IGHV Mutation Research Articles

Ibrutinib plus fludarabine, cyclophosphamide, and rituximab as initial treatment for younger patients with chronic lymphocytic leukaemia: a single-arm, multicentre, phase 2 trial

Fludarabine, cyclophosphamide, and rituximab (FCR) can improve disease-free survival for younger (age ≤65 years) fit patients with chronic lymphocytic leukaemia with mutated IGHV. However, patients with unmutated IGHV rarely have durable responses. Ibrutinib is active for patients with chronic lymphocytic leukaemia irrespective of IGHV mutation status but requires continuous treatment. We postulated that time-limited ibrutinib plus FCR would induce durable responses in younger fit patients with chronic lymphocytic leukaemia. We did a multicentre, open-label, non-randomised, single-arm phase 2 trial at seven sites in the USA. We enrolled patients aged 65 years or younger with previously untreated chronic lymphocytic leukaemia. Our initial cohort (original cohort) was not restricted by prognostic marker status and included patients who had del(17p) or TP53 aberrations. After a protocol amendment (on March 21, 2017), we enrolled an additional cohort (expansion cohort) that included patients without del(17p). Ibrutinib was given orally (420 mg/day) for 7 days, then up to six 28-day cycles were administered intravenously of fludarabine (25 mg/m2, days 1-3), cyclophosphamide (250 mg/m2, days 1-3), and rituximab (375 mg/m2 day 1 of cycle 1; 500 mg/m2 day 1 of cycles 2-6) with continuous oral ibrutinib (420 mg/day). Responders continued on ibrutinib maintenance for up to 2 years, and patients with undetectable minimal residual disease in bone marrow after 2 years were able to discontinue treatment. The primary endpoint was the proportion of patients who achieved a complete response with undetectable minimal residual disease in bone marrow 2 months after the last cycle of ibrutinib plus FCR. Analyses were done per-protocol in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov (NCT02251548) and is ongoing. Between Oct 23, 2014, and April 23, 2018, 85 patients with chronic lymphocytic leukaemia were enrolled. del(17p) was detected in four (5%) of 83 patients and TP53 mutations were noted in three (4%) of 81 patients; two patients had both del(17p) and TP53 mutations. Median patients' age was 55 years (IQR 50-58). At data cutoff, median follow-up was 16·5 months (IQR 10·6-34·1). A complete response and undetectable minimal residual disease in bone marrow 2 months after the last cycle of ibrutinib plus FCR was achieved by 28 (33%, 95% CI 0·23-0·44) of 85 patients (p=0·0035 compared with a 20% historical value with FCR alone). A best response of undetectable minimal residual disease in bone marrow was achieved by 71 (84%) of 85 patients during the study. One patient had disease progression and one patient died (sudden cardiac death after 17 months of ibrutinib maintenance, assessed as possibly related to ibrutinib). The most common all-grade toxic effects were haematological, including thrombocytopenia in 63 (74%) patients, neutropenia in 53 (62%), and anaemia in 41 (49%). Grade 3 or 4 non-haematological serious adverse events included grade 3 atrial fibrillation in three (4%) patients and grade 3 Pneumocystis jirovecii pneumonia in two (2%). The proportion of patients who achieved undetectable minimal residual disease in bone marrow with ibrutinib plus FCR is, to our knowledge, the highest ever published in patients with chronic lymphocytic leukaemia unrestricted by prognostic marker status. Ibrutinib plus FCR is promising as a time-limited combination regimen for frontline chronic lymphocytic leukaemia treatment in younger fit patients. Pharmacyclics and the Leukemia & Lymphoma Society.

S105 GENETIC MARKERS AND OUTCOME IN THE CLL14 TRIAL OF THE GCLLSG COMPARING FRONT LINE OBINUTUZUMAB PLUS CHLORAMBUCIL OR VENETOCLAX IN PATIENTS WITH COMORBIDITY

Background:Genomic aberrations, IGHV mutation status and mutations in genes such as TP53 are established prognostic factors in CLL in the context of chemoimmunotherapy. Their role is less‐well established when using chemo‐free regimens such as obinutuzumab (GA‐101) plus venetoclax (Ven‐G).Aims:This study evaluates the prognostic impact of genetic risk factors on obinutuzumab+chlorambucil (G‐Clb) vs. obinutuzumab+Venetoclax (Ven‐G).Methods:We assessed the incidence and impact of genetic factors in the phase 3 CLL14 trial comparing G‐Clb vs.Ven‐G in patients with CIRS>6 or creatinine clearance < 70 ml/min. Genomic aberrations were assessed by FISH, IGHV by sequencing with a threshold of 98% homology and mutations via amplicon‐based targeted NGS for TP53, NOTCH1, SF3B1, ATM, MYD88, FBXW7, POT1, BIRC3, XPO1, NFKBIE, EGR2 and RPS15 for variants with allele fraction ≥10%. Of the intention to treat population (n = 432) FISH/IGHV/NGS was assessable in 418/408/421 cases. PFS and OS were estimated via Kaplan‐Meier method and compared by non‐stratified log‐rank test. Hazard ratios (HRs) were calculated via Cox proportional hazards regression modeling.Results:The incidence of genomic aberrations considering the hierarchical model were del(17p) 7%, del(11q) 18%, +(12q) 18% and del(13q) 35%. IGHV was unmutated in 61% of patients. The incidence of gene mutations was NOTCH1 23% (exon 34 and/or 3’UTR), SF3B1 16%, ATM 13%, TP53 10%, XPO1 6%, RPS15 5%, POT1 5%, BRAF 4%, BIRC3 4%, NFKBIE 4%, EGR2 4%, MYD88 2%, FBXW7 1%. High coincidence was found for del(17p) and TP53 mut in 77% of all del(17p) cases and 57% of all TP53 mut cases.None of the parameters impaired overall response rate (ORR) to Ven‐G at treatment completion. For G‐Clb, the ORR rate was lower in patients with del(17p) (36% vs. 73%), del(11q) (58% vs. 74%), TP53 mut (58% vs. 74%), ATM mut (56% vs. 75%) and BIRC3 mut (33% vs. 74%).At a median follow‐up of 29 months, there were 107 events for PFS and 37 for OS in the intention to treat population. Del(17p) was the only genomic abnormality with impact on PFS in G‐Clb (HR 4.6, p < 0.001) and Ven‐G (HR 4.4, p = 0.001; Fig 1a). Similarly, TP53 mutations affected PFS in both treatment arms (G‐Clb HR 2.7, p = 0.001; Ven‐G HR 3.1, p = 0.01). However, TP53 mut without del(17p) was not associated with shorter PFS. None of the other evaluated factors affected Ven‐G efficacy, while for G‐Clb del(11q) (HR 2.3, p = 0.002), BIRC3 (HR 4.0, p = 0.001), NOTCH1 (HR 1.8, p = 0.03) and unmutated IGHV (HR 3.4, p < 0.001) were adverse factors.In several genetic subgroups including del(17p), del(11q), TP53 mut, NOTCH1 mut, SF3B1 mut, ATM mut Ven‐G was superior to G‐Clb in PFS. Regarding IGHV, only patients with unmutated status had a significant PFS benefit from Ven‐G in comparison to G‐Clb (IGHVunm HR 0.2, p < 0.001; IGHVmut HR 0.6, p = 0.29; Fig.1b). Multivariable testing for interaction between treatment and IGHV mutational status was significant (p = 0.03) indicating unmutated IGHV as a predictive factor for increased benefit from Ven‐G. OS was lower with del(17p) in both treatment arms (G‐Clb: HR 11.0, p < 0.001; Ven‐G: HR 3.4, p = 0.03) and with TP53 mut, BRAF mut and IGHVunmut in the G‐Clb arm (HR 5.5, p = 0.002; HR 6.6, p < 0.01; HR 5.4, p = 0.03), while none of the other factors were significantly associated with OS.Summary/Conclusion:Prognostic value of genomic aberrations, IGHV and gene mutations were confirmed for G‐Clb, while with Ven‐G only del(17p) and TP53 mut were associated with short PFS and only del(17p) with short OS. Unmutated IGHV was identified as a predictive factor characterizing a group of patients with particular benefit from Ven‐G.image

S107 FIVE‐YEAR FOLLOW‐UP OF PATIENTS RECEIVING IBRUTINIB FOR FIRST‐LINE TREATMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Ibrutinib (ibr), a first‐in‐class, once‐daily inhibitor of Bruton tyrosine kinase (BTK), is approved in the EU and other regions for treatment of chronic lymphocytic leukemia (CLL). RESONATE‐2 is a phase 3 study comparing the efficacy and safety of first‐line ibr vs chlorambucil (chl) in older patients (pts) with CLL/small lymphocytic lymphoma (SLL). As ibr is given as continuous therapy, long‐term efficacy and safety data in pts receiving ibr are critical to inform clinical practice.Aims:To report long‐term data over a median of 5 years of follow up from the RESONATE‐2 study of first‐line ibr in CLL/SLL.Methods:RESONATE‐2 is a phase 3, open‐label, international, randomized study (PCYC‐1115/1116; NCT01722487, NCT01724346). Pts ≥65 years old with previously untreated CLL/SLL without 17p deletion (N = 269) were randomly assigned 1:1 to receive ibr 420 mg once daily continuously until disease progression or unacceptable toxicity or chl 0.5–0.8 mg/kg for up to 12 cycles. Endpoints included progression‐free survival (PFS), overall survival (OS), overall response rate (ORR), and safety. In long‐term follow up, efficacy was assessed by investigator per International Workshop on Chronic Lymphocytic Leukemia (iwCLL) 2008 criteria with modification. Adverse event (AE) prevalence rates are reported.Results:Baseline characteristics were well balanced across treatment arms, as previously described. After a median follow‐up of 5 years (range, 0.1–66 months), the PFS benefit was sustained for ibr vs chl (hazard ratio [HR] 0.15 [95% confidence interval (CI): 0.10–0.22]). PFS estimates at 60 months were 70% for ibr vs 12% for chl. Ibr also resulted in improved OS vs chl; 83% vs 68% at 60 months, respectively, even with 57% of pts crossing over from chl to ibr after progression. Ibr improved PFS compared to chl in pts with unmutated immunoglobulin heavy chain variable region (IGHV) (HR 0.11 [95% CI: 0.06–0.19]) and in pts with 11q deletion (HR 0.03 [95% CI: 0.01–0.11]). As a composite, pts with high‐risk genomics (unmutated IGHV, 11q deletion, and/or TP53 mutation) had superior outcomes with ibr compared with chl (PFS: HR 0.08 [95% CI: 0.05–0.15]; OS: HR 0.37 [95% CI: 0.18–0.74]). With ibr, ORR including partial response with lymphocytosis was 92% and complete response (CR/CRi) rate increased over time to 30% (increased from 11% CR/CRi at primary analysis [median follow up 18 months]). The most common grade ≥3 AEs included neutropenia (13%), pneumonia (12%), hypertension (8%), anemia (7%), hyponatremia (6%), atrial fibrillation (5%), and cataract (5%); rates of most events decreased over time. Dose reductions due to grade ≥3 AEs decreased over time (5% of pts in years 0–1, 2% in years 1–2, 3% in years 2–3, 1% in years 3–4, and 0 in years 4–5). AEs of any grade leading to ibr discontinuation occurred in 7% of pts in year 0–1, 6% in years 1–2, 5% in years 2–3, 6% in years 3–4, and 1% in years 4–5. Pts responded to subsequent CLL therapies, including chemoimmunotherapy and alternate kinase inhibitors following ibr discontinuation. Ibr benefit continues in 58% of pts who remained on therapy.Summary/Conclusion:Single‐agent ibr had sustained PFS and OS benefit, including for pts with high‐risk genomic features, in the longest follow‐up to date from a phase 3 study of first‐line BTK‐directed therapy. Responses to ibr improved over time with nearly three‐fold more pts achieving CR/CRi with long term follow up. With up to 66 months follow up, more than half of pts remain on long‐term continuous ibr treatment. No new safety signals emerged.image

PB1868 B‐CELL PROLYMPHOCYTIC LEUKEMIA (B‐PLL) AND PROLYMPHOCYTOID MANTLE CELL LYMPHOMA (PMCL) (MORE THAN 55% OF PROLYMPHOCYTES) ARE CLOSED BUT DISTINCT ENTITIES. ON BEHALF GFCH AND FILO GROUPS

Background:B‐PLL is a rare entity that was debated. It is now recognized in the WHO classification and defined by the presence of prolymphocytes in peripheral blood (PB) exceeding 55% of lymphoid cells, different from transformed chronic lymphocytic leukemia (CLL) and cases with translocation t(11;14) that are classified as MCL. We previously showed that B‐PLL have a high frequency of MYC activation by translocation [t(MYC)] or gain, and frequent del17p, patients with both abnormalities (abs) having the worst prognosis. It has been suggested that B‐PLL should be considered as a subgroup of MCL.Aims:In order to evaluate the relationship between B‐PLL and MCL, we compared cytogenetics and molecular data.Methods:We studied 35 B‐PLL and 4 pMCL (≥55% of prolymphocytes in PB) (multicentric study by the Groupe Francophone de Cytogénétique Hématologique and the FILO group), 24 MCL and 16 CLL as control, using karyotype (K), FISH, whole‐exome sequencing (WES), IGHV and RNA‐sequencing. All MCL had a t(11;14), while the presence of a CCND1, CCND2 or CCND3 rearrangement was excluded in B‐PLL cases.Results:A morphological review, performed by 3 independent experts, did not show any distinctive features between B‐PLL and pMCL. In B‐PLL, we found a complex K (CK) (≥3 abs) in 71%, and a highly complex K (HCK ≥ 5) in 44%. The most frequent chromosomal abs were: MYC abs (n = 27, 77%): t(MYC) (n = 22, 63%) or MYC gain (n = 5, 14%) and del17p (n = 13, 37%). Among the 19/24 MCL with an informative K, we found CK in 13 (68%), including 11 HCK (58%) and del11q (ATM) in 3 (16%). By FISH, we found 9 (38%) del17p and 7 (29%) MYC abs (6 gains and 1 t(MYC)). All 4 pMCL had a CK, including 3 HCK, 2 had a t(MYC), 1 a MYC gain, 2 a del17p and 2 a del11q (Figure A). Using WES on 23 B‐PLL, the most frequent mutated genes were TP53 (n = 12, 52%), MYC, BCOR (n = 5, 22% each), SETD2, MYD88 (n = 4, 17% each), SF3B1 and CHD2 (n = 3, 13% each). WES performed on 3 pMCL showed 2 cases ATMmut, and 1 CCND1mut/TP53mut (Figure B). Overall, ATM inactivation was present in 1/35 (2.9%) B‐PLL vs 3/4 (75%) pMCL (p = .001). The majority of B‐PLL used a VH3 or VH4 gene (17/19, 89%), but none of the 4 pMCL (VH1, n = 3, VH2, n = 1). The level of somatic hypermutation was higher in B‐PLL: 16/19 (84%) had ≤98% of homology vs 1/4 pMCL (p = .04). Principal component analysis of gene expression data in 12 B‐PLL, 3 pMCL and 16 CLL analyzed by RNA‐Seq showed that B‐PLL and pMCL clustered together, distinctively from CLL (Figure C). However, 386 genes were differently expressed between B‐PLL and pMCL, with enrichment in cell cycle genes in pMCL, including CCND1 and CCND2. The level of SOX11 expression was significantly lower in B‐PLL than in pMCL (p = .02). Comparing B‐PLL and CLL, GSEA analysis revealed enrichment in MYC signaling pathway, and cell metabolism signatures in B‐PLL.Summary/Conclusion:In conclusion, our results show that B‐PLL have a genomic profile distinct from MCL and CLL, with frequent t(MYC), TP53, MYC and BCOR mutations. However, B‐PLL have similarities with the rare subgroup of pMCL: morphology, high frequency of MYC abs and gene expression profiles related to cell cycle and proliferation. Nevertheless, we highlighted distinctive features in addition to t(11;14): IGHV repertoire and mutation level, SOX11 expression and frequency of ATM abs. Our data advocate that pMCL could be an aggressive form of MCL, which have to be definitely distinguished from B‐PLL with a routine test, the karyotype.image

PF392 MONOCLONAL GAMMOPATHY AND HYPOGAMMAGLOBULINEMIA AS INDEPENDENT PROGNOSTIC FACTORS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A RETROSPECTIVE MULTICENTRIC EXPERIENCE

Background:Chronic Lymphocytic Leukemia (CLL) is an indolent B‐cell lymphoproliferative disorder. Several prognostic factors such as IGHV mutation status and chromosomal aberrations as trisomy 12, del11q, del13q or del17p have been detected so far. More recent genetic mutations such as BIRC3, SF3B1, NOTCH1 and TP53 stratifies even further the prognosis and outcome in CLL patients (pts). However all data above reported, because of expensive techniques and experience typical of big laboratories, are not available to all medical centers while data concerning the presence of hypogammaglobulinemia, IgM or IgG monoclonal gammopathy are easily achievable, even though the prevalence and the impact on natural history of CLL pts is controversial and contradictory.Aims:The aim of the study is to evaluate the prevalence and the outcome of monoclonal IgM/CLL, IgG/CLL and hypogammaglobulinemia compared with CLL pts with normal immunoglobulin (Ig) levels.Methods:We collected from four different Italian centers 1359 pts diagnosed with typical CLL from 1987 to 2017 with a baseline assessment of serum Ig, immunofixation, chromosomal aberrations and clinical features; evaluating their impact on time to progression (TTP), time to treatment (TTT) and overall survival (OS).Results:Once assessment was made, pts included in our study who met eligibility criteria were 1359. Median age was similar in all the groups and data gathered from the four centers showed an overlapping rate about prevalence and time‐dependent‐parameters in CLL pts and their related subclasses.The overall prevalence of monoclonal gammopathy is 14% of whom 128 pts (9%) with IgG/CLL, 68 pts (5%) with IgM/CLL. Hypogammaglobulinemia was detected in 172 pts (13%) while 991 pts (73%) had no evidence of paraprotein. Among the groups, serum IgG paraprotein showed a correlation with a poorer and shorter overall survival (OS) and an inferior clinical outcome.Summary/Conclusion:Our study provided data about prevalence and prognosis of monoclonal gammopaty and hypogammaglobulinemia in CLL patients. Serum Ig paraprotein was present in 14% of all CLL pts with a worst outcome in IgG/CLL group. Thus, making of serum Ig, a simple data to obtain, useful to assess CLL prognosis.image

PF370 SIGNAL-OMICS ANALYSIS OF THE IMPACT OF THERAPY ON BCR SIGNALLING IN CHRONIC LYMPHOCYTIC LEUKAEMIA

Background: Induction of signalling via cell surface receptor activation is a critical driver of chronic lymphocytic leukaemia (CLL) pathobiology, especially via the B-cell receptor (BCR), which promotes tumour survival and progression. The vital nature of BCR signalling has been recognized through the development of kinase inhibitors (KI), most notably ibrutinib, that target key nodes within this pathway. Current efforts to monitor signalling investigate expression of a highly confined series of kinases and phosphoproteins. While generating key insights, full appreciation of their wider significance to malignant pathology and therapy response remains an unresolved issue. Here, we describe a ‘signal-omics’ approach, involving a kinobead-based protocol used in conjunction with mass-spectrometry (MS) or immunoblotting. Kinobead binding is related to the availability of a kinase's active site and thus can be used to determine signalling within cells. We have employed this approach characterise surface-IgM (sIgM) signalling within primary CLL cells. Aims: Validate signal-omic profiling for investigating BCR signalling in primary CLL. Methods: Kinobead Signalling Analysis of BCR Signalling Response Whole cell lysates from anti-IgM and control F(ab’)2-treated primary CLL cells underwent kinobead isolation. Elutions from these were processed and were tested using mass-spectrometry (MS). MaxQuant software analysis of MS- data was used to identify kinases and relative binding to kinobeads. Immunoblotting Immunoblotting was used to screen whole cell lysates and kinobead elutions for kinase expression and activation change in all conditions. Results: Employing this approach, it was possible to isolate over 100 kinases from primary CLL cells and identified a ‘fingerprint’ of over 50 kinases which displayed unique, patient-specific response to sIgM stimulation. Signal strength was correlated to sIgM expression but not IGHV mutation status, calcium flux, karyotype or staging. Interestingly, our analysis recognised greater activation change in patients who had undergone prior chemoimmunotherapy compared to those from untreated/treatment-naïve patients. Comparison of matched samples was also able to illustrate kinomic changes in patients who have failed chemoimmunotherapy or have developed resistance to ongoing ibrutinib treatment. Summary/Conclusion: These data represent the first comprehensive, high-resolution investigation into BCR signalling response within CLL, where our probing of kinase active sites reveals unique evidence of adaptive reprogramming in response to therapy.

PF374 IBRUTINIB PLUS FLUDARABINE, CYCLOPHOSPHAMIDE, AND RITUXIMAB (IFCR) AS INITIAL THERAPY FOR YOUNGER PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA: A SINGLE‐ARM, MULTICENTER, PHASE 2 TRIAL

Background:Fludarabine, cyclophosphamide, rituximab (FCR) can provide prolonged disease free survival for young, fit chronic lymphocytic leukemia (CLL) patients with mutated IGHV; however, patients with unmutated IGHV rarely have durable response. The oral Bruton Tyrosine Kinase inhibitor, ibrutinib, is highly effective for patients irrespective of IGHV mutation status, but requires continuous therapy. We hypothesized that a time‐limited regimen of ibrutinib plus FCR (iFCR) would have curative potential for a broad population of young, fit CLL patients.Aims:The primary objective was to determine the rate of complete remission (CR) with undetectable bone marrow minimal residual disease (BM‐uMRD) two months after completing iFCR combination therapy. Secondary objectives were to assess clinical response rates, progression‐free and overall survival, rates of best response and BM‐uMRD, safety/tolerability, ibrutinib pharmacokinetics (PK), and association of established CLL prognostic factors with clinical response.Methods:We performed a multicenter, open‐label, non‐randomized, phase 2 investigator sponsored trial of iFCR, and enrolled CLL patients age ≤65 years without restriction by IGHV mutation status, all of whom met iwCLL criteria for initial treatment. Ibrutinib 420 mg daily was given for 7 days, followed by combination ibrutinib with FCR for up to 6 cycles. Responders continued on ibrutinib maintenance, and patients with BM‐uMRD after 2 years of maintenance discontinued therapy. Response was assessed by 2008 iwCLL criteria, and toxicity was assessed by CTCAE v4.03 and iwCLL criteria. This trial is registered with ClinicalTrials.gov (NCT02251548).Results:Between October, 2014, and April, 2018, 85 patients were enrolled at 9 US sites. The median age was 55 years (range 38–65). IGHV was unmutated in 46/79 patients (58.2%). Deletion of 17p and TP53 mutation were present in 4/83 (4.8%) and 3/81 (3.7%) patients, respectively; 2 of these patients had both. The median number of FCR cycles completed was 6 (range 1–6). The median AUC of ibrutinib in combination with FCR was 337.2 (IQR 198.6–553.9), and the median Cmax was 97.3 (IQR 65.1–168.8). These PK parameters are similar to ibrutinib monotherapy in prior studies, and did not appear to be altered by FCR. The rate of CR‐BM‐uMRD 2 months post‐FCR was 33% (28/85 patients, 95% CI: 0.25–1.0)), with a best rate at any point on trial that increased to 41% (35/85), and a best rate of CR/CRi irrespective of MRD status of 66%. The best rate of BM‐uMRD irrespective of IW‐CLL response status was 84% (71/85), and did not differ by IGHV mutation status (Figure 1). With a median follow‐up of 16.5 months (range 3.1–48.9), one patient progressed and one died due to a presumed cardiac event. The most common treatment‐emergent grade 3/4 adverse events were hematologic, including neutropenia 35%, thrombocytopenia 32%, and anemia 11%. Eight patients (9%) experienced grade ≥3 febrile neutropenia. No cases of Richter's Syndrome have been observed.Summary/Conclusion:The rate of BM‐uMRD induced by iFCR is, to our knowledge, the highest ever reported for a regimen treating a population of CLL patients unrestricted by prognostic marker status. The safety profile of iFCR was generally favorable, with typical toxicities of ibrutinib and FCR observed, and no additional toxicities discernable with the combination. These data establish iFCR as a highly effective novel‐agent‐containing time‐limited combination for initial therapy for young, fit CLL patients.image

PS1122 RECURRENT XPO1 MUTATIONS IDENTIFIED AS PREDICTIVE MARKER FOR ADVANCED CLL PROGRESSION

Background:Exportin 1 (XPO1/CRM1) is responsible for nuclear‐to‐cytosol export of RNAs and protein cargos, including various tumor suppressors (e.g. p53, IκB) and growth regulators (e.g. RB, p21). Our previous work has shown that XPO1 is upregulated in chronic lymphocytic leukemia (CLL), leading to mislocalization of critical cargos, which may contribute to activation of pathways involved in leukemogenesis and disease progression. Whole exome sequencing has revealed recurrent mutations to a highly conserved amino acid (E571G/E571K) near the XPO1 cargo binding groove in ∼5% of CLL cases and in ∼25% of CLL patients that develop a clinical complication of their disease with a very abysmal prognosis known as Richter's transformation. Despite inherent association with advanced hematologic malignancies, conflicting reports describe the merit of XPO1 mutations as a predictive marker for poor clinical outcome in CLL.Aims:We aimed to assess the significance of recurrent XPO1 E571 mutations on CLL progression and overall outcome by concurrent analysis of a human CLL clinical cohort and a large scale evaluation of a novel XPO1 transgenic mouse model.Methods:Retrospective analysis of an independent sample set of CLL cases (N = 466) was used to correlate XPO1 mutation status (N = 18, >30% clonal population CLL cells) with clinical data. These included time to first treatment, overall survival, IgHV mutation status, ZAP‐70 expression (%), and status of epigenetic programming. Differential gene expression analysis in newly diagnosed untreated CLL patient lymphocytes was constructed from RNA‐sequencing, with downstream pathway analysis performed by Ingenuity Pathway Analysis software. A novel XPO1 transgenic mouse model (Eμ‐XPO1) was developed with human XPO1 (WT/G/K) gene under the heavy chain promoter/enhancer of the B cell expression, and was then crossed with the Eμ‐TCL1 mouse to assess contribution of XPO1 mutations in concordance with murine CLL. Disease onset and progression was determined via flow cytometry (% CD19/CD5 double positive cells) analysis of peripheral blood.Results:In the CLL cohort we analyzed, presence of XPO1 E571 mutations (18/466, 4%) confers disease characteristics consistent with poor clinical prognosis; including association with low epigenetic programming (18/18), unmutated IgHV region (17/17), elevated ZAP‐70 expression (p = 0.012), and reduction in treatment free progression (p = 0.002) and overall survival (p = 0.068) compared with XPO1 un‐mutated CLL. Similarly, overexpression of E571WT, K or G as the sole genetic abnormality (Eμ‐XPO1) resulted in the development of lymphoid neoplasms at advanced ages (>20 mo) compared to age‐matched B6 mice. Furthermore, overexpression of E571G (N = 174, p = 0.001) and E571K (N = 188, p = 0.093), but not E571WT (N = 151, p = 0.713) XPO1 showed a trend of accelerated disease onset in the Eμ‐XPO1xTCL1 model consistent with a role of mutant XPO1 in disease progression. Lastly, RNA‐seq in XPO1 E571 or wild‐type CLL cells revealed unique clustering of differentially expressed genes specific to lymphocyte activation, signaling, and regulation, suggesting amplification of pro‐survival and anti‐apoptotic intracellular pathways.Summary/Conclusion:We report evidence for XPO1 E571 mutations as a predictive marker for poor CLL clinical prognosis warranting further investigation for selective targeting of mutated XPO1 as a personalized cancer therapy in CLL.

PF381 FACTORS IMPACTING TREATMENT SELECTION IN TREATMENT‐NAÏVE PATIENTS WITH CLL: A MULTICENTER STUDY

Background:The approval of novel targeted agents (TA) has revolutionized the treatment landscape for patients (pts) with chronic lymphocytic leukemia (CLL). Guidelines suggest that treatment selection between chemo‐immunotherapy (CIT) and TA is driven by several factors including age, disease stage, patient's fitness, comorbid conditions, IGHV mutation status, del(17p) and/or TP53 mutation, CLL‐associated symptoms and patient preference. However, it is not well understood to what extent other external factors influence treatment selection in real‐world clinical practice.Aims:To identify characteristics (clinical, demographic, and socioeconomic) associated with treatment selection between CIT and TA in first‐line CLL based on real‐world data collected from multiple cancer centers in the US.Methods:The CLL Collaborative Study of Real‐World Evidence (CORE) is a large multicenter retrospective observational study of CLL pts treated at either community or academic centers in the US. Pts were eligible for inclusion in this analysis if they started first‐line CLL therapy after January 1st, 2016 (post‐approval of novel TA in the first‐line). Descriptive statistics were used to compare sociodemographic and clinical characteristics (collected prior to initiation of treatment) between pts on CIT and TA. Clinical and sociodemographic covariates were included in multivariable logistic regression model to evaluate factors associated with first‐line treatment selection.Results:Of the 386 treatment‐naïve pts with CLL, 39% received CIT and 61% received TA. Approximately 46% of the pts receiving TA were younger than 65 years of age, and 44% had commercial insurance. More than half of the pts (59%) receiving CIT were younger than 65 years of age and only 36% had commercial insurance. Majority (82%) of pts receiving TA were initiated on Ibrutinib monotherapy. BR (45%) and FCR (27%) were the most commonly administered CIT regimens. About 93% and 80% of all pts underwent testing for chromosomal abnormalities and IGHV mutation status, respectively. Del(17)p/TP53 mutation status was observed in 5% (n = 8) and 26% (n = 60) of pts initiating CIT and TA, respectively. Complex karyotype (3+) was observed in 1% of CIT versus (vs) 7% of TA pts. In addition, 36% of CIT pts and 50% of TA pts had creatinine clearance <80 mL/min. Results from multivariable logistic regression model showed that pts (age >65yrs) were two times more likely to receive TA than pts younger than 65 (OR: 1.95; 95%CI: 1.04–3.66). Clinical characteristics such as del(17p)/TP53 (OR: 7.28; 95%CI: 3.18–16.66) and creatinine clearance <80 ml/min (OR: 1.83; 95%CI: 1.07–3.11) were significantly associated with higher likelihood of TA treatment selection. Other clinical characteristics such as IGHV mutation status and ECOG score were not associated with treatment selection. Pts with commercial insurance (OR: 2.55; 95%Cl: 1.28–5.08) and pts having Medicare with supplemental plans (OR: 2.84; 95%Cl: 1.19–6.77) were more likely to receive TA versus those with Medicare only.Summary/Conclusion:Findings from this study demonstrated that older age, insurance coverage, del(17p)/TP53 mutation status, and creatinine clearance were associated with treatment selection preferences in real‐world clinical practice. As TAs and novel combinations continue to change the treatment paradigm in first‐line CLL, their utilization patterns and associated outcomes need to be continuously evaluated.

PF368 DYSREGULATION OF BACH2 AND FOXP1 GENES IN T AND B CELLS IS MORE PRONOUNCED IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS COMPARED TO AGED‐MATCHED HEALTHY DONORS WITH AN IMPACT ON SURVIVAL

Background:We previously investigated BACH2/RPDM1 gene expression in B/T cells from chronic lymphocytic leukemia (CLL) patients and aged‐matched healthy donors (HD) to understand the immunosenescence with aging, which showed that BACH2/PRDM1 are strongly correlated with aging. Recent data show that candidate immune suppressor gene FOXP1 is an important regulator of CLL pathogenesisAims:We further investigated the significance of BACH2, PRDM1, FOXP1 and inhibitory molecule expression in T/B cells from CLL patients.Methods:Peripheral blood mononuclear cells were isolated from untreated CLL patients and age‐matched HD using Lymphoprep density gradient centrifugation. T/B cells were purified by magnetic isolation or FACS sorting. BACH2, PRDM1, FOXP1, PD1, PD‐L1 and TIM3 transcripts were quantified using RT‐qPCR. Nuclear labeling of BACH2/PRDM1 and FOXP1 was accomplished using fixed and permeabilized cells, pre‐labeled for membrane markers and acquired by flow cytometry. Prognostic value of BACH2/PRDM1 and FOXP1 expression in purified CD19+ B cells was analyzed retrospectively in a cohort of CLL patients (n = 270), and correlated with clinical parameters.Results:BACH2 gene expression in HDs is significantly downregulated while PRDM1 gene expression increased in T and B cells according to age groups. BACH2 expression is further reduced in T/B cells from CLL patients compared to age‐matched HDs. Also, PRDM1 is significantly upregulated in T cells from CLL patients but not in leukemic‐B cells. We didn’t observe any age‐related changes in FOXP1 expression. However, among BACH2, FOXP1 and PRDM1 protein expression in lymphocytes from CLL patients, we observe a significant increase of BACH2 and FOXP1 protein expression in CD19+ B cells compared to T cell subsets (n = 21). We analyzed PD1, TIM3 transcript/protein expression in T cells and PD‐L1 expression in B cells to evaluate the exhaustion phenotype associated with CLL and aging. This analysis showed us that PD1 expression is significantly upregulated in CD4+/CD8+ T cells in the older vs younger HDs (n = 60) and also in T cells from CLL patients (n = 41) when compared with age‐matched HDs both at transcript and protein level. High PD‐L1 mRNA and protein expression were also strongly correlated with increased age in HD B cells with a further increase detected in leukemic B cells. In addition, we found a strong inverse correlation between BACH2 and PD1 in CD4+/CD8+ T cells; and between BACH2 and PDL1 in CD19+ B cells. TIM3 transcript/protein expression in T cells was very low in CLL patients and not detected in HD T cells. Prognostic value of BACH2/PRDM1 and FOXP1 expression in purified CD19+ B cells was analysed by RT‐qPCR retrospectively in a cohort of CLL patients, and correlated with clinical stage and IGHV mutation status. In this retrospectively analysed CLL cohort, increased BACH2 expression was correlated with improved treatment‐free survival (p = 0.0352). On the contrary, increased FOXP1 expression was correlated with worsened overall survival of CLL patients in the same cohort (p = 0.0165). However, there was no correlation between BACH2/FOXP1 and Binet stage or IGHV mutation status. We didn’t observe any significant correlations with PRDM1 in terms of survival of CLL patients.Summary/Conclusion:The decrease of BACH2 and increase of FOXP1 in leukemic‐B cells impact on CLL patients’ survival. PD1 and PD‐L1 expression in T/B cells from CLL patients and aged‐matched HDs are significantly correlated with aging‐associated exhaustion of immune cells. The role of BACH2 and FOXP1 in tumor microenvironment is thus further investigated.

Fc receptor-like 2 (FCRL2) is a novel marker of low-risk CLL and refines prognostication based on IGHV mutation status

Fc receptor-like 2 (FCRL2) is a novel marker of low-risk CLL and refines prognostication based on IGHV mutation status

Impact of gene mutations and chromosomal aberrations on progression-free survival in chronic lymphocytic leukemia patients treated with front-line chemoimmunotherapy: Clinical practice experience

The impact of genetic aberrations on rituximab-based therapeutic regimens has been intensely studied in chronic lymphocytic leukemia (CLL). According to the current consensus chemoimmunotherapy consisting of rituximab and DNA-damaging drugs is not suitable for patients with TP53 defects. In our study, we focused on CLL patients with an intact TP53 gene and investigated four recurrently mutated genes in CLL, genomic aberrations by FISH, and IGHV status with the aim of analyzing their impact on progression-free survival (PFS) after front-line therapy with FCR (fludarabine, cyclophosphamide, rituximab) or BR (bendamustine, rituximab) regimens. Using next-generation sequencing, we analyzed 120 patients treated with FCR and 57 patients treated with BR at a university hospital. We used a 10% cut-off for variant allele frequency and recorded the following mutation frequencies in the pre-therapy samples: ATM 23%, SF3B1 20%, NOTCH1 19% and BIRC3 11%. The data on cytogenetic aberrations (11q22, 13q14, trisomy 12) and IGHV mutation status were also considered in PFS analyses. In univariate analyses, we observed a negative impact of BIRC3 mutations and 11q22 deletion in both regimens, while the unmutated IGHV status was associated with a significantly shorter PFS only in the FCR-treated cohort. In a multivariate analysis, only deletion 11q22 in both regimens, and the unmutated IGHV in the FCR cohort maintained an independent association with the reduced PFS. Notably, sole 11q22 deletion, without an ATM mutation on the other allele, manifested the shortest PFS of all analyzed markers. Deletion 11q22 and IGHV status predict PFS in previously untreated CLL patients.

Prognostic and Predictive Effect of IGHV Mutational Status and Load in Chronic Lymphocytic Leukemia: Focus on FCR and BR Treatments

BackgroundMost important markers in chronic lymphocytic leukemia (CLL) are TP53 abnormalities, including mutations and deletions, and the mutational status of immunoglobulin heavy chain (IGHV) genes. However, some recent publications suggest that the IGHV mutational load could have a prognostic effect on CLL patients. Patients and MethodsWe performed a single-center retrospective study on 459 patients with productive rearrangement of the B-cell receptor to evaluate the prognostic and predictive role of IGHV mutational status and burden within the germline sequence. In particular we focused on FCR (fludarabine with cyclophosphamide, and rituximab)- (64 naive and 30 relapsed) and BR (bendamustine with rituximab)-treated patients (17 naive and 61 relapsed). A cutoff value of 2% of difference within the IGHV germline was used to define the IGHV mutational status. ResultsWe reported that unmutated IGHV (U-IGHV) patients were characterized by a significant shorter progression-free survival (PFS) and overall survival (P < .0001) compared with mutated IGHV (M-IGHV) patients. Moreover, treatment-naive M-IGHV patients experienced a long-term disease control after FCR or BR, with PFS reaching a plateau regardless of mutational load. In our series the extent of IGHV gene mutation did not provide further relevant prognostic data over the mutational status. Relapsed patients showed dismal outcome with chemoimmunotherapy regardless of IGHV status or load. ConclusionOur data, together with from those from the literature, confirmed the cutoff value of 2% to define the mutational status of IGHV gene and suggest that FCR/BR are good first-line treatment strategies for M-IGHV patients, whereas U-IGHV patients should be managed with B-cell receptor and/or B-cell lymphoma 2 (BCL2) inhibitors.

Telomere length predicts for outcome to FCR chemotherapy in CLL

We have previously shown that dividing patients with CLL into those with telomeres inside the fusogenic range (TL-IFR) and outside the fusogenic range (TL-OFR) is powerful prognostic tool. Here, we used a high-throughput version of the assay (HT-STELA) to establish whether telomere length could predict for outcome to fludarabine, cyclophosphamide, rituximab (FCR)-based treatment using samples collected from two concurrent phase II studies, ARCTIC and ADMIRE (n = 260). In univariate analysis, patients with TL-IFR had reduced progression-free survival (PFS) (P < 0.0001; HR = 2.17) and shorter overall survival (OS) (P = 0.0002; HR = 2.44). Bifurcation of the IGHV-mutated and unmutated subsets according to telomere length revealed that patients with TL-IFR in each subset had shorter PFS (HR = 4.35 and HR = 1.48, respectively) and shorter OS (HR = 3.81 and HR = 2.18, respectively). In addition, the OS of the TL-OFR and TL-IFR subsets were not significantly altered by IGHV mutation status (P = 0.61; HR = 1.24 and P = 0.41; HR = 1.47, respectively). In multivariate modeling, telomere length was the dominant co-variable for PFS (P = 0.0002; HR = 1.85) and OS (P = 0.05; HR = 1.61). Taken together, our data suggest that HT-STELA is a powerful predictor of outcome to FCR-based treatment and could be used to inform the design of future risk-adapted clinical trials.

Analysis of LPL gene expression in patients with chronic lymphocytic leukemia

The IGHV mutational status is one of the most important markers for chronic lymphocytic leukemia(CLL) prognostication. Lipoprotein lipase(LPL) gene expression was found to correlate with IGHV status and was suggested as its surrogate marker. Recent data reported that LPL expression might be influenced by pivotal signalling pathways in CLL. This study aimed to assess LPL gene expression in relation to key immunogenetic and molecular markers of CLL, including IGHV mutational status, B-cell receptor (BCR) stereotypy, TP53, NOTCH1, and SF3B1gene mutations. Materials andMethods: Expression of LPL mRNA was measured in peripheral blood mononuclear cells of 73CLL patients by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). IGHV, NOTCH1, TP53, and SF3B1 gene mutation analysis was performed by PCR amplification and direct sequencing. 44of 73 (60%) CLL cases were categorized as LPL-positive based on the cut-off value established by ROC (receiver operating characteristic) curve analysis. LPL expression was significantly associated with IGHV mutation status (r=0.684; p < 0.0001) and tended to correlate with presence ofNOTCH1 gene mutations (p = 0.113). BCR stereotyped cases showed higher LPL expression values in comparison to unstereotyped cases in the LPL-positive group of patients (p=0.041). LPL expression was associated with a shorter overall survival in the entire СLL group (median 107vs 143, p=0.048) as well as in Binet A patients, albeit with borderline significance (median 139vs not reached, p=0.086). LPL expression was found to be closely correlated with IGHV gene mutational status and overall survival, proving LPL as prognostic marker in CLL. Our results also indicate a possible relationship between aberrant expression of LPL and BCR- and NOTCH1-dependent signalling pathways.

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (iFCG) for Firstline Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

Ibrutinib, Fludarabine, Cyclophosphamide, and Obinutuzumab (iFCG) for Firstline Treatment of Patients with CLL with Mutated IGHV and without TP53 Aberrations

ROR1 Expression Is Associated with Oncogenic Dedifferentiation in Chronic Lymphocytic Leukemia

An integrated analysis of transcriptomic signatures applied to almost 12,000 primary human tumors of 33 different cancer types from The Cancer Genome Atlas (TCGA) datasets defined a signature to quantify various degrees of stemness and assigned a stemness index to each tumor included in the analysis. Stemness indices were lowest in normal cells, increased in primary tumors, and were highest in metastatic disease, consistent with the notion that tumor progression generally involves oncogenic dedifferentiation. Higher values for stemness indices were associated with biological processes active in cancer stem cells, which had the highest levels of oncogenic dedifferentiation (Malta TM, et al, Cell 173:338, 2018). We examined whether such oncogenic dedifferentiation stemness indices could distinguish different prognostic subgroups of patients with chronic lymphocytic leukemia (CLL). For this, we performed gene set enrichment analysis (GSEA) on array-based transcriptomes (GSE49896 in Mraz M, et al, Blood 124(1):84, 2014) of 100 CLL samples, that expressed unmutated IGHV (U-IGHV) (N=44) or mutated IGHV (M-IGHV) (N=56). We found that the oncogenic dedifferentiation stemness signature was enriched in CLL cells with U-IGHV relative to that of cases with M-IGHV (FDR q = 0.03). Included in the oncogenic dedifferentiation stemness signature is ROR1, which encodes an oncoembryonic orphan receptor for Wnt5a that can contribute to tumor-cell survival, proclivity to relapse, and cancer metastases (Zhang S, et al, PNAS 111:17266, 2014 and Cui B, et al, Cancer Res 73(12):3649, 2013). Moreover, ROR1 may contribute to disease progression in CLL (Cui B, et al, Blood 128(25):2931, 2016). Hence, we interrogated whether ROR1 expression is associated with the oncogenic dedifferentiation stemness signature. We performed GSEA on the transcriptomes of: 98 CLL cases (Ferreira PG, et al, Genome Research 24:212, 2014) and 448 CLL cases available through GEO database (GSE13204) in Kohlmann A, et al, BJH 142:802, 2008, segregated based on ROR1 expression in ROR1Hi or ROR1Lo. We also performed GSEA on the transcriptome of: 90 CLL cases in GSE13204 dataset segregated into two groups representing the 10% of patients who had CLL cells with highest levels of ROR1 mRNA (ROR1>90%), and the 10% of patients who had CLL cells with lowest levels of ROR1 mRNA (ROR1<10%), 24 CLL cases with leukemic cells either expressing ROR1 (ROR1pos) or with low to negligible ROR1 (ROR1neg)CLL (Cui B, et al, Blood 128(25):2931, 2016), and isolated CD5+B220low splenic leukemia cells that developed in ROR1xTCL1 transgenic (Tg) mice or TCL1 Tg mice (Widhopf II GF, et al, PNAS 111(2):793, 2014). We found that the oncogenic dedifferentiation stemness signature was significantly enriched in ROR1Hi CLL in the cohort of 98 CLL cases and in the cohort of 448 CLL cases (FDR q values of 0.02 and <0.001, respectively). In addition, the oncogenic dedifferentiation stemness signature was significantly enriched in ROR1>90% relative to ROR1<10% CLL, ROR1pos relative to ROR1neg CLL, and in the ROR1+ leukemia of ROR1xTCL1 Tg mice relative to ROR1Neg leukemia that develops in TCL1 mice (FDR q values of 0.01, 0.01 and <0.001, respectively). To explore whether ROR1 signaling is involved in inducing oncogenic dedifferentiation, we compared the transcriptomes of matched sets of CLL cells obtained from patients before and after treatment with cirmtuzumab, a humanized monoclonal antibody (mAb) that can inhibit ROR1-signaling (Choi MY, et al, Cell Stem Cell 22:951, 2018). GSEA showed that, compared with pre-treatment CLL cells, the post-treatment leukemia cells had a highly significant reversal of the oncogenic dedifferentiation stemness signatures (FDR q < 0.001). Collectively these findings indicate that oncogenic dedifferentiation stemness signature may be enriched in subgroups of CLL patients that have more aggressive disease and enhanced by ROR1 signaling. Moreover, treatment with agents such as cirmtuzumab that can inhibit ROR1-signaling may reverse the expression of genes associated with stemness and oncogenic dedifferentiation in patients with CLL. DisclosuresChoi:Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Genentech: Speakers Bureau; Rigel: Consultancy; Gilead: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau.

The Role of Molacular Genetic Examination in Differential Diagnosis and Prognostic Prediction in Patints with B-Cell Chronic Lymphoproliferative Disorders: A Restrospective Study of 1592 Patients

Background: B-cell chronic lymphoproliferative disorders (BCLPD) are a heterogeneous group of mature B-cell malignancies characterized by monoclonal proliferation in the bone marrow (BM) or peripheral blood (PB). The overlapped clinical features and the lack of individual diagnostic markers make diagnosis challenging, especially in patients who have difficulty to get adequate non-BM biopsy specimens or patients manifest as leukemic onset without nodes-involvement. It is necessary to explore novel methods to make precise diagnostic classification and prognostic prediction through simply PB/BM examination.Methods and results: A total of 1592 patients with newly diagnosed BCLPD were enrolled from January 1998 to December 2016, of which chronic lymphocytic leukemia (CLL) accounted for 39%, leukemic marginal zone lymphoma (l-MZL)occupied 13%, and Waldenstrom's macroglobulinemia/ lymphoplasmacytic lymphoma (LPL/WM), leukemic follicular lymphoma (l-FL) and mantle cell lymphoma (l-MCL) were 13%, 9% and 8%, respectively. There are 14% patients failed to fit into any of the categories and remaining unclassified which defined as BCLPDU. The median age at diagnosis was 58 years old. The male/female ratio of all BCLPD patients was 1.84:1. Male predominance was pronounced in WM/LPL, l-MCL and HCL with a M/F ratio of 2.5-2.7, while with balance gender in l-MZL and l-FL.FISH analyses were performed in 1234 patients. 17p and 11q deletion were most common in l-MCL (36% and 17%), while 13q deletion and trisomy 12 were most frequent in CLL (35% and 21%). Patients with 17p deletion showed significantly adverse survival in CLL and l-MCL (p<0.05), but the phenomenon was not found in other types of BCLPD. Instead, complex karyotype showed better predictive capability of worse survival than 17p deletion in patients with l-FL, l-MZL and BCLPDU.A total of 665 patients had been successfully amplified the IGHV-IGHD-IGHJ rearrangements. Based on a cut-off value of 98% homology, the IGHV mutated proportion in patients with l-MCL was 48.6%, which was higher than other BCLPDs, following CLL 33.3% and l-MZL 28.6%, while most of WM and l-FL were unmutated. The most frequent rearrangement in l-MZL were IGHV1-2*01, which was seldom found in other subtypes. Also, DH2-2 and VH3-7 were mainly found in CLL and l-MCL respectively. Besides, we have identified several stereotyped IGH CDR3 which were specific for the certain BCLPD subtype. About 23% unclassified patients could acquire further diagnosis according to the special mutation status, VDJ rearrangements or stereotyped CDR3 sequencing.With a median follow-up of 50.3 months, 522 patients (38.0%) had disease progression or died. Patients with lMCL have a significant worse survival than other entities, with a 3-year overall survival (OS) rate of 58%. Following , patients with WM/LPL had relatively poorer prognosis with a 5-year OS rate of 68.2%, while the prognosis for l-MZL, l-FL and CLL were relatively favourable, with a 5-year OS of 80%.The OS of the B-CLPD had improved over time due to the emergence of immunotherapy and novel (3-year OS improved from 82.1% to 92.2%). The improvement of l-MCL and WM was obvious but not for CLL and l-MZL.Conclusion: Non-BM biopsy specimens are not available for part of BCLPD patients. Overlapped magnification of BM/PB brings on difficulties in differential diagnosis. Besides morphology and immunophenotype, comprehensive analysis of FISH, karyotype, IGHV, MYD88 or BRAF mutation and other molecular examination might contribute to further differential diagnosis and prognostic prediction. Moreover, it might provide advice for the initial time and strategy of treatment. DisclosuresNo relevant conflicts of interest to declare.

A Scoring System to Predict the Risk of Atrial Fibrillation in Chronic Lymphocytic Leukemia and Its Validation in a Cohort of Ibrutinib-Treated Patients

BACKGROUD. The B-cell receptor inhibitor ibrutinib has significantly improved treatment and overall management of chronic lymphocytic leukemia (CLL). Although several data derived from clinical trials suggest that ibrutinib increases the risk of atrial fibrillation (AF), the incidence of AF in a real-life cohort of CLL patients is unknown. Furthermore, it would be clinically relevant to identify patients at high risk of AF during ibrutinib. The aim of this study is to report the prevalence and risk factors of AF in ibrutinib-naive CLL, in order to define a predictive model for the development of AF and to test it in a cohort of subjects receiving ibrutinib. METHODS. We retrospectively analyzed data from 860 ibrutinib-naive CLL patients, referred to the Padua University hospital. Comorbidities, clinical and biological prognostic markers were analyzed using the Mann-Whiney, Fisher exact or Chi-square tests, when appropriated. Time to AF (TTAF) and overall survival (OS) were evaluated with Kaplan-Meier method. Univariate and multivariate Cox models were run to identify independent factors associated with AF. Then, risk values were obtained based on the hazard ratios. The score for AF was calculated as the sum of each risk values. Subsequently, the model was evaluated in a cohort of 354 ibrutinib-treated patients referred from 8 Italian hematological centers. RESULTS. Among the 860 patients from Padua hospital, 60% were male, 49% were older than 65 years, 73% were Binet A stage at diagnosis and 41% underwent at least one line of treatment. A prior history of AF was present in 21 patients (2.4%) at CLL diagnosis, while, among the remaining 839 patients without a previous history of AF, 47 (5.6%) developed it after a median follow-up of 9.4 years, resulting in an estimated incidence of almost 0.8% cases/year. Moreover, the median OS for patients with AF was significantly shorter than that patients without AF (12 vs 22 years, p 65 years (p=0.001, 1 point), male gender (p=0.003, 1 point), valvular hearth diseases (p=0.001, 2 points), cardiopathy (p Subsequently, we applied our AF model to a cohort of 354 ibrutinib-treated patients, 64% were male, the median age was 69 years, 88 were treatment-naive, 70% U-IGHV and 39% harbored TP53 abnormalities. Forty-four subjects developed AF after a median observation of 25 months, with an estimated 2-year TTAF of 12%. Only 9 out of the 44 patients (20%) discontinued ibrutinib. Sixteen patients (4%) were classified as AF score 0, 218 (62%) score 1-2, 73 (21%) score 3-4 and 46 (13%) at least score 5. Our model was also able to identify patients at a higher risk AF during ibrutinib, in fact the 2-year risk of AF was 0%, 5%, 17% and 40% for patients with score 0, 1-2, 3-4, and ≥5 respectively (p CONCLUSIONS. In this study, variables associated with an increased risk of developing AF were identified and recapitulated into a scoring system. Our model proved to be a valid tool to identify patients at a higher risk of developing AF, including ibrutinib-treated patients. Taking these data into account, patients with a score ≥5 should be carefully monitored during ibrutinib treatment given the very high-risk of developing AF or should be considered for alternative therapies. Disclosures Visentin:janssen: Consultancy, Honoraria. Mauro:abbvie: Other: board member; janssen: Other: board member. Reda:Janssen and Cilag: Consultancy; ABBVIE: Consultancy; Gilead: Consultancy; Celgene: Consultancy. Molica:Jansen: Other: Advisory board; Gilead: Other: Advisory board; Roche: Other: Advisory board; AbbVie: Other: Advisory board. Rigolin:Gilead: Research Funding. Tedeschi:Gilead: Consultancy; Janssen: Consultancy, Speakers Bureau; AbbVie: Consultancy. Cortelezzi:novartis: Consultancy; abbvie: Consultancy; roche: Consultancy; janssen: Consultancy. Coscia:Abbvie, Gilead, Shire: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Janssen, Karyopharm: Research Funding. Cuneo:Abbvie: Other: advisory board, Speakers Bureau; Roche: Other: advisory board, Speakers Bureau; Gilead: Other: advisory board, Speakers Bureau; janssen: Other: advisory board, Speakers Bureau. Foa:CELGENE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; NOVARTIS: Speakers Bureau; AMGEN: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau. Trentin:Roche: Membership on an entity9s Board of Directors or advisory committees; Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding.

Detection of Clinically Relevant Molecular Alterations in Chronic Lymphocytic Leukemia (CLL) By Nanopore Sequencing

IntroductionChronic Lymphocytic Leukaemia (CLL) is the most prevalent leukaemia in the Western world and characterised by clinical heterogeneity. IgHV mutation status, mutations in the TP53 gene and deletions of the p-arm of chromosome 17 are currently used to predict an individual patient's response to therapy and give an indication as to their long-term prognosis. Current clinical guidelines recommend screening patients prior to initial, and any subsequent, treatment. Routine clinical laboratory practices for CLL involve three separate assays, each of which are time-consuming and require significant investment in equipment. Nanopore sequencing offers a rapid, low-cost alternative, generating a full prognostic dataset on a single platform. In addition, Nanopore sequencing also promises low failure rates on degraded material such as FFPE and excellent detection of structural variants due to long read length of sequencing. Importantly, Nanopore technology does not require expensive equipment, is low-maintenance and ideal for patient-near testing, making it an attractive DNA sequencing device for low-to-middle-income countries.MethodsEleven untreated CLL samples were selected for the analysis, harbouring both mutated (n=5) and unmutated (n=6) IgHV genes, seven TP53 mutations (five missense, one stop gain and one frameshift) and two del(17p) events. Primers were designed to amplify all exons of TP53, along with the IgHV locus, and each primer included universal tails for individual sample barcoding. The resulting PCR amplicons were prepared for sequencing using a ligation sequencing kit (SQK-LSK108, Oxford Nanopore Technologies, Oxford, UK). All IgHV libraries were pooled and sequenced on one R9.4 flowcell, with the TP53 libraries pooled and sequenced on a second R9.4 flowcell. Whole genome libraries were prepared from 400ng genomic DNA for each sample using a rapid sequencing kit (SQK-RAD004, Oxford Nanopore Technologies, Oxford, UK), and each sample sequenced on individual flowcells on a MinION mk1b instrument (Oxford Nanopore Technologies, Oxford, UK). We developed a bespoke bioinformatics pipeline to detect copy-number changes, TP53 mutations and IgHV mutation status from the Nanopore sequencing data. Results were compared to short-read sequencing data obtained earlier by targeted deep sequencing (MiSeq, Illumina Inc, San Diego, CA, USA) and whole genome sequencing (HiSeq 2500, Illumina Inc, San Diego CA, USA).ResultsFollowing basecalling and adaptor trimming, the raw data were submitted to the IMGT database. In the absence of error correction, it was possible to identify the correct VH family for each sample; however the germline homology was not sufficient to differentiate between IgHVmut and IgHVunmut CLL cases. Following bio-informatic error correction and consensus building, the percentage to germline homology was the same as that obtained from short-read sequencing and nanopore sequencing also called the same productive rearrangements in all cases.A total of 77 TP53 variants were identified, including 68 in non-coding regions, and three synonymous SNVs. The remaining 6 were predicted to be functional variants (eight missense and two stop-gains) and had all been identified in early MiSeq targeted sequencing. However, the frameshift mutation was not called by the analysis pipeline, although it is present in the aligned reads. Using the low-coverage WGS data, we were able to identify del(17p) events, of 19Mb and 20Mb length, in both patients with high confidence.ConclusionsHere we demonstrate that characterization of the IgHV locus in CLL cases is possible using the MinION platform, provided sufficient downstream analysis, including error correction, is applied. Furthermore, somatic SNVs in TP53 can be identified, although similar to second generation sequencing, variant calling of small insertions and deletions is more problematic. Identification of del(17p) is possible from low-coverage WGS on the MinION and is inexpensive. Our data demonstrates that Nanopore sequencing can be a viable, patient-near, low-cost alternative to established screening methods, with the potential of diagnostic implementation in resource-poor regions of the world. DisclosuresSchuh:Giles, Roche, Janssen, AbbVie: Honoraria.