutations were assessed by Sanger sequencing. Clones with mutations that encoded truncated proteins were used for further experiments. Characteristic features of both mutated and wild-type (WT) clones were examined: cell morphology by Wright-Giemsa staining; karyotype analysis by G-banding; and cell cycle, apoptosis, and cell differentiation by flow cytometry. RNA sequencing (RNA-Seq) was performed to screen differentially expressed genes between WT and ASXL1-knockout clones, followed by validation of gene expression using reverse transcription quantitative PCR (RT-qPCR).Results.Of 88 single cell clones, 23 clones exhibited frame-shift and nonsense mutations of ASXL1, resulting in truncated proteins. Among them, 17 clones with single nucleotide insertion (c.594insA, heterozygous or homozygous) that encoded truncated proteins Ser199Glufsx55 were used for further experiments. Karyotype analysis revealed no significant differences between WT and ASXL1-knockout clones, suggesting that neither CRISPR/Cas9 itself nor ASXL1 mutations caused chromosomal instability. No significant differences between WT and ASXL1-knockout clones were observed in cell morphology, cell proliferation, cell cycle, or 5-fluoruracil-induced cell apoptosis. When monocyte/macrophage differentiation was induced chemically by exposure to phorbol 12-myristate 13-acetate, CD11b cell surface expression was much lower in ASXL1-knockout clones than in WT. By RNA-Seq, several genes (BIRC7, CACNA2D3, CTSG, CYBB, NAIP, NTNG1, OXR1, and ACTL8)involved in pathways related to cell death and survival (including TNFR1 signaling pathway, TNFR2 signaling pathway, TWEAK signaling pathway, apoptosis signaling pathway, death receptor signaling pathway, Rac signaling pathway, and phagosome maturation pathway) were down-regulated, but were not correlated to functional deficits in cell growth or apoptosis, which was indistinguishable between ASXL1-knockout and WT clones. RT-qPCR confirmed down-regulation of genes in ASXL1-knockout compared to WT clones, including CYBB (restrictedly expressed in terminally differentiating myeloid cells) and CLEC5A (involved in granulocytic differentiation), which may contribute to disturbance in myeloid differentiation of ASXL1-knockout U937 cells. Downregulaton of NAIP, which inhibits CASP3, CASP7, and CASP9 activities, may be related to deregulation of apoptosis in ASXL1-knockout U937.Conclusion.ASXL1 mutations are frequent in both malignant and non-malignant blood diseases, and may be involved in myeloid neoplastic transformation. Using gene editing of a human leukemic cell line, we show that ASXL1-knockout perturbs myeloid differentiation and down regulates multiple genes associated with myeloid development such as CYBB and CLEC5A, providing potential mechanisms for its role in bone marrow failure and neoplastic transformation. DisclosuresYoung:Novartis: Research Funding.

Analysis of the PD-1/PD-L1 Axis Points to Association of Unfavorable Recurrent Mutations with PD-L1 Expression in AML

BackgroundOne of the potential mechanisms responsible for leukemic cells evading cytotoxic T lymphocytes might be the pathway of programmed death-1 receptor (PD-1). PD-1 and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, promoting tumor development.AimsAs PD-L1 expression was broadly described in many types of human cancer cells including AML, we were focused on PD-1 expression, which until now was mostly characterized for T cells, where it inhibits proliferation and cytotoxic capabilities. Recently, Wang et al. described PD-L1 expression regulation through miR-34a molecules in AML patients. Overexpression of miR-34a blocked PD-L1 and reduced its surface expression. Moreover, Cortez et al. for the first time identified novel, complete mechanism of PD-L1 regulation by p53 via miR-34a in non-small cell lung cancer (NSCLC). In this study, our comprehensive analyses of PD-1, PD-L1, TP53and miR-34a expression in AML patients shed new light on the complex regulation of PD-1/PD-L1 axis during development of this disease.MethodsWe performed analysis of TP53, PD-1, PD-L1 and miR-34a expression in 197 AML patients available from The Cancer Genome Atlas (TCGA) database. Moreover, we assessed mRNA expression of TP53 and PD-1 in independent cohort of 54 primary AML patient samples using qRT-PCR method. We also characterized several SNPs for PD-1that demonstrate relevant associations with a higher risk of developing autoimmune diseases: PD-1.1 (rs36084323), PD-1.3 (rs11568821), PD-1.5 (rs2227981), PD-1.6 (rs10204525), PD -1.7 (rs41386349), PD-1.9 (rs2227982) in 54 AML, 64 MDS and 100 HVs samples.ResultsTCGA data analysis showed higher expression of PD-L1 in the AML group with low expression of TP53 (p=0.008). In contrast, PD-L1 expression was elevated in the group with TP53 mutations compared to unmutated TP53 (p<0.001).We also found a negative correlation of miR-34a and PD-L1 expression (r=-0.2; p=0.005), while there were no differences in PD-L1 expression between groups with or without following mutations: IDH1, TET2, RUNX1, NRAS, CEBPA, PTPN11, KIT, KRAS, FLT3, DNMT3, NPM1 and IDH2. The highest expression of PD-L1 was found in the poor prognosis group according to cytogenetic risk and molecular risk markers. We also observed an association between the expression level of PD-L1 and the number of recurrent mutations present in an AML case. Patients with more than 4 recurrent mutations were characterized with higher expression of PD-L1 compared to the group of patients with 0-3 recurrent mutations (p=0.01). Next, we observed significant differences in PD-1 expression in the group of 54 AML patients compared to HVs (p<0.001), and there were differences in the PD-1expression level regarding the PD-1. 5 polymorphism (p=0.07). Moreover, analysis of the PD-1. 3 polymorphism in HVs and MDS revealed that genotype GG was associated with nearly fivefold lower risk of disease (OR=4.93, p=0.009). We observed significant differences in OS in AML patients in case of presence of certain genotypes of PD-1. 6. Genotype AA was significant associated with higher risk of shorter OS compared to the rest of the genotypes (58 vs 333 days, HR=35; p=0.02).ConclusionsOur analyses indicate that p53 might specifically modulate the tumor immune response by regulating PD-L1 via miR-34a which directly binds to the PD-L13'-UTR and blocks its expression. Moreover, we found that high PD-L1 expression is associated with the higher numbers of recurrent mutations as well as poor cytogenetic and molecular risk groups. We found significant differences in PD-1expression in AML patients compared to HVs that further support a deregulation of a signal transduction through the PD-1/PD-1L axis in AML.While our SNP analysis in AML patients suggested a prognostic impact of PD-1. 6 polymorphism, further studies are warranted to evaluate the impact of the PD-1/PD-L1 axis in AML.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313). DisclosuresGrzasko:Janssen: Honoraria; Munipharma: Honoraria; Celgene: Honoraria.

Impact of Gender on Outcome after Chemoimmunotherapy with Fludarabine, Cyclophosphamide and Rituximab (FCR) or Bendamustine Plus Rituximab (BR) in Patients with Chronic Lymphocytic Leukemia (CLL): A Meta-Analysis of Three Phase II/III Studies of the German CLL Study Group (GCLLSG)

Impact of Gender on Outcome after Chemoimmunotherapy with Fludarabine, Cyclophosphamide and Rituximab (FCR) or Bendamustine Plus Rituximab (BR) in Patients with Chronic Lymphocytic Leukemia (CLL): A Meta-Analysis of Three Phase II/III Studies of the German CLL Study Group (GCLLSG)

Estimation of the role of single nucleotide polymorphism in lymphotoxin beta gene during pig domestication based on the bioinformatic and experimental approaches

The existence of a tradeoff between the reproductive success and immunity was demonstrated in the works conducted on wild and laboratory animals. Therefore, individuals with an increased reproductive capacity but with decreased immunity could be selected in the process of domestication. A decreased reactivity of the immune system could subsequently become inheritable by fixation of the genes with unfavorable mutations in the population. The aim of the investigation was to study (1) the genotype and allele frequencies of the rs340283541 single nucleotide polymorphism (SNP) in the lymphotoxin beta (LTB) cytokine gene in domestic pigs and wild boars; (2) the mRNA expression for this gene in mini pigs with different genotypes; and (3) to perform the bioinformatic analysis of a potential functional role of this SNP. The GG genotype frequency in the boar sample was significantly lower than this genotype frequency in the combined sample from different domestic pig breeds and populations. The level of the LTB gene mRNA expression in a lymph node in mini pigs with GG genotype had a trend towards an increase (p < 0.06) as compared with the A allele carriers. The rs340283541 SNP is located within the conservative sequence motif revealed in 12 mammalian species (that indirectly indicates important functional role of SNP). By means of the context analysis, it was detected that the A allele contains potential binding sites for the BRN-2 and AP-1 transcription factors, while the G allele contains those for the RFX1, ISGF3 (ISRE site), and USF (that are expressed in the immune system cells). Thus, an increase in the frequency of the rs340283541 SNP (located in the LTB gene 3'-region) GG genotype occurred in the process of pig domestication. The GG genotype is probably associated with an increased level of the LTB gene mRNA expression in the lymph node tissue. An increase in the expression level in pigs with the GG genotype can be associated with generation of the RFX1, ISRE, and USF binding sites and/or damage of the BRN-2 and AP-1 binding sites. It is also possible that the rs340283541 polymorphism is in linkage disequilibrium with another functionally significant mutation.

The rs9909659G/A polymorphisms of the STAT3 gene provide prognostic information in acute myeloid leukemia

Background: The signal transducer and activator of transcription 3 (STAT3) is one of the most prominent predictable factors for cancer and leukemia. This study aimed to investigate the relationship between STAT3 gene polymorphisms and the treatment outcome of acute myelogenous leukemia (AML) in a Chinese population. Methods: A total of 152 AML patients were included in our study, and the therapeutic effects were evaluated. Two single nucleotide polymorphisms (SNPs) in the STAT3 gene (rs9909659G/A and rs17886724T/C) were genotyped. SNP genotyping was performed using the MassARRAY system (Sequenom) via matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Results: The results illustrated that the frequency of the TC/CC genotype of rs17886724 was higher in the unfavorable group than that of the T/T genotypes (P=0.004). Meanwhile, the C allele in rs17886724 was more frequently classified into the unfavorable group (P=0.023). Age and gender classifications were similar for different rs9909659 genotypes. Patients with M7, low hemoglobin, unfavorable cytogenetic factor, unfavorable gene mutation factor, and high lactate dehydrogenase (LDH) were associated with the GA/AA genotype in rs9909659 (P=0.031, P=0.000, P=0.000, P=0.019, and P=0.001, respectively). Patients with the GA/AA genotype of rs9909659 suffered from poor treatment, including unfavorable cytogenetic and poor response (P=0.001 and P=0.038, respectively). Clearly, AML patients with the GA/AA genotype of rs9909659 were not sensitive to standard chemotherapy. Conclusions: The rs9909659G/A polymorphisms of the STAT3 gene could be used to predict the treatment outcome in AML patients.

Effects of TP53 and PIK3CA mutations in early breast cancer: a matter of co-mutation and tumor-infiltrating lymphocytes.

The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p<0.001) and PIK3CA with lower (p=0.004) TILs in an ER /PgR-specific manner (p<0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6% of all tumors) conferred worst DFS (HR 0.59; 95% CI 0.44-0.79; p=0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p=0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95% CI 0.47-0.88; p=0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95% CI 1.07-2.24; p=0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials.