Primary Chemoresistance Research Articles

Assessment of the Expression of Genes Related to Epigenetic and Chromatin Modifications in Diffuse Large B-Cell Lymphoma Patients Resistant to R-CHOP

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. The mainstay of frontline therapy remains R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or R-CHOP-like immunochemotherapy. Patients' responses to R-CHOP therapy vary depending on the stage and/or molecular subtypes of DLBCL. Approximately 40% of advanced-stage DLBCL patients suffer from a refractory or relapsed disease[1]. The outcome for patients with early relapses after rituximab-containing first-line therapy remains poor[2]. DLBCL poses a marked degree of genetic heterogeneity. Here, we compare the expression of genes related to epigenetics and chromatin modifications in DLBCL patients resistant and non-resistant to R-CHOP therapy. Objective The aim of this study is to assess the differences in the expression of genes related to epigenetics and chromatin modification in DLBCL patients that could lead to chemoresistance and failure of R-CHOP treatment. Method The gene expression profile (GEP) was obtained from Gene Expression Omnibus (GEO) database (Accession number: GSE173263) submitted by Dlouhy et al[3]. The GEP was analyzed in DLBCL cases, including 10 early failure cases (defined as refractory to induction treatment or early relapsing (<12 months from diagnosis)) and 29 responding patients, to identify features associated to primary chemoresistance. The comparison was done using the list of genes proposed by Bakhshi, T. et. al[4] , where he selected the genes that were identified to potentially cause epigenetic and chromatin modifications in DLCBL. All the comparisons were done using a T-test. Results There were significant differences in the gene expression related to epigenetics and chromatin modifications between DLBCL patients in the early failure arm versus the remission arm. Patients who had early failure to R-CHOP tended to have higher expression of these genes: SMARCA4 (p=0.010), SETD1B (p=0.018), SETD2 (p=0.024), EP300 (p=0.012), MLL3 (p=0.049), INO80 (p=0.001), CHD8 (p=0.007), NCOR1 (p=0.020), ARID1B (p=0.034), and SETD5 (p=0.007). Compared to the patients who were in remission after R-CHOP therapy, these patients had significantly higher expression of HIST1H1E (p=0.007). Conclusion Gene expression related to epigenetics and chromatin modifications significantly impacts the prognosis of DLBCL patients treated with R-CHOP. Differences in gene expression may lead to chemoresistance, and higher expression levels are associated with a worse prognosis. This study sheds light on the importance of chromatin and epigenetic modifications in DLBCL progression, guiding the development of future therapies.

Neoadjuvant Chemoradiotherapy Followed by Radical Cystectomy for Muscle-Invasive Bladder Cancer: Analysis of Efficacy and Safety in 119 Patients

IntroductionCisplatin-based systemic chemotherapy is recommended as neoadjuvant treatment for muscle-invasive bladder cancer (MIBC) before radical cystectomy (RC). However, clinical challenges include the possibility of primary chemoresistance and limited feasibility in patients with renal impairment. This study investigated the efficacy and safety profiles of neoadjuvant chemoradiotherapy (NCRT) followed by RC. Materials and MethodsWe retrospectively analyzed 119 patients with nonmetastatic MIBC, who were pathologically diagnosed with urothelial carcinoma and underwent NCRT before RC. The pathological response to NCRT was evaluated using RC specimens. Recurrence-free survival (RFS) and cancer-specific survival (CSS) were compared according to pathological responses to NCRT. ResultsOf the 119 patients, 111 (93%) underwent RC; ypT0 and downstaging to ≤ypT1 were observed in 42 (38%) and 76 (68%) patients, respectively. In the multivariable analysis, smaller tumor size was independently associated with ypT0. During a median follow-up of 5.2 years, 28 (25%) patients developed recurrence and 22 (20%) died of bladder cancer after RC. The 5-year RFS and CSS rates were 75% and 80%, respectively. The 5-year RFS rates in patients with ypT0, ypTa/is/1, and ≥ypT2 were 87%, 87%, and 46%, respectively. Similarly, patients with ypT0 and ypTa/is/1 had more favorable CSS (90% and 87% at 5 years, respectively) than those with ≥ypT2 (60%, P = .001). None of the patients experienced ≥grade 4 adverse events related to NCRT or ≥grade 4 complications of RC. ConclusionsThis study demonstrated sufficient efficacy and safety profile of NCRT followed by RC. Chemoradiotherapy may be a helpful alternative for neoadjuvant treatment before RC.

Therapeutic targeting in pediatric acute myeloid leukemia with aberrant HOX/MEIS1 expression

Despite advances in the clinical management of childhood acute myeloid leukemia (AML) during the last decades, outcome remains fatal in approximately one third of patients. Primary chemoresistance, relapse and acute and long-term toxicities to conventional myelosuppressive therapies still constitute significant challenges and emphasize the unmet need for effective targeted therapies. Years of scientific efforts have translated into extensive insights on the heterogeneous spectrum of genetics and oncogenic signaling pathways of AML and identified a subset of patients characterized by upregulation of HOXA and HOXB homeobox genes and myeloid ecotropic virus insertion site 1 (MEIS1). Aberrant HOXA/MEIS1 expression is associated with genotypes such as rearrangements in Histone-lysine N-methyltransferase 2A (KMT2A-r), nucleoporin 98 (NUP98-r) and mutated nucleophosmin (NPM1c) that are found in approximately one third of children with AML. AML with upregulated HOXA/MEIS1 shares a number of molecular vulnerabilities amenable to recently developed molecules targeting the assembly of protein complexes or transcriptional regulators. The interaction between the nuclear scaffold protein menin and KMT2A has gained particular interest and constitutes a molecular dependency for maintenance of the HOXA/MEIS1 transcription program. Menin inhibitors disrupt the menin-KMT2A complex in preclinical models of KMT2A-r, NUP98-r and NPM1c acute leukemias and its occupancy at target genes leading to leukemic cell differentiation and apoptosis. Early-phase clinical trials are either ongoing or in development and preliminary data suggests tolerable toxicities and encouraging efficacy of menin inhibitors in adults with relapsed or refractory KMT2A-r and NPM1c AML. The Pediatric Acute Leukemia/European Pediatric Acute Leukemia (PedAL/EUPAL) project is focused to advance and coordinate informative clinical trials with new agents and constitute an ideal framework for testing of menin inhibitors in pediatric study populations. Menin inhibitors in combination with standard chemotherapy or other targeting agents may enhance anti-leukemic effects and constitute rational treatment strategies for select genotypes of childhood AML, and provide enhanced safety to avoid differentiation syndrome. In this review, we discuss the pathophysiological mechanisms in KMT2A-r, NUP98-r and NPM1c AML, emerging molecules targeting the HOXA/MEIS1 transcription program with menin inhibitors as the most prominent examples and future therapeutic implications of these agents in childhood AML.

Clinical significance of L1CAM expression in metastatic tubo-ovarian high-grade serous carcinoma

ObjectiveTo analyze the expression and prognostic role of L1CAM in tubo-ovarian high-grade serous carcinoma (HGSC). MethodsL1CAM protein expression by immunohistochemistry was analyzed in 644 HGSC (413 effusions, 231 surgical specimens). Expression was analyzed for association with clinicopathologic parameters and survival. ResultsL1CAM protein expression was found in 401/413 (97%) effusions and 209/231 (90%) surgical specimens, with significantly higher staining extent in effusions (p < 0.001). L1CAM protein expression in effusions was unrelated to clinicopathologic parameters (p > 0.05). In surgical specimens, higher L1CAM expression was significantly related to primary (intrinsic) chemoresistance (p = 0.017). High (>25%) L1CAM expression in HGSC effusions (p = 0.02), older patient age (p = 0.013), FIGO stage IV disease (p < 0.001) and larger residual disease volume (p = 0.001) were significantly associated with shorter overall survival (OS) in univariate analysis. In Cox multivariate analysis, only FIGO stage (p = 0.001) and residual disease volume (p = 0.003) were independent prognosticators of OS. L1CAM expression in effusions was unrelated to progression-free survival (PFS). There was no association between L1CAM expression in surgical specimens and survival. ConclusionL1CAM is overexpressed in HGSC effusions compared to surgical specimens. Its overexpression in effusions is significantly associated with shorter OS, but not independently of established prognostic factors such as FIGO stage and residual disease volume.

TIPE2 sensitizes breast cancer cells to paclitaxel by suppressing drug-induced autophagy and cancer stem cell properties.

Drug resistance is a great obstacle to the clinical application of paclitaxel (PTX) in breast cancer treatment. Chemoresistance can be either primary or acquired. Multifarious factors are related to drug resistance. Among these factors, drug-induced autophagy has been shown to contribute to acquired chemoresistance in cancer cells. Additionally, cancer stem cells (CSCs) drive primary chemoresistance. Recent advances regarding TIPE2 demonstrate that TIPE2 enhances osteosarcoma and non-small cell lung cancer cell sensitivity to cisplatin. However, the role of TIPE2 in PTX resistance in breast cancer cells has not been elucidated. Here, the in vitro and in vivo study demonstrated that TIPE2 sensitized breast cancer cells to PTX by suppressing drug-induced autophagy and CSC properties. Mechanistically, we found that TIPE2 activated the AKT/mTOR signalling pathway and inhibited the TAK1/MAPK signalling pathway to suppress drug-induced autophagy. Moreover, TIPE2 inhibited TAK1/NF-κB activation to reduce breast CSC properties. Collectively, our results first elucidated the inhibitory role of TIPE2 in breast cancer chemoresistance. Thus, TIPE2 may be a new target for breast cancer chemotherapy.

The Clinico-Genomic Landscape of Induction Failure in Childhood and Young Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy comprising 10-15% of childhood ALL. Previously, we found that Induction Failure (IF) occurs in approximately 10% of T-ALL cases, 3-fold more than in B-ALL, with only 50% achieving long-term survival (O'Connor et al, JCO 2017). Despite the preponderance of IF in T-ALL, little is known about the factors that predict treatment resistance and influence outcome, or how best to treat these patients. To address this, we present the largest cohort of T-ALL IF reported to date, comprising all IF cases on two large multi-national consecutive randomized trials, UKALL2003 and UKALL2011. Methods The UKALL2003 and UKALL2011 trials recruited 711 patients with T-ALL between 2004-2019; 688 of these patients were assessable at the end of induction (EOI). We supplemented existing trial data with new clinical data, and combined whole genome sequencing (WGS) and RNA-sequencing (RNAseq), to comprehensively characterize the clinical and genetic landscape of T-ALL IF. Results Of 688 T-ALL patients, 10.3% experienced IF. IF was significantly associated with increasing age occurring in 5.6% of patients below 10 years, 12.9% of those aged 10-16 years and 20% of patients over 16 years (p<0.001). Survival was significantly worse in IF (5yr Event-free survival (EFS) 47.9% (95%CI: 35.8 - 59.1) vs. 84.0% (95%CI: 80.7 - 86.8); HR:5.14 (95% CI: 2.82-6.07), p<0.001; 5yr Overall survival (OS) 52.1% (95%CI: 39.7 - 63.1) vs. 90.2% (95%CI: 87.4 - 92.5); HR: 6.38 (95% CI: 4.17-9.76), p<0.001). There was a clear trend towards increasing use of nelarabine over the study period; IF patients treated on UKALL2003 commonly remained on standard trial chemotherapy (74.2%; 26/35), while IF patients treated on the later UKALL2011 trial were more frequently treated with nelarabine and HSCT (68.6%; 24/35, p=0.0007). However, this did not translate into an improved outcome for IF patients, with no clear benefit of either HSCT or Nelarabine therapy. WGS revealed a picture of remarkable genetic heterogeneity, with 25 different initiating lesions converging on 10 subtype-defining T-ALL genes, rather than a single unifying driver of IF (Figure 1). Allocation of cases to conventional T-ALL subtypes found almost half were of the HOXA subtype, significantly more than in responsive T-ALL (p<0.001). This was consistent with immunophenotypic characterisation showing a significant enrichment of the Early T-cell precursor (ETP) subtype, present in 48% of IF cases vs. 10% of non-IF cases (p<0.001), indicating a less differentiated, more stem cell-like leukemia. Although the overall proportion of TAL1 cases was lower in IF, there was an unexpected dominance of non-coding TAL1 enhancer mutations, accounting for 87.5% of TAL1 cases. Importantly, TAL1 cases had a particularly dismal outcome with an OS of 12.5% (0.6-42.3). Notably, none of the 7 patients with a TAL1 non-coding driver lesion survived, and 2 never achieved remission, identifying this as a very high-risk genetic lesion in the context of IF. Mutational profiling found a reduction in mutations in several highly recurrent T-ALL genes (NOTCH1, FBXW7, CDKN2A, LEF1) coupled with an increase in mutations in WT1 and MED12, both of which have been associated with disease resistance in other leukemias. In addition, 2 genes not previously reported in T-ALL were recurrently mutated in IF with mutations in CHD4 in 5 cases and KAT6A in 4 cases, suggesting they may be novel drivers of refractory disease. Survival analyses also showed a significantly poorer survival in patients with mutations in MYCN and NRAS. WGS of samples collected at the end of induction showed no new lesions demonstrating that disease at presentation is representative of true refractory leukemia and that IF does not occur as a result of chemotherapy-induced mutagenesis. Conclusions Our results confirm the poor prognosis of IF in T-ALL and highlight a lack of progress in treatment of this high-risk leukemia, with no clear improvement in outcome despite increased use of nelarabine and HSCT. Genomic analyses identified a marked enrichment for non-coding TAL1 enhancer mutations, characterizing an ultra-high-risk group of patients in the context of IF, in which there were no survivors. The lack of a readily targetable pathway of primary chemoresistance supports alternative strategies, such as those employing immunotherapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 2359: Identifying synergistic master regulators of pancreatic ductal adenocarcinoma tumorigenesis through CRISPR/Cas9 combinatorial genetic screens

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related mortality and is highly resistant to cytotoxic, targeted, and immune therapies. Compared to other cancers, PDAC is remarkable for its relatively uniform set of DNA alterations; unfortunately, these hallmark events are not currently targetable and other mutations known to confer sensitivity to specific drugs are uncommon. Consequently, cytotoxic combinations remain the standard of care, with most patients quickly exhibiting primary or secondary chemoresistance. Previous studies have shown that transcriptional regulatory proteins can form small, highly interconnected and autoregulated modules that can initiate and maintain malignant tumor cell state, and that the Master Regulator (MR) protein they comprise represent a novel class of non-oncogene dependencies. We used the VIPER algorithm to identify MR proteins representing mechanistic regulators of the transcriptional state for 200 laser microdissected PDAC epithelial compartment samples, collected at Columbia University Irving Medical Center (CUIMC). To systematically identify synergistic pairs of MRs, we designed a combinatorial CRISPR-Cas9 screen based on the Big Papi dual SaCas9/SpCas9 system which was used to systematically target all possible pairs of the top 50 MRs identified by VIPER analysis across 6 PDAC cell lines (KP4, PATU8988S, PANC1, HPAFII, BXPC3, MIAPACA2) with three guide-RNAs per gene, thus differentiating between potentially idiosyncratic, cell line-specific and highly conserved genetic interactions. We also developed a novel nonparametric statistical method for inferring genetic interactions from this data based on the use of conditional Mann-Whitney U tests; our new analytical method significantly improved over a previously developed parametric model of synergy inference, demonstrating increased correlation between genetic interactions inferred from biological replicates of the same cell line while simultaneously prioritizing four established synthetic lethal gene pairs in the top 10 gene pairs following integration across all 6 cell lines: BCL2L1-MCL1, BCL2L1-BCL2L2, MAPK1-MAPK3, and SEC23A-SEC23B. The methods also identified 314 additional highly significant (FDR &amp;lt; 0.05), yet never previously reported synthetic lethal interactions that are being validated using an array-based, low-throughput screening platform. We expect that the results of this screen will help elucidate mechanisms controlling the state of PDAC tumor cells and identify potentially druggable gene pairs. Citation Format: Aaron T. Griffin, Mikko Turunen, Lorenzo Tomassoni, Kenneth P. Olive, Pasquale Laise, Andrea Califano. Identifying synergistic master regulators of pancreatic ductal adenocarcinoma tumorigenesis through CRISPR/Cas9 combinatorial genetic screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2359.

Chemosensitivity Evaluation using Circulating Tumor Cells Predicts Chemotherapy Outcomes in Patients with Advanced Cancer.

Chemotherapy is a clinically recognized effective technique for systemic treatment of malignant tumors. However, the tumor heterogeneity and multiple drug resistance (MDR) to the chemotherapeutic agents often lead to a failure of response to chemotherapy. We utilized a novel in vitro chemosensitivity test to identify sensitive and effective chemotherapeutic drugs and further elucidated the correlation between the in vitro chemosensitivity and clinical outcomes. We developed a circulating tumor cell-based in vitro drug sensitivity test to evaluate the sensitivity of different chemotherapeutic agents. High glucose uptake combined with negative CD45 marker were exploited to distinguish the CTCs from leukocytes. The altered glucose metabolism of single cell was measured by custom-designed computational algorithm, and the toxicity of different drug combinations was assessed by different fluorescent intensity on CTCs in the treated and control group. We analyzed the potential of CTCs in predicting chemotherapy response in 92 patients with different cancer types. Our data showed that the isolated CTCs accurately predicted chemotherapy outcomes, especially in patients with late-stage cancer. CTC-based chemosensitivity evaluation can help guide clinical decision making and identify patients who are likely to benefit from chemotherapy. CTC-based chemosensitivity evaluation is an effective methodology to study the chemosensitivity of tumor cells in vitro. Our results using CTC-based chemosensitivity evaluation method were well correlated with the clinical outcomes of chemotherapy. The clinical implementation of our CTC-based chemosensitivity evaluation method can help spare patients with primary chemoresistance from the unnecessary toxicities of chemotherapy and improve chemotherapy outcomes.

SOX8 Affects Tumoral SPARC Expression by Regulating EZH2 to Attenuate Effectiveness of albumin-bound paclitaxel in PDAC.

Pancreatic cancer is a dismal malignancy with poor prognosis. In spite of progress in surgical technology, chemotherapy is still the cornerstone in the multi-disciplinary treatment. Albumin-bound paclitaxel is a first-line treatment for PDAC patients. Yet the response rate of the drug is far from satisfying. SOX8 is a member of the sex determining region Y-boxes family, which is potentially related to the chemoresistance of tumor. Patient with high expression of SOX8 were insensitive to albumin-bound paclitaxel. SOX8 reduced apoptosis and G2/M cell cycle arrest caused by albumin-bound paclitaxel. SOX8 transcriptionally regulated EZH2, which reduced expression of SPARC by promoting the methylation of SPARC, thereby reducing the transport of albumin-bound paclitaxel in pancreatic cancer cells. EZH2 inhibitor, UNC1999, can reverse the effect of SOX8 on chemo-resistance of albumin-bound paclitaxel. Collectively, our data revealed SOX8/EZH2/SPARC signaling induced primary chemo-resistance of albumin-bound paclitaxel in pancreatic ductal adenocarcinoma.

High Toxicity and Poor Survival with Association CHOP Plus Etoposide Compared to CHOP Regimen in 124 Brazilian Patients with Nodal PTCL Lymphomas (nPTCL): A Real-Life Experience

Introduction: Nodal PTCL constitute a heterogeneous group of rare malignancies derived from CD4+ T-helper, CD8+ T-cytotoxic or follicular T-helper lymphocytes. It presents aggressive clinical-biological behavior and distinct outcomes [1]. These tumors have significant geographic variation, making important studies of clinical and epidemiological characteristics and outcomes of patients in specific areas of the word. Latin American data on nPTCL are scarce in the literature [2] and its first-line treatment is still controversial and ineffective, due to high rates of primary chemo-resistance. Therefore, this study aims to describe clinical, laboratory and epidemiological characteristics, identify prognostic factors and analyze the outcomes of patients with nPTCL treated with CHOP/CHOP-like regimens as first-line therapy in Brazil.Methods: This is a retrospective, observational and unicentric study involving 124-Brazilian patients with nPTCL treated at HC/FMUSP from January 2000 to December 2017. All cases were submitted to centralized histopathological review and classified according to WHO-2016 criteria on PTCL/NOS, AITL, ALK+/ALCL or ALK-/ALCL. Clinical-laboratory and outcomes data were obtained from digital medical records. Descriptive variables were arranged in absolute numbers and relative frequencies. OS and PFS curves were estimated by the Kaplan-Meier method. Univariate Cox analysis was used to determine factors with prognostic impact through the association between categorical variables and survival curves. Variables that were significant in the univariate analysis were tested in a multivariate analysis to establish independent variables. Statistical analysis was performed using SPSS V.22 software and p-values ≤ 0.05 were considered statistically significant.Results: The clinical-laboratory characteristics of 124 nPTCL patients were summarized in Table 1. With a median age of 48.5 years (18-87 years) and 57.3% of male, about 81.5% had B-symptoms, 88.7% with CS III/IV and 58.1% had IPI ≥ 3. ORR to first-line treatment was 58.9%, 37.9% (47/124) were treated with CHOP regimen and 35.5% (44/124) with CHOEP, 30.1% (37/124) were submitted to radiotherapy and 32.3% (40/124) were consolidated with ASCT. We observed a higher 2-year OS for patients treated with CHOP versus CHOEP (78.7% vs. 61.4%; p=0.05), as well as a better 2-year PFS for the same regimen (69.7% vs. 25.0%; p<0.0001) - Figure 1. CHOEP treatment was associated with higher rates of G3-4 neutropenia, febrile neutropenia and G3-4 thrombocytopenia (57% x 88% p=0.001, 38% x 70% p=0.003 and 27% x 63% p=0.0007, respectively). Overall mortality was 55.6% (69/124) during all follow-up, with disease progression being the major cause of death (29/69 - 42.0%). With a median follow-up of 23.7 months (0.10-278.6 months), medians of OS and PFS were 48.0 months (95% CI: 9.0-86.9) and 8.8 months (95% CI: 3.9-13.7), respectively. Estimative of 2-year OS and PFS for the global cohort were 61.3% and 41.5%, respectively. In the univariate analysis, factors with a favorable prognostic impact on OS were: IPI < 3 (HR: 0.30; 95% CI: 0.15-0.58; p<0.0001), absence of bone marrow infiltration (HR: 0.39; 95% CI: 0.20-0.75; p=0.005), LDH < 480 U/L (HR: 0.36; 95% CI: 0.19-0.68; p=0.002), radiotherapy (HR: 0.23; 95% CI: 0.10-0.55; p=0.001) and ASCT (HR: 0.28; 95% CI: 0.004-0.30; p<0.0001). Factors associated with better 2-year PFS were: IPI < 3 (HR: 0.36; 95% CI: 0.18-0.71, p=0.004), absence of bone marrow infiltration (HR: 0.30; 95% CI: 0.26-0.55; p=0.03 ), LDH < 480 U/L (HR: 0.36; 95% CI: 0.19-0.67; p=0.001), radiotherapy (HR: 0.17; 95% CI: 0.06-0.44; p<0.0001) and ASCT (HR: 0.03 ; 95% CI: 0.004-0.23); p=0.001). In the multivariate analysis, factors associated with better 2-year OS were: LDH < 480 U/L (HR: 0.40; 95% CI: 0.21-0.76); p=0.005) and ASCT (HR: 0.47; 95% CI: 0.006-0.34; p=0.003). LDH < 480 U/L (HR: 0.45; 95% CI: 0.23-0.87; p=0.01) and ASCT (HR: 0.07; 95% CI: 0.01-0.54; p=0.01) were also associated with higher 2-year PFS.Conclusion: This is the largest real-life Latin American nPTCL cohort published to date. Patients with nPTCL have poor survival and high rate of chemo-resistance. In our cohort, adding etoposide to the CHOP regimen showed no survival benefit and was associated with high toxicity. Normal values of LDH and consolidation with ASCT were independent factors associated with better outcomes in Brazilian patients with nPTCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Myeloablative Conditioning With PBSC Grafts For T-Replete Haploidentical Donor Hematopoietic Cell Transplantation Using Post-Transplant Cyclophosphamide Results In Universal Engraftment, Low Rates Of Gvhd, NRM and Excellent Survival Outcomes: An Analysis Of Two Consecutive Phase II Studies From a Single Center

Lack of a matched sibling or suitably matched unrelated donor can be a significant barrier to allogeneic hematopoietic cell transplantation in patients who stand to benefit from this procedure. HLA-haploidentical donors are readily available for nearly all such patients. Whereas previous attempts at haploidentical donor hematopoietic stem cell transplantation (haploSCT) using standard immunosuppression or ex-vivo T-cell depletion have had relatively poor outcomes, a recent approach using a T-replete bone marrow graft with non-ablative conditioning and post-transplant cyclophosphamide (ptCy) for control of alloreactivity has proved to be remarkably well tolerated (Luznik et al BBMT 2008). However, relapse of malignancy remains a major challenge with the use of this non-myeloablative regimen. The use of myeloablative conditioning and a PBSC graft may help reduce the relapse rate following T-replete haploSCT but this has not been well studied. We enrolled a total of 41 patients on two consecutive prospective phase II trials of myeloablative haploSCT and ptCy using a T-replete G-CSF mobilized PBSC graft. The first trial (NSH 864) employed a high-dose busulfan-based preparative regimen (fludarabine 30 mg/m2/d d-6 to -2, Busulfan 110-130 mg/m2/d d-7 to -3 , Cy 14.5 mg/kg/d d-6&-5) [n=20], the second (NSH 922) utilized a TBI-based regimen (fludarabine 25 mg/m2/d x 3 d and TBI 150 cGy bid on d -4 to -1 [total dose 1200cGy]) [n=21]. Post-grafting immunosuppression consisted of Cy 50mg/kg/day on days 3 and 4, MMF through d 35, and tacrolimus through d 180 on both protocols. Eligibility was limited to patients ≤60 years of age with aggressive/advanced hematologic malignancies perceived to be at high risk of relapse following nonmyeloablative transplant. Leukemia patients in CR1 were eligible if they had either poor-risk cytogenetics/molecular studies or primary chemoresistance. Median patient age was 45 years (range 24-60). Twenty-five patients were transplanted in complete remission (CR) or in chronic phase (CP), while 16 patients were transplanted with advanced disease. No patient experienced graft failure, with a median time to neutrophil and platelet recovery of 16 and 25 days, respectively. All evaluable patients achieved sustained complete donor T cell and myeloid chimerism by Day +30. Median follow-up for survivors is 20 months (NSH 864 - 39 months; NSH 922 – 11months). The cumulative incidence of grades II-IV and III-IV acute GVHD at day +100 was 30% and 17% respectively. The cumulative incidence of chronic GVHD at one year post transplant was 41% (severe in 9%). Non-relapse mortality (NRM) at 12 and 24 months was 8% and 17% respectively. Cumulative incidence of relapse was 31% at both 12 and 24 months post-transplant. The relapse rate was lower in patients transplanted in CR/CP (18%) vs. those transplanted with advanced disease (49%, p=0.044). BK virus-associated hemorrhagic cystitis (HC) occurred in 56% of patients, and was severe (requiring hospital admission for bladder irrigation and/or pain management) in 19%. The incidence of HC and severe HC was significantly lower in patients receiving TBI-containing conditioning than those receiving Busulfan (38% vs. 75%, p=0.028 and 10% vs. 30%, p=0.13, respectively). With a median follow-up of 20 months, the estimated 2 year overall survival (OS) was 51% with a 52% estimated disease-free survival (DFS). In patients transplanted in CR/CP, 2-year DFS was 64% vs. 35% in patients transplanted with advanced disease (p=0.077). There was an encouraging trend to better 2-year DFS in patients receiving TBI-based conditioning than those receiving Busulfan, 75% vs. 40%, p=0.072. Myeloablative haploSCT with PBSC grafts and ptCy allows reliable engraftment with early donor T cell chimerism; low rates of GVHD, infectious complications, and NRM; and promising survival in patients with high-risk hematologic malignancies. Outcomes are particularly good in patients treated in CR/CP (18% relapse, 64% DFS). When compared with Busulfan-based conditioning, TBI-based conditioning is associated with less risk of HC and improved DFS. Myeloablative haploSCT is a valid option for patients with aggressive/advanced hematologic malignancies who lack timely access to a conventional donor.▪▪ Disclosures:No relevant conflicts of interest to declare.

Promoter Methylation of the MGRN1 Gene Predicts Prognosis and Response to Chemotherapy of High-Grade Serous Ovarian Cancer Patients.

Aberrant DNA methylation is considered to play a critical role in the chemoresistance of epithelial ovarian cancer (EOC). In this study, we explored the relationship between hypermethylation of the Mahogunin Ring Finger 1 (MGRN1) gene promoter and primary chemoresistance and clinical outcomes in high-grade serous ovarian cancer (HGSOC) patients. The MALDI-TOF mass spectrometry assays revealed a strong association between hypermethylation of the MGRN1 upstream region and platinum resistance in HGSOC patients. Spearman’s correlation analysis revealed a significantly negative connection between the methylation level of MGRN1 and its expression in HGSOC. In vitro analysis demonstrated that knockdown of MGRN1 reduced the sensitivity of cells to cisplatin and that expression of EGR1 was significantly decreased in SKOV3 cells with low levels of MGRN1 expression. Similarly, EGR1 mRNA expression was lower in platinum-resistant HGSOC patients and was positively correlated with MGRN1 mRNA expression. Kaplan-Meier analyses showed that high methylation of the MGRN1 promoter region and low expression of MGRN1 were associated with worse survival of HGSOC patients. In multivariable models, low MGRN1 expression was an independent factor predicting poor outcome. Furthermore, low expression of EGR1 was also been confirmed to be significantly related to the poor prognosis of HGSOC patients by Kaplan-Meier. The hypermethylation of the MGRN1 promoter region and low expression of MGRN1 were associated with platinum resistance and poor outcomes in HGSOC patients, probably by altering EGR1 expression.

Mutation Profiling Reveals Novel Epigenetics and Splicing Mutations in Chemorefractory Pediatric AML

Despite substantial improvement of treatment outcome in pediatric acute myeloid leukemia (AML), chemotherapy resistance remains to be a major concern. While molecular basis behind therapeutic failure was intensely interrogated, genomic alteration of chemotherapy resistance remains poorly characterized in pediatric AML. Here, we performed whole exon sequencing (WES) to identify gene mutations associated with primary and secondary chemo-resistance in pediatric AML.Taking advantage of the available WES dataset from pediatric AML patients in two hospital centers, we compared mutation frequency of total 45 pediatric AML cases, including 24 cases of induction remission and 21 cases of chemo-resistance, to that of a cohort of 200 adult AML in TCGA dataset. We observed mutation frequency of signaling molecules were much higher in pediatric AML than in adult AML. The most frequent mutated genes are NRAS (27%, 12/45) and KIT (20%, 9/45) in pediatric AML, which has significant higher mutation frequency than in adult AML (NRAS, P=0.0002; KIT, P=0.0001). In consistent with previous findings, epigenetic regulators such as DNMT3A (0%, 0/45), TET2 (0%, 0/45), IDH1/2 (2%, 1/45) and ASXL1/2 (2%,1/45) are rarely mutated in pediatric AML patients, while they are commonly found mutated in adult AML. It's worth noting that we uncovered several novel epigenetic mutations which were not found in TCGA dataset, such as KDM5C, SIRT6, CHD4, SS18 and SETD8.We assessed whether any mutated genes are associated with primary chemo-resistance group as comparing to primary chemo-sensitive group in studies cohorts. The chemo-sensitive group contained 24 primary induction remission samples, and 3 primary chemo-sensitive diagnosis samples of secondary chemo-resistance patients. Besides high recurrence of FLT3-ITD, we observed none of signaling molecule mutations are significantly enriched in primary chemo-resistance group. It's been reported that KIT mutations are frequently mutated in pediatric AML, and associated with adverse outcome. In our studied cohort, KIT mutations are more frequently found in relapsed AML (57%, 4/7), than primary chemo-resistance (28%, 2/7). Thus, poor clinical outcome of KIT mutations is more likely attributed to disease recurrence but not primary chemo-resistance.Furthermore, epigenetic mutations were enriched at primary chemo-resistance group (7/18 vs 2/27, P=0.009), including histone modified enzyme KDM5C, SIRT6, SETD8, splisomal factors PRPF6, PRPF8 and other epigenetic regulators CHD4, and IDH2. We found high allele frequency of KDM5C point mutation (35%) and SIRT6 frameshift mutation (42%) in one chemorefractory t (8:21) translocated AML patient. In addition, we also identified splicing mutation PRPF6 and PRPF8 in chemorefractory AML patients. In light of recent discovery of epigenetic alteration mediating chemo-resistance in adult AML, we thought these epigenetic mutations found in our cohort may also play a critical role in chemo-refractory pediatric AML.The development of new treatment strategies for pediatric AML must be based on an improved understanding of the molecular mechanisms underlying chemotherapy resistance. Previously, Fiona et al, shown that SETBP1 and ASXL1 and RELN mutations were associated with primary chemo-resistance in adult and pediatric AML patients by utilizing targeted sequencing of 670 genes. We have, for the first time, performed whole exon sequencing into 21 pediatric chemo-resistance AML. We identified novel epigenetic and splicing alterations such as KDM5C, SIRT6, SS18, CHD4 and PRPF8, PRPF6, which may play critical role in leukemogenesis and chemo-resistance in pediatric AML patients. Taking together, our study shed light into genetic lesions and evolution patterns underlying chemotherapy resistance and failure in pediatric AML. Larger cohort of patients and functional studies are needed to further understand molecular spectrum underlying chemo-resistance in pediatric AML. DisclosuresNo relevant conflicts of interest to declare.

NUP-98 Rearrangements Led to the Identification of Candidate Biomarkers for Primary Induction Failure in Pediatric Acute Myeloid Leukemia.

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for “Differentially Expressed Genes” (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2, TMA7, SPCS2, CDCP1, CAPZA1, FGFR1OP2, MAN1A2, NT5C3A and SRP54. In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.

Outcomes for relapsed versus resistant low risk gestational trophoblastic neoplasia following single-agent chemotherapy

ObjectiveTo compare outcomes for relapsed versus resistant low risk gestational trophoblastic neoplasia (GTN) following single-agent chemotherapy. MethodsThis was a single center retrospective study of low risk GTN. Cases failing to achieve a normal hCG with first-line therapy were defined as chemotherapy resistance. Cases achieving hCG remission, but recurring, were defined as relapse. Primary endpoints were remission rate with second-line therapy and time to remission. Univariate and multivariate analyses were performed to define prognostic factors. ResultsAmong 877 low risk GTN patients there were 124 (14.8%) chemotherapy resistant and 22 (2.6%) relapse cases. Complete remission rates with second-line therapy were similar between relapse (77.3%) and resistant (76.6%) cases (p = 0.95), but resistance was associated with a longer time to reach complete remission with second-line therapy (median 8.3 vs 4.9 weeks; p = 0.024). In multivariate analysis, the significant prognostic factors for second-line therapy remission and time to second-line therapy remission were use of multi-agent chemotherapy (OR of 9.45; 95%CI, 2.13–41.97; p = 0.003) and primary chemo-resistance (HR of 0.27; 95%CI, 0.12–0.59; p = 0.001), respectively. With additional therapies, sustained remission rates rose to 90% (18/20) for relapse and 99.2% (120/121) for chemo-resistance (p = 0.053). ConclusionsAlthough second-line therapy for resistant or relapsed low risk GTN is able to achieve complete remission in most cases, time to complete remission for relapsed disease was shorter than for resistant disease. Further studies on the biologic differences between resistant and relapsed disease may clarify the optimal treatment for these clinical situations.

Targeting an autocrine IL-6-SPINK1 signaling axis to suppress metastatic spread in ovarian clear cell carcinoma.

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor cell expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Expression of Wnt pathway molecules is associated with disease outcome in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and clinical role of Wnt pathway molecules in metastatic high-grade serous carcinoma (HGSC). mRNA expression by qPCR of 20 molecules related to Wnt signaling (WNT1, WNT2, WNT3, WNT4, WNT5A, WNT6, WNT7, WNT11, FZD1, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, LRP5, LRP6, DKK, CCND, RUNX2) was analyzed in 87 HGSC effusions. Thirty-nine surgical specimens (19 ovarian, 20 from otherintra-abdominal sites) were analyzed for comparative purposes. Protein expression of YAP and LRP and their phosphorylated forms by western blotting were analyzed in 52 tumors. Significant differences in mRNA expression as a function of the anatomic site were observed for WNT3 (p = 0.005), WNT5A (p = 0.008), WNT7 (p < 0.001), FRZ5 (p = 0.04), and FRZ6 (p < 0.001). YAP and LRP and their phosphorylated forms were detected in HGSC specimens. FZD10 was overexpressed in effusions from patients who had complete response to chemotherapy compared with those with less favorable response (p = 0.037). WNT4 (p = 0.005), WNT7 (p = 0.047), RUNX2 (p = 0.038), LRP5 (p = 0.022), LRP6 (p = 0.011), FZD6 (p = 0.036), FZD7 (p = 0.004), and FZD10 (p = 0.015) levels were inversely related to primary chemoresistance. High FZD5 levels in pre-chemotherapy effusions tapped at diagnosis and high WNT2 levels in post-chemotherapy disease recurrence effusions were related to shorter overall survival (p = 0.018 and p = 0.011, respectively), whereas high RUNX2 (p = 0.031) and FZD1 (p = 0.029) in post-chemotherapy effusions were associated with longer overall survival. In multivariate analysis of post-chemotherapy cases, WNT2 (p = 0.002), RUNX2 (p = 0.017), FZD1 (p = 0.036), and FZD4 (p = 0.013) were independent prognosticators. In conclusion, expression of Wnt pathway molecules is anatomic site dependent. In HGSC effusions, it is informative of chemoresponse and survival.

Long-Term Outcomes of Nivolumab Therapy in Patients with Relapsed/Refractory Classic Hodgkin’s Lymphoma after High-Dose Chemotherapy with Autologous Hematopoietic Stem Cell Transplantation in Real Clinical Practice

Aim. To assess prognostic factors and to analyze the outcomes of nivolumab therapy in patients with relapsed/re-fractory classic Hodgkin's lymphoma (cHL) after autologous hematopoietic stem cell transplantation (auto-HSCT). Materials &amp; Methods. The retrospective analysis included 42 patients treated with nivolumab 3 mg/kg after auto-HSCT in the period from 2016 to 2020. The response to nivolumab therapy was assessed every three months by whole-body PET/CT based on LYRIC criteria. Toxicity profile was assessed by establishing adverse events (AE) based on NCI CTCAE 4.03 criteria. Results. The study included 42 patients with relapsed/re-fractory cHL: 21 (50 %) men and 21 (50 %) women. The median age was 32.5 years (range 22-43 years). At diagnosis the following cHL stages were identified: stage II in 14 pts (33.3 %), stage III in 12 pts (28.6 %), and stage IV in 16 pts (38.1 %). Primary chemoresistance after the first-line therapy was observed in 26 pts (61.9 %) and early relapse in 4 pts (9.52 %). The median follow-up was 38 months, 3-year overall survival was 97 % (95% confidence interval, 95% CI, 83.2-99.6 %), 3-year progression-free survival (PFS) was 34.8 % (95% CI 20.3-49.9 %; median 12.9 months). Objective response was reported in 69 % of patients, complete response (CR) in 33.3 %, partial response in 35.7 %, stable disease in 7.1 %, indeterminate response in 14.3 %, and progression in 9.5 % of patients. The analysis of factors affecting PFS revealed significant differences in patients who reached CR after 6 nivolumab cycles: 3-year PFS 56.2 % (95% CI 24.4-79.1 %) vs. 25.2 % (95% CI 10.46-43.1 %) in patients who did not reach CR (p = 0.054). If extranodal lesions were identified at nivolumab therapy onset, PFS was 29 % (95% CI 7.8-37.5 %) vs. 68 % (95% CI 35.9-86.8 %) in their absence (p = 0.0079). The overall rate of AEs on nivolumab therapy was 92.9 %, severe AEs of grade 3-4 were observed in 19.1 % of patients. Conclusion. Nivolumab shows high efficacy in the treatment of patients with relapsed/refractory cHL after the failure of auto-HSCT and considerably improves prognosis compared with historical control. The efficacy of nivolumab is independent of brentuximab vedotin use and duration of prior therapy. Throughout the follow-up period the toxicity level of nivolumab was acceptable and controlled. Clinical factors that affect prognosis for patients on immunotherapy were identified.

Influence of nivolumab on epidemiology of infectious complications in patients with relapse or refractory Hodgkin’s lymphoma

Abstract Background The clinical development of checkpoint inhibitor-based immunotherapy has ushered in an exciting era of anticancer therapy. Despite many reports on anti PD-1 antibody therapy for the treatment of Hodgkin’s lymphoma (HL), the risk of infection among patients receiving nivolumab (nivo) is still unknown. Methods Between 2016 and 2018, 112 patients with r/r HL were observed and treated with nivo (3 mg/kg) in CIC 725. The median number of nivo courses received was 20 (range, 1-30). 18 patients underwent allo-HSCT after therapy with nivo. The median follow-up period was 1.4 years (1 month-2.6 year). All patients had a standard anti-infective prophylaxis and treatment according to the international guidelines. Results During salvage treatment with nivo of r/r HL 11 (10%) patients had documented infection episodes (n = 16): bacterial infections – 37.5% (n = 6), invasive fungal diseases (IFD) – 25% (n = 4) and viral infections – 37.5% (n = 6). The median time to infection episodes was 98 days (12-365) after first nivo administration. Incidence of bacterial infections in study cohort was 5.3% (n = 6). Two patients developed pneumonia, others with one: sinusitis, meningitis cause by Listeria meningitis, colitis and gonitis. Incidence of viral infections was 5.3% (n = 6): pneumonia associated with HHP-6 and CMV in 50% and generalized infections in 50% caused by HSV-1,2 and HHV-6. IFD were diagnosed in 3.6% patients (n = 4). The main etiology agent was Aspergillus spp. in 50%. Primary chemoresistant disease before nivo therapy was the only risk factor of infection complications during treatment of r/r HL (p = 0,029). Overall survival (OS) at 1 year after first nivo administration in study cohort was 96.5%. Only one death was attributed to infection; the patient died due to sepsis of unknown etiology. Conclusion Incidence of infectious complications in r/r HL treated with nivo was 10%. Etiology of infectious complications presented by bacterial infections –37.5%, invasive fungal diseases – 25% and viral infections – 37.5%. Primary chemoresistance was a risk factor for infection complications. Wherewith infections could be managed successfully and carry favorable prognosis. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

1750P - Multi-level proteomics identifies FABP5 as a primary chemoresistance mediator in extensive-stage small cell lung cancer

BackgroundSmall cell lung cancer (SCLC), a highly invasive neuroendocrine tumor, accounts for approximately 15% of all new diagnosed lung cancer. Primary disease is highly sensitive to chemotherapy as well as radiotherapy, recurrences occur quickly, and less effective to subsequent therapies than the initial chemotherapy, the response rate of second-line therapy topotecan is only 22%. Drug resistance, is the main reason for failure of SCLC chemotherapy. Here, we quantitatively analyzed the proteomes of Cisplatin/Etoposide-resistant and -sensitive ES-SCLC patients from minute amounts of formalin-fixed and paraffin-embedded (FFPE) tissues. FABP5 was identified to be related with primary chemosensitivity, and may be an important mediator of Cisplatin/Etoposide-induced cell death. Methods51 tissue samples were collected from ES-SCLC patients who underwent Cisplatin and Etoposide chemotherapy in Zhe Jiang Cancer Hospital from January 2015 to November 2017. The 51 cases included 23 chemosensitive patients and 28 chemoresistant patients. System-wide quantitative proteomics approach was applied on the cohort of chemo-resistant and -sensitive tissues, and label free protein quantification was performed. Realtime-PCR was performed to assess the mRNA expression in H69 and multi-drug resistant H69AR cells. ResultsA total of 4,485 proteins were identified and quantified, more than 50% proteins were 10-70KD, Proteins with a fold difference greater than 1.5 (up/down) and a P value (t test) less than 0.05 were considered differentially expressed proteins, few overall proteome changes across the two groups. Comparison of the two-group found that FABP5 was significantly decreased in drug-resistant patients. Consistent with the protein level in tumor tissues, FABP5 mRNA was down-regulated in H69AR cells. FABP5 expression strongly correlated with decreased disease-free survival and overall survival in ES-SCLC patients. ConclusionsThese data revealed FABP5 may serve as a potential biomarker for primary chemoresistance and prognostic factor for patients with ES-SCLC, which need further research. Legal entity responsible for the studyYamei Chen. FundingNational Natural Science Foundation of China (grant numbers 81672972). DisclosureAll authors have declared no conflicts of interest.