ALBERT

Description: It is commonly assumed that asexual lineages are short-lived evolutionarily, yet many asexual organisms can generate genetic and phenotypic variation, providing an avenue for further evolution. Previous work on the asexual plant pathogen Phytophthora ramorum NA1 revealed considerable genetic variation in the form of Structural Variants (SVs). To better understand how SVs arise and their significance to the California NA1 population, we studied the evolutionary histories of SVs and the forest conditions associated with their emergence. Ancestral state reconstruction suggests that SVs arose by somatic mutations among multiple independent lineages, rather than by recombination. We asked if this unusual phenomenon of parallel evolution between isolated populations is transmitted to extant lineages and found that SVs persist longer in a population if their genetic background had a lower mutation load. Genetic parallelism was also found in geographically distant demes where forest conditions such as host density, solar radiation, and temperature, were similar. Parallel SVs overlap with genes involved in pathogenicity such as RXLRs and have the potential to change the course of an epidemic. By combining genomics and environmental data, we identified an unexpected pattern of repeated evolution in an asexual population and identified environmental factors potentially driving this phenomenon.

Description: Neuroinflammation is considered a key pathological process in neurodegenerative diseases of aging, including Alzheimer’s disease (AD). Many studies have defined phenotypes of reactive microglia, the brain-resident macrophages, with different antigenic markers to identify those potentially causing inflammatory damage. We took an alternative approach with the goal of characterizing the distribution of purinergic receptor P2RY12-positive microglia, a marker previously defined as identifying homeostatic or non-activated microglia. We examined the expression of P2RY12 by dual-color light and fluorescence immunohistochemistry using sections of middle temporal gyrus from AD, high plaque and low plaque non-demented cases in relation to amyloid beta (Aβ) plaques and phosphorylated tau, markers of pathology, and HLA-DR, IBA-1, CD68, and progranulin, microglial phenotype markers. In low plaque cases, P2RY12-positive microglia mostly had non-activated morphologies, while the morphologies of P2RY12-positive microglia in AD brains were highly variable, suggesting its expression could encompass a wider range of phenotypes than originally hypothesized. P2RY12 expression by microglia differed depending on the types of plaques or tangles they were associated with. Areas of inflammation characterized by lack of P2RY12-positive microglia around mature plaques could be observed, but many diffuse plaques showed colocalization with P2RY12-positive microglia. Based on these results, P2RY12 expression by microglia should not be considered solely a marker of resting microglia as P2RY12 immunoreactivity was identifying microglia positive for CD68, progranulin and to a limited extent HLA-DR, markers of activation.

Description: Background Patients (pts) with DLBCL who have received rituximab (R) antibody in the first-line setting, have poorer treatment outcomes when they are re-treated with R-chemotherapy in the salvage setting. Resistance to R may have developed. Ofatumumab (O) is a human monoclonal antibody against CD 20 that targets a different epitope than R. We hypothesized that replacing R with O in second line setting may overcome R resistance. The primary end-point of this ongoing phase II study is the objective response rate (ORR) following two cycles of O in combination with ICE (Ifosfamide, Carboplatin and Etoposide) or O-ICE. Secondary end points include the assessments of the safety and tolerability of the combination, progression-free (PFS) and overall survival (OS). Methods This was a multi-center, non-randomized phase II open label study. Pts with histologically confirmed DLBCL who failed or were refractory to first line R-based anthracycline therapy were eligible. Pts were accrued according to a 2-stage Simon’s Optimal Design at the National Cancer Centre Singapore and 3 medical centres in South Korea; Samsung Medical Centre, Ajou University Hospital and Soonchunhyang University Hospital. Stage 1 will enrol 24 pts and if 14 or more pts respond, we will proceed to stage 2 where another 37 pts would be recruited. Pts receive 3-weekly ICE (Ifosfamide 5g/m2 on day 2, Carboplatin AUC = 5 on day 2, Etoposide 100 mg/m2 day 1-3) and O (1000 mg), infused on day 1 and 8 of cycle 1, and thereafter only on day 1 of each cycle. A restaging scan is performed after cycle 2. Pts who are candidates for high dose chemotherapy (HDC) and autologous stem cell rescue (ASCR) would receive additional 1 or 2 more cycles of O-ICE before stem cell mobilization. Pts who are not candidates for HDC and ASCR would receive up to 6 cycles of O-ICE. Results A total of 24 pts with a median age of 54.5 years (27-75) were enrolled. Among them, 14 (58%) were male and 14 (58%) had stage III/IV disease. A median of 4 (2-6) courses of O-ICE was administered and 11 pts underwent HDC+ASCR. The ORR after 2 cycles was 58.3%; complete response (CR) 29.2% and partial response (PR) 29.2%. On treatment completion, the best ORR became 62.5% (CR = 37.5%, PR = 25%). Grade 1/2 non-hematologic toxicities, independent of relation to study drug, occurring in at least 20% of pts included nausea (50%), pain (50%), fatigue (42%), rash (42%), anorexia (33%), vomiting (33%), diarrhea (25%), edema (29%), fever (21%), pruritus (21%), cough (21%) and paresthesia (21%). Grade 3/4 toxicities included neutropenia (50%), thrombocytopenia (71%) and 4 anemia (54%). Febrile neutropenia occurred in 12.5% and no treatment related deaths were reported. The median follow up time was 8.2months (95% CI 5.3-14.0). The median PFS was 8.2months (95% CI 4.2-17.8) and the median OS was not reached (95% CI 8.6-not reached). Conclusion The study regimen met the predefined threshold of 14 responding pts in stage 1. The ORR of O-ICE was comparable to that of RICE in the CORAL study and this result is significant considering that all pts in this study had received R prior to study entry. Together with the tolerability data and promising activity, continuation of study enrolment in stage 2 has started. Disclosures Tao: Medscape: Honoraria; Takeda: Honoraria. Quek:Novartis: Consultancy, Honoraria; Bayer: Honoraria. Kim:Novartis: Research Funding; Cellgene: Research Funding; Takeda: Research Funding.

Description: Background: Peripheral T-cell lymphomas (PTCL) typically have a dismal outcome when treated with conventional combination chemotherapy. However, the role of HDC with ASCT in patients who attained CR1 following induction chemotherapy remains controversial. Methods: This is a retrospective analysis performed using a prospective database of newly diagnosed PTCL patients treated at our centres from 1993 to 2015. After excluding patients with NK/T cell lymphoma, ALK-positive anaplastic large cell lymphoma, cutaneous T cell lymphoma and composite lymphomas, we identified 164 PTCL patients who were treated with curative intent. Among them, 73 patients achieved CR1 following induction chemotherapy. We compared the characteristics and outcomes of those who went on to receive further ASCT with those who did not receive further consolidation ASCT. Fisher's exact test and Student's T test was used to compare the baseline characteristics such as age, stage, international prognostic index risk group (IPI), prognostic index for PTCL (PIT), lactate dehydrogenase (LDH) and treatments between the two cohorts. Progression-free survival (PFS) and overall survival (OS) were compared using the log-rank test. Cox proportional hazard regression analysis was performed to estimate the hazard ratios (HR). Results: Of the 164 patients treated with curative intent, the median OS of patients who achieved CR1 was not reached compared to 1.23 years among those who did not achieve CR1 with a HR of 5.24 (95% CI: 3.11-8.83, p

Description: Background A recent genome-wide association study (GWAS) including 253 Chinese individuals from Singapore with B-cell Non-Hodgkin Lymphoma (B-NHL), a new susceptibility locus, rs6773853 was identified between BCL6 (B-cell lymphoma protein 6) and LPP (lipoma preferred partner) on chromosome 3q27. This was significantly associated with an increased risk of B-NHL; Odds ratio (OR) per-copy of the risk allele = 1.44 and diffuse large B-cell lymphoma (DLBCL) OR = 1.47. The aims of this study were to determine the prognostic significance of rs6773853 in a case-only analysis of B-NHL patients and to determine if there were any differences in patients and tumor characteristics in B-NHL patients with rs6773853 locus present and absent. Methods Genome-wide genotyping to identify germline mutations from DNA of patients' blood samples have been described. Patient, tumour and treatment data were obtained from clinical and pathology databases as well as electronic and hardcopy of medical records. Fluorescent in-situ hybridization testing for BCL6 translocations using breakapart probes were performed on tumor samples whenever possible. Patients were compared in a two-way (homozygous wild-type [WT] versus presence of 1 or more copies of the risk allele of rs6773853) and three-way (homozygous WT versus heterozygous carrier versus homozygous variant of rs6773853) manner. Prognostic factors were analyzed first by univariate analysis. A multivariate Cox regression model was then fitted including all factors showing statistical significance on univariate analysis to identify the best predictors of overall survival. Results Clinical and pathologic data were available for 245 B-NHL patients in whom GWAS was performed. The minor allele of rs6773853 locus was identified in 127 patients: 100 were homozygous and 27 were heterozygous carriers. The WT genotype was present in 118 patients. There were no statistically significant differences in the clinical and tumor characteristics or treatment received in both 2-way (Table 1) and 3-way analyses. On univariate analysis, age 〉60 years, ECOG 2–4, B symptoms, advanced stage, aggressive B-NHL, 2 or more extra-nodal sites, elevated LDH, high-risk IPI and a personal history of another cancer were associated with a poorer OS. On multivariate analysis the presence of rs6773853 locus was independently associated with a poorer OS. (Table 2) The frequencies of BCL6 translocations were 17.9% (7/39) and 20.8% (10/48) in the B-NHL with rs6773853 locus absent and present respectively. Conclusions The rs6773853 susceptibility locus to B-NHL is independently prognostic of a poorer OS. This may be related to its close proximity to BCL6, the transcriptional repressor that tightly regulates the germinal centre reaction, although exploratory analysis showed that rs6773853 did not confer a greater risk of BCL6 translocations. Further functional studies and fine mapping will investigate the role of BCL in the tumorigenesis of these B-NHL patients with rs6773853. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Natural-killer/T-cell lymphoma (NKTL) is a rare subset of non-Hodgkin's lymphoma that is more prevalent in Asia than in the West. We report a case of two brothers who were diagnosed with NKTL and died of their disease at 23 and 37 years old respectively, and further investigations performed to determine the underlying genetic basis to their disease. Methods Both patients were recruited into the Singapore lymphoma study where retrieval of their clinical data and biospecimens had been approved by the Singhealth instititutional review board. Their clinical courses are described using information from electronic medical records. For the older brother we extracted tumor DNA from a patient-derived xenograft (PDX), and DNA from a buccal swab and peripheral blood (PB) sample. In the younger brother DNA was extracted from normal and tumor tissue from archived formalin fixed paraffin embedded (FFPE) samples. DNA from the unaffected mother, older brother and paternal aunt were obtained from PB and buccal swab samples. We performed whole exome sequencing (WES) on the younger affected brother's tumor, the older affected brother's, mother's and unaffected brother's blood sample. Subsequent analysis of variants took into account autosomal recessive (AR), X-linked recessive (XR) and autosomal dominant (AD) inheritance models. Candidate genes are validated using Sanger sequencing. Microarray on the older affected brother and 12 sporadic NKTL cases were analyzed. Results The older affected brother was 35 years old when he presented with left nasal blockage in March 2013. He was diagnosed with stage IIA NKTL and treated with 4 cycles of bortezomib-GIFOX as part of a clinical trial followed by radiotherapy to the nasal region. He had primary refractory disease and was further treated with 4 cycles of SMILE followed by high dose chemotherapy and autologous stem cell transplantation (HDC/ASCT). He progressed and received off-label ruxolitinib, and everolimus/HDAC inhibitor as part of a clinical trial. He then had radiotherapy to an ulcerating penile lesion before he died of progressive disease 27months after diagnosis. The younger affected brother was 18 years old when he was diagnosed with NKTL in 1998. He received 6 cycles of CHOP with intravenous high-dose methotrexate and intrathecal methotrexate and went into remission. Three years later, he was diagnosed with chronic myeloid leukemia and 6 years later, he had a relapse of NKTL for which he received ESHAP, TBI and allogeneic bone marrow transplant. He died of transplant related complication 6 years after diagnosis. They both have an older unaffected brother. Their father died of non-malignant condition in his fifties and their mother remains well. They have no other relatives with hematologic malignancy. We have identified several candidate variants and genes on WES that are being validated and functional studies are ongoing. GEP shows 234 genes that are significantly upregulated in the affected brother's tumor compared to 12 sporadic NKTL tumors including p53 and 12 genes that were significantly downregulated including CTLA4 and virus entry pathways. Conclusion: We describe a case of two brothers with a rare lymphoma and the ongoing efforts in elucidating the genetic basis of their disease. To the best of our knowledge this is the first study of its kind to be presented. Disclosures No relevant conflicts of interest to declare.

Description: Aim Composite follicular lymphoma with diffuse large B-cell lymphoma (FL/DLBCL) is an uncommon hematological neoplasm. The aim of this study was to examine clinico-pathological features of patients with FL/DLBCL and investigate relevant predictors of survival outcome. Methodology Patients with histologically-proven FL/DLBCL at diagnosis (n=106) and who were subsequently treated with Rituximab-based chemotherapy from 2002-2017 at the National Cancer Centre Singapore were retrospectively analyzed. Survival analyses were performed using the Kaplan-Meier method and multivariate Cox proportional models. Results The cohort consisted of 72 men and 34 women with a median age of 59 years (range, 24-82). The cell of origin by Han's algorithm was GCB in 37.7%, ABC in 58.5% and unknown in 3.8%. Eight patients (7.5%) were double-hit for c-MYC, BCL2 and/or BCL6 rearrangements. In a multivariate model inclusive of known clinico-pathological parameters at diagnosis, presence of B symptoms (p = 0.0122), stage 3 or 4 lymphoma (p = 0.0166) and double-hit genotype (p = 0.0045) were independently prognostic for worse overall survival (OS). These factors, excluding B symptoms, were similarly prognostic for progression-free survival (PFS). Including first-line treatment data in the multivariate model, lack of complete response (p 〈 0.0001) and use of chemotherapy regimens other than R-CHOP (p = 0.0360) alongside presence of B symptoms (p = 0.0022), were the only remaining independent prognostic variables for worse OS. Classification by cell of origin was not prognostic. A Clinico-Genotypic Index derived from point-wise addition of all five adverse parameters (score of 0, 1, 2, 3-4) revealed four prognostic risk groups accounting for 25%, 30%, 25% and 20% of the cohort, with a predicted 5-year OS of 100%, 95%, 57% and 19% respectively (p 〈 0.0001). Conclusion A Clinico-Genotypic Index derived from clinical and molecular factors can classify patients with composite FL/DLBCL into distinct prognostic groups. Han's algorithm has no prognostic value in this disease entity. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Background Hodgkin lymphoma (HL) accounts for 15% of all cancer diagnosis in young adults. Standard of care is ABVD (Adriamycin, Bleomycin, Vinblastine, Dacarbazine), which has resulted in overall cures of 70-90%. In the setting of HL, bleomycin is still one of the cornerstones in its treatment with the omission of bleomycin in the randomized trial of GHSG HD13 showing poorer outcomes. A major complication of HL treatment is Bleomycin induced pneumonitis (BIP) occurring in 0.4-28% of patients, with up to 10% mortality. Currently, we are unable to predict which patients are more prone. Given that human genomic variations underlie both disease susceptibility and drug response, uncovering genetic biomarkers predisposing to BIP is imperative to predicting and preventing BIP. Methods In this first pharmacogenomics study of BIP in South-East Asian patients, we perform both candidate gene and GWAS analyses. 96 HL patients were recruited from the National University Cancer Institute, Singapore (NCIS). BIP was clinically characterized and graded by CTCAEv4. Genetic association analysis was performed by regression (additive model) accounting for non-genetic predictor variables. Results Age at the start of bleomycin treatment, sex, genetic ancestry, concomitant chemotherapy treatment, treatment with G-CSF, stage of HL disease, pretreatment creatinine clearance, weight, chest irradiation and smoking history did not differ significantly between patients with and without BIP. The total number of treatment cycles (P=0.006) , cumulative exposure (P=0.01) and total exposure (P=0.028) were significantly lower in the BIP group as Bleomycin was omitted from further chemotherapy regimens once BIP was diagnosed. We independently replicated the association of HFE rs1799945 (H63D) with BIP (Cases: 15.38% vs Controls: 1.72%, P=0.0069, OR(95%CI)=2.89 (1.88-88.59)), while BLMH rs1050565 was not associated with BIP (P=0.92, OR(95%CI)=1.07 (0.29-3.93)). H63D carriers have 12.44 (1.98-78.17) times the odds of developing BIP compared to non-carriers (P=0.0088). We uncover a significant association of H63D with the severity of BIP (P=0.00087). HIST1H1T rs198846, a downstream variant in LD with H63D was also associated with BIP and its severity. H63D was predicted to be highly pathogenic (CADD Score=24.40) and at the top 1% of the most deleterious mutations in the human genome. The GWAS uncovered 4 novel genetic biomarkers with suggestive evidence of association with BIP (Unknown locus on Chr 17, MRC2 rs8072984, TMEM260 rs1124062 and KIF26B rs12747330). MRC2 rs8072984 is involved with remodeling of extracellular matrix, and defects in this could cause poor lung remodeling after bleomycin insult. TMEM260 rs1124062 and KIF26B rs12747330 are both involved with renal anomalies and development suggesting that defects in these could affect bleomycin clearance. Conclusion This is the first pharmacogenomics study of BIP in South East Asian patients, looking at both candidate gene and GWAS analyses. Our results suggest that iron regulation could be the driver in the etiopathogenesis of BIP, supporting the utility of H63D testing in the risk stratification of patients receiving Bleomycin. Moreover, GWAS analyses have discovered 4 novel markers that suggest an association with BIP, which will be further evaluated and should be independently replicated in a larger cohort. Disclosures Chng: Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Merck: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding.

Description: Aim: Composite histologies of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) may be present synchronously at diagnosis or metachronously at the time of transformation of a formerly diagnosed FL. The aim of this study was to examine their clinico-pathological characteristics and treatment outcomes. Method: Patients who were consecutively diagnosed with composite FL/DLBCL (n=120) and FL (n=346) from 2001-2017 at the National Cancer Centre Singapore were retrospectively analyzed. Chi-squared tests and multivariate logistic regression were performed to evaluate clinico-pathological associations between the two cohorts. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Results: Amongst the FL cases, 21 patients (6.1%) with metachronous transformed FL/DLBCL were identified. The median lag time from diagnosis of FL to DLBCL transformation was 47 months (range, 7.8-168). Clinico-pathological features in synchronous and metachronous FL/DLBCL were similar, with both entities demonstrating a male preponderance (67% male and 33% female). Median age at diagnosis was 67 years (range, 41-81) and 60 years (range, 24-90) for metachronous and synchronous FL/DLBCL, respectively. The cell-of-origin by Han's criteria was similar (metachronous: GCB 52%, ABC 43%, unknown 5%; synchronous: GCB 38%, ABC 57%, unknown 5%; p = 0.21), as were the occurrence of C-MYC/BCL2/BCL6 double-hit rearrangements. However, survival from the time of DLBCL development was significantly worse (median, 3 vs 12 years) for metachronous compared to synchronous FL/DLBCL (HR 2.20, 95%CI 0.88-5.49, p = 0.022). Double-hit, advanced stage, and use of non-RCHOP regimens (OR 7.54, 95%CI 2.84-20.1, p = 0.0001) were associated with lack of complete response to chemotherapy. In metachronous FL/DLBCL, the R-CHOP regimen was less commonly used (77% vs 56%, p = 0.049). Correspondingly, complete response to chemotherapy was less likely in metachronous cases (38% vs 63%, p = 0.037). Conclusion: Metachronous and synchronous FL/DLBCL share similar clinico-pathological characteristics. A preceding diagnosis of FL however, predicts for significantly worse survival outcomes and suboptimal responses to chemotherapy. Disclosures No relevant conflicts of interest to declare.