Heavy Chain Variable Region Gene Research Articles

Long-Term Studies Assessing Outcomes of Ibrutinib Therapy in Patients With Del(11q) Chronic Lymphocytic Leukemia

BackgroundCertain genomic features, such as del(11q), expression of unmutated immunoglobulin heavy-chain variable region (IGHV) gene, or complex karyotype, predict poorer outcomes to chemotherapy in patients with chronic lymphocytic leukemia (CLL). Patients and MethodsWe examined the pooled long-term follow-up data from PCYC-1115 (RESONATE-2), PCYC-1112 (RESONATE), and CLL3001 (HELIOS), comprising a total of 1238 subjects, to determine the prognostic significance of these markers in patients treated with ibrutinib. ResultsWith a median follow-up of 47 months, ibrutinib-treated patients had longer progression-free survival (PFS) than patients treated in the comparator arm, regardless of genomic risk factors. Among patients treated with ibrutinib, we found that high-risk genomic features were not associated with shorter PFS (63-75% across all subgroups at 42 months) or overall survival (79-83% across all subgroups at 42 months). Surprisingly, we observed that ibrutinib-treated patients with del(11q) actually had a significantly longer PFS than ibrutinib-treated patients without del(11q) (42-month PFS rate 70% vs. 65%, P = .02). ConclusionThese analyses not only demonstrate that genomic risk factors previously associated with poor outcomes lose their adverse prognostic significance but also that del(11q) can be associated with a superior PFS with ibrutinib therapy.

Elevated Lactate Dehydrogenase Has Prognostic Relevance in Treatment-Naïve Patients Affected by Chronic Lymphocytic Leukemia with Trisomy 12.

Chronic Lymphocytic Leukemia (CLL) patients with +12 have been reported to have specific clinical and biologic features. We performed an analysis of the association between demographic; clinical; laboratory; biologic features and outcome in CLL patients with +12 to identify parameters predictive of disease progression; time to treatment; and survival. The study included 487 treatment-naive CLL patients with +12 from 15 academic centers; diagnosed between January 2000 and July 2016; and 816 treatment-naïve patients with absence of Fluorescence In Situ Hybridization (FISH) abnormalities. A cohort of 250 patients with +12 CLL followed at a single US institution was used for external validation. In patients with +12; parameters associated with worse prognosis in the multivariate model were high Lactate DeHydrogenase (LDH) and β-2-microglobulin and unmutated immunoglobulin heavy-chain variable region gene (IGHV). CLL patients with +12 and high LDH levels showed a shorter Progression-Free-Survival (PFS) (30 months vs. 65 months; p < 0.001), Treatment-Free-Survival (TFS) (33 months vs. 69 months; p < 0.001), Overall Survival (OS) (131 months vs. 181 months; p < 0.001) and greater CLL-related mortality (29% vs. 11% at 10 years; p < 0.001) when compared with +12 CLL patients with normal LDH levels. The same differences were observed in the validation cohort. These data suggest that serum LDH levels can predict PFS; TFS; OS and CLL-specific survival in CLL patients with +12.

Distinct immunoglobulin heavy chain variable region gene repertoire and lower frequency of del(11q) in Taiwanese patients with chronic lymphocytic leukaemia.

SummaryChronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries but very rare in Asia. Peripheral blood or bone marrow mononuclear cells obtained at initial diagnosis from 194 patients with CLL were analysed to determine the ethnic difference in genetic abnormalities. Mutated IGHV was detected in 71·2% of Taiwanese CLL and IGHV3‐23 was the most frequently used gene. Stereotyped BCR was present in 18·3% with subset 8 being the most frequent. All cases with subset 8 belonged to IGHV 4‐39 and were exclusively associated with un‐mutated IGHV and poor outcome. Mutation frequencies of SF3B1 (9·7%), NOTCH1 (8·6%), BIRC3 (1·1%), ATM (16·9%) or TP53 (8·1%), and frequencies of cytogenetic abnormalities including trisomy 12 (18·6%), del(17p) (10·4%), del(13q) (43·7%) and IGH translocation (10·1%) were comparable to those reported from Western countries, except del(11q) (6·9%) which was lower in our patients. Patients with un‐mutated IGHV, subset 8, disrupted TP53, trisomy 12, and SF3B1 mutations had a worse outcome compared to patients without these mutations. In conclusion, IGHV3‐23 usage, stereotyped subset 8 and lower frequency of del(11q) show an ethnicity‐dependent association in Taiwanese CLL patients.

Clinical characteristics and outcomes of Richter transformation: experience of 204 patients from a single center.

The natural history, prognostication and optimal treatment of Richter transformation developed from chronic lymphocytic leukemia (CLL) are not well defined. We report the clinical characteristics and outcomes of a large series of biopsy-confirmed Richter transformation (diffuse large B-cell lymphoma or high grade B-cell lymphoma, n=204) cases diagnosed from 1993 to 2018. After a median follow up of 67.0 months, the median overall survival (OS) was 12.0 months. Patients who received no prior treatment for CLL had significantly better OS (median 46.3 vs. 7.8 months; P<0.001). Patients with elevated lactate dehydrogenase (median 6.2 vs. 39.9 months; P<0.0001) or TP53 disruption (median 8.3 vs. 12.8 months; P=0.046) had worse OS than those without. Immunoglobulin heavy chain variable region gene mutation, cell of origin, Myc/Bcl-2 double expression and MYC/BCL2/BCL6 double-/triple-hit status were not associated with OS. In multivariable Cox regression, elevated lactate dehydrogenase [Hazard ratio (HR) 2.3, 95% Confidence Interval (CI): 1.3-4.1; P=0.01], prior CLL treatment (HR 2.0, 95%CI: 1.2-3.5; P=0.01), and older age (HR 1.03, 95%CI: 1.01-1.05; P=0.01) were associated with worse OS. Twenty-four (12%) patients underwent stem cell transplant (20 autologous and 4 allogeneic), and had a median post-transplant survival of 55.4 months. In conclusion, the overall outcome of Richter transformation is poor. Richter transformation developed in patients with untreated CLL has significantly better survival. Stem cell transplant may benefit select patients.

PF365 PROGNOSTIC IMPLICATIONS OF IGHV GENE REARRANGEMENTS WITH “BORDERLINE” PERCENTAGE OF MUTATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Background:The somatic hypermutation (SHM) of immunoglobulin heavy chain variable (IGHV) region genes classifies chronic lymphocytic leukemia (CLL) patients into two subgroups: unmutated CLL (UM‐CLL, ≥98%) associated with an adverse prognosis and mutated CLL (M‐CLL, &lt;98%) showing a good outcome. In cases with a % of IGHV identity close to the 98% cut‐off, defined as “borderline” (BL‐CLL), the prognosis remains controversial.Aims:To investigate the clinico‐biologic features and prognosis of BL‐IGHV CLL in view of their undefined outcome.Methods:We retrospectively analyzed 811 untreated CLL patients, including 327 at diagnosis, sequenced at our Institution. BL‐CLL were defined as cases with a 97–97.9% IGHV identity, according to the 2017 European Research Initiative on CLL (ERIC) recommendations. IGHV were amplified using family‐specific VH primers (framework region 1 [FR1] or leader primers) and sequenced using the 3500 Series Genetic Analyzer. Following the ERIC recommendations, we updated the IGHV analysis with two different approaches in order to accurately determine the SHM rate: 1) re‐sequencing of 82 old FR1 cases with leader primers; 2) realignment of all cases to the updated IMGT/V‐QUEST tool. From the re‐analysis with leader primers of 82 old FR1 sequenced cases, none shifted the UM/M IGHV category, while 6 (7.3%) were reclassified as follows: 4 M‐CLL as BL‐CLL and 2 BL‐CLL as UM‐CLL. No case shifted across the three categories after the realignment to the updated IMGT/V‐QUEST tool. Major stereotyped subset analysis was determined according to the ARResT/AssignSubsets tool (bat.infspire.org/arrest/ericll.org/pages/services/tool). Time‐to‐first treatment (TFT) was evaluated as a clinical endpoint. Two further independent cohorts of 308 and 465 newly diagnosed CLL patients were used to validate the data.Results:In the 811 untreated patients, 418 (51.5%) UM‐CLL, 39 (5%) BL‐CLL and 354 (43.5%) M‐CLL were identified, with a higher frequency of UM‐CLL due to the enrichment of cases enrolled in first line clinical trials. Sixteen cases (2%) belonged to stereotyped subset #2. BL‐CLL showed a similar proportion of TP53 deletion and mutation compared to UM‐CLL, but a significant lower frequency of other unfavorable prognostic factors (stage, CD38/ZAP70 expression) (Table 1). Moreover, BL‐CLL were enriched in subset #2 (5/39, 13%), in agreement with the higher incidence of subset #2 among these cases. Applying the conventional 98% IGHV cut‐off, TFT was significantly shorter in UM‐CLL than M‐CLL (p &lt; 0.0001), as well as in subset #2 CLL vs M‐CLL (p = 0.017), as expected. When analyzing the 97–97.9% IGHV subgroup, BL‐CLL showed a significantly longer TFT than UM‐CLL (p &lt; 0.0001), similar to that of M‐CLL (Fig. 1a). The TFT of BL‐CLL cases remained comparable to that of M‐CLL, also when the analysis was restricted to the 327 CLL patients at diagnosis (80% Binet stage A) (125 UM‐CLL, 19 BL‐CLL and 183 M‐CLL) (Fig. 1b), as well as excluding subset #2 cases. These findings were validated in two independent cohorts of CLL at diagnosis (105 UM‐CLL, 16 BL‐CLL, 187 M‐CLL; TFT BL vs UM‐CLL: p &lt; 0.0001 and 143 UM‐CLL, 13 BL‐CLL, 309 M‐CLL; p = 0.024), that included 719 Binet stage A CLL.Summary/Conclusion:Our data suggest that the prognosis of BL‐CLL is better than that of UM‐CLL and similar to that of M‐CLL, thus supporting the 98% cut‐off for clinical purposes. The issue of BL‐CLL remains open to further investigations.image

Gain of the short arm of chromosome 2 (2p gain) has a significant role in drug-resistant chronic lymphocytic leukemia.

The different types of drug resistance encountered in chronic lymphocytic leukemia (CLL) cannot be fully accounted for by the 17p deletion (and/or TP53 mutation), a complex karyotype (CK), immunoglobulin heavy‐chain variable region genes (IGHV) status and gene mutations. Hence, we sought to assess the associations between recurrent genomic abnormalities in CLL and the disease's development and outcome. To this end, we analyzed 64 samples from patients with CLL and gain of the short arm of chromosome 2 (2p+), which is frequent in late‐stage and relapsed/refractory CLL. We found that fludarabine/cyclophosphamide/rituximab (a common first‐line treatment in CLL) is not effective in removing the 2p+ clone ‐ even in samples lacking a CK, the 17p deletion or unmutated IGHV. Our results suggest strongly that patients with CLL should be screened for 2p+ (using karyotyping and fluorescence in situ hybridization) before a treatment option is chosen. Longer follow‐up is now required to evaluate bendamustine‐rituximab, ibrutinib, and idelalisib‐rituximab treatments.

A high mutation rate of immunoglobulin heavy chain variable region gene associates with a poor survival and chemotherapy response of mantle cell lymphoma patients.

Immunoglobulin heavy chain variable region (IGHV) gene mutation status is a biomarker for the prognosis of chronic lymphocytic leukemia, whether it is associated with the diagnosis, staging, and prognosis of patients with mantle cell lymphoma (MCL) remains to be determined.The IGHV gene mutations of 52 MCL patients were determined by DNA sequencing and compared with published IGHV germline sequences.DNA sequence alignment of IGHV variable regions with published IGHV germline sequences showed that the coincidence rate was 94% to 100%. Ten cases (21%) were significantly mutated with the rate of 96.9% to 94.0%. The overall survival time of patients was negatively correlated with the degree of IGHV gene mutation. Further survival analysis with log-rank test demonstrated that the patients with significant IGHV gene mutations showed a trend towards poor survival.The mutation rate of the IGHV variant region may be determined to assess the prognosis and overall survival time of MCL patients.

Survival analysis of 118 chronic lymphocytic leukemia patients with abnormal TP53 gene in the era of traditional immunochemotherapy

目的分析传统免疫化疗（CIT）时代，TP53基因异常[TP53缺失和（或）突变]慢性淋巴细胞白血病（CLL）患者一线CIT疗效及生存情况。方法收集2003年1月至2017年8月在江苏省人民医院血液科诊断的118例TP53异常CLL患者基线资料[性别、年龄、分期、B症状（发热、盗汗、体重减低）、β2微球蛋白（β2-MG）、免疫球蛋白重链可变区（IGHV）突变状态、染色体核型、一线CIT]进行生存分析，分析101例接受一线CIT患者的疗效。结果118例CLL患者均通过电话、门诊及住院等方式随访至2018年3月10日，中位无进展生存（PFS）时间为12（95%CI 10.148~13.852）个月，中位总生存（OS）时间为53（95%CI 41.822~64.178）个月。分析患者基线资料对患者生存影响，β2-MG<3.5 mg/L患者具有较长的PFS时间（P=0.027），女性患者以及Binet A期患者具有更长OS时间（P=0.011）。118例患者中，有17例（14.4%）自诊断至死亡/随访终点未接受任何抗肿瘤治疗，101例接受了一线及以上的CIT（85.6%），中位至首次治疗时间（TTFT）仅为1（0~62）个月，Binet A期较Binet B/C期患者具有更长TTFT（P<0.001）。进一步分析101例患者初治方案，按治疗方案分为四组，分别是温和治疗组（主要为单用苯丁酸氮芥或单用利妥昔单抗治疗）30例（29.7%），含氟达拉滨化疗组32例（31.7%），含大剂量激素化疗组23例（22.8%），其他化疗组16例（15.8%）。含大剂量激素化疗方案可获得较长PFS时间（P<0.001），四组间OS差异无统计学意义。结论TP53基因异常的CLL患者对免疫化疗反应差，临床进展迅速，一线使用含大剂量激素方案化疗可以延长PFS时间，为患者后续加入复发难治新药临床试验创造机会。

SF3B1 mutation predicts unfavorable treatment-free survival in Chinese chronic lymphocytic leukemia patients.

Splicing factor 3b subunit 1 (SF3B1), a splicing factor modulating RNA alternative splicing, is frequently mutated in multiple hematological malignancies including myelodysplastic syndromes and chronic lymphocytic leukemia (CLL). The clinical impact of SF3B1 mutation on CLL remains controversial especially for patients of Asian descent. We retrospectively analyzed the frequency of SF3B1 mutation by Sanger sequencing in 399 newly diagnosed Chinese CLL patients. SF3B1 mutation was detected in 5.5% (22/399) of the studied cohort with 59.1% of them being c.A2098G (p.K700E). SF3B1 mutation was common in patients with unmutated immunoglobulin heavy chain variable region gene, positive CD38 and positive ZAP-70. Survival analysis showed that SF3B1 mutation was associated with short treatment-free survival (TFS), but not overall survival (OS). We then developed 2 new risk models, named CLL-IPI-S and CLL-PI, according to the SF3B1 mutation status and CLL-international prognostic index (CLL-IPI); CLL-PI showed greater power to predict TFS than CLL-IPI in Chinese CLL patients. Our data suggest a low incidence and adverse clinical significance of SF3B1 mutation in newly diagnosed Chinese CLL patients.

Long-term and high dose dexamethasone injection decreases the expression of Immunoglobulin Heavy (Light) Chain Variable Region Genes (IGH(L)Vs) in the mouse spleen

Glucocorticoid hormones have been widely used in clinical practice as potent anti-inflammatory and immunosuppressive agents. However, the underlying mechanisms of how they work remain unaddressed. Here, we used RNA-set to profile spleen gene expression in adult mice after consistent intraperitoneal injection of dexamethasone. We identified 13565 genes in control (injected with 0.9% NaCl) and 13702 genes in dexamethasone-injected group, 12920 genes were expressed in both, but 645 genes were identified only in control and 782 genes were identified only in the dexamethasone group. In the dexamethasone-injected group 101 and 67 genes were down-and up-regulated, respectively. Among these, 129 were coding genes, 19 were identified as non-coding genes or pseudogenes, and the remaining 20 were TEC (to be experimentally confirmed) genes. Gene ontology (GO) and KEGG pathway analysis revealed that the cytokine-cytokine receptor interaction pathway was highly enriched in these differentially expressed genes, and that a majority of the 129 identified coding genes were involved in immune system and cell adhesion-associated processes. Moreover, systemic lupus erythematosus, renin-angiotensin system, fat digestion and absorption, and glycerolipid metabolism pathways were significantly affected in the dexamethasone-treatment group. No obvious signaling pathway was enriched in the control group. Additionally, 20 immunoglobulin heavy or light chain variable region genes (IGH(L)Vs) were down regulated in the dexamethasone-injected group. IGH(L)Vs encode the variable region of immunoglobulin heavy chain and determine the diversity and specificity of antibodies. We were unable to determine the function of the 19 non-coding genes with differential expression following dexamethasone treatment. Our findings indicate that the expression of IGH(L)Vs and non-coding genes play an important role in the anti-inflammatory and immunosuppressive effects of dexamethasone and could be developed as potential agents in clinical practice.

Somatic MDC1 Mutation in Putative Pre-Leukaemic Stem Cell of a Biclonal Case of Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is an inherently heterogeneous disease in which founding lesions and cell of origin remain a subject of debate. This case of truly biclonal CLL provides evidence of the presence of a pre-leukaemic stem cell, supporting other lines of evidence that pathogenic genetic and epigenetic lesions in CLL arise early in haematopoiesis.An individual with trisomy 12 containing CLL, present at a frequency of 30%, was identified. Two distinct CD5/19-positive CLL clones were separated by flow cytometry, based on bimodal expression of CD49d. Each leukaemic clone was subjected to targeted immunoglobulin heavy chain variable region gene (IGHV) sequencing, whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray. CD5-positive T cells and a non-malignant superficial skin biopsy sample from the same individual were analysed as comparators.The CD49d-positive and CD49d-negative fractions harboured completely different IGHV rearrangements and mutational statuses (V4-34*02, hypermutated, and V3-21*02, unmutated, respectively) indicative of two unique leukaemic clones. Furthermore, trisomy 12, a relatively common aneuploidy in CLL, was present at high frequency (89.9%) in the CD49d-positive fraction but was absent in the CD49-negative and T-cell fractions. A mutation in the known CLL driver SF3B1 (c.1866G>T) was present in the CD49-negative, unmutated clone alone, along with a 17p deleted subclone in around 20% of this fraction. The presence of a 17p deletion is highly clinically relevant in this disease, and it is noteworthy that this subclone was not detected by routine fluorescence in situ hybridisation (FISH) at diagnosis. Two putative driver mutations in KMT2D and BCL11B were detected in the CD49d-positive, trisomy 12 clone alone (c.15256C>T and c.1387G>A respectively). A TET2 mutation (c.3609C>G) was found in all three fractions. The mutation in this epigenetic regulator has previously been described in clonal haematopoiesis of indeterminate potential (CHIP) and was hypothesised to have been acquired in a haematopoietic stem cell predisposing the individual to the development of haematological malignancy. However, the presence of this mutation was confirmed by Sanger sequencing of genomic DNA extracted from the skin sample, and is therefore of germline origin. The role of germline TET2 variants has not been described in lymphoid malignancy.Importantly, eight coding mutations were found common to both leukaemic clones but were absent from T-cells, implicating a common cell of origin prior to IGHV rearrangement but following B-lineage commitment (that is, a putative pre-leukaemic stem cell). One of these mutations was a disruptive 123 base pair deletion (c.4824_4946del) in MDC1 (mediator of DNA damage checkpoint 1) and was present at a variant allele frequency (VAF) of approximately 50% in both CLL clones, suggesting it was a founder mutation prior to subclone divergence. MDC1 is a mediator in the Ataxia Telangiectasia Mutated (ATM)-dependent DNA damage repair pathway and has not been previously implicated in CLL pathogenesis unlike ATM, which is frequently lost in del11q CLL. The proposed evolution of CLL in this case is presented in Figure 1.In summary, this highly unusual case of CLL provides insights into the evolution of CLL and adds another line of evidence to support the proposed early origins of CLL in haematopoiesis. The role that MDC1 may contribute to CLL pathogenesis, in particular its association with ATM dysregulation, remains to be elucidated. Further work to delineate critical pathways based on the discovered mutations is underway, and includes global transciptomic analysis of the purified leukaemic clones by RNAseq. DisclosuresNo relevant conflicts of interest to declare.

A Clinical Review of the Co-Occurrence of Myeloproliferative and Lymphoproliferative Neoplasms

The co-occurrence of myeloproliferative (MPN) and lymphoproliferative neoplasms (LPN) is rare and many publications have been limited to small case reports. Others have involved a considerable number of patients, but the coexistence remains underreported and inadequately studied. A recent retrospective review reported that a MPN patients have a 2.8-fold higher relative risk of developing LPN.A database developed at Weill Cornell Medicine (WCM) was queried for patients with ≥3 visits between 1998-2018 with a diagnostic code for a MPN and lymphoma or myeloma subtype. Patients identified were verified to ensure that study inclusion criteria were satisfied. Observed co-occurrence was compared to nation-wide reported prevalence.Demographic and clinical details of 24 patients with a MPN and LPN were recorded (Table 1). The ratio of males to females was 1.7. Essential thrombocythemia and polycythemia vera, and chronic lymphocytic leukemia (CLL) were the leading MPN and LPN subtypes, respectively. Patients were assigned to risk or staging categories at diagnosis based on subtype-specific criteria (Table 2).Median values for diagnostic bone marrow biopsy findings in 14 patients were 2% [0-5] for myeloblasts and 80% [15-100] for cellularity. Additionally, 10 patients had evaluated reticulin fibrosis: 5 (50.0%) presented as MF-0, 4 (40.0%) as MF-1, and 1 (10.0%) as MF-2.Progression to myelofibrosis was confirmed by morphology in 1 (4.2%) patient 10.1 years after a polycythemia vera diagnosis and 5.4 years after a diffuse large B-cell lymphoma (DLBCL) diagnosis. Progression to acute myeloid leukemia was confirmed by morphology in 1 (4.2%) patient 2.4 years after a chronic myelomonocytic leukemia diagnosis and 1.2 years after a smoldering myeloma (SM) diagnosis.Interphase fluorescence in situ hybridization (iFISH) detected cytogenetic abnormalities in 5/8 (62.5%) CLL patients: 2/5 (40.0%) and 5/5 (100.0%) patients harbored deletions in trisomy 12 and 13q14, respectively. Immunoglobulin heavy chain variable region gene (IGVH) status was unmutated in 2 (25.0%) patients. One (12.5%) patient was CD38+ and 2/6 (33.0%) patients were ZAP-70+. At diagnosis, all 8 patients presented with early stage disease (Rai stage 0-II). Based on the CLL-specific international prognostic index (IPI), 3/8 (37.5%) and 5/8 (62.5%) presented as low-risk and intermediate-risk, respectively.Of the 6 lymphoma patients: 5 (83.0%) patients presented with Ann Arbor stage-IV disease at diagnosis. Four (66.7%) patients presented as low/intermediate-risk, and 2 (33.3%) presented as high-risk based on disease-specific IPIs. One patient presented with -17p by iFISH. All 4 patients that were evaluated for Ki-67 had moderate/high expression.Of the 7 multiple myeloma (MM) patients, 6 (85.7%) presented as stage 1 and 1 (14.3%) as stage 3. Of the 3 SM patients, all 3 presented as low risk [12]. In addition, these patients were categorized as IgG-K (4; 40.0%), IgG-L (2; 20.0%), IgA-K (1; 10.0%), IgA-L (1; 10.0%), IgM (1; 10.0%), and biclonal IgG-L/IgA-L (1; 10.0%) [13].Mutation statuses were identified by commercially tested myeloid or lymphoid molecular panels. As expected in this MPN subtype distribution, 11 (45.8%) are JAK2+, 2 (8.3%) are MPL+, 1 (4.2%) is BCR-ABL+, and 1 (4.2%) is CALR+ (Table 3).The risk of a co-occurrent MPN and LPN is higher than expected if they are mutually exclusive (Table 4A-4B, 5). Of interest, 13 (54.2%) patients were diagnosed with a MPN 11.8±18.8 years prior to a LPN; conversely, 11 (45.9%) were diagnosed with a LPN 6.5±6.2 years prior to a MPN. In addition, MPN therapy was started 2.0±2.3 years after a MPN diagnosis, and LPN therapy was started 2.6±4.0 years after a LPN diagnosis. A review of survival analysis requires larger subtype populations since the degree of survival can vary greatly, but it has been reported that patients with a MPN or LPN have significantly reduced life expectancy when compared to the general population. Median follow-up for our patient is 8.2 years (1.5-28.0) with 17/24 (70.8%) patients still being actively followed at our institution, 6 (25.0%) are been lost to follow-up, and 1 (4.2%) is deceased.The significant prevalence of these hematologic malignancies in combination emphasizes the importance of performing a bone marrow biopsy, which we espouse at our institution, cytogenetic analysis, and myeloid and lymphoid molecular testing to identify mutations. [Display omitted] DisclosuresFurman:Loxo Oncology: Consultancy; Gilead: Consultancy; Verastem: Consultancy; Acerta: Consultancy, Research Funding; TG Therapeutics: Consultancy; Incyte: Consultancy, Other: DSMB; Pharmacyclics LLC, an AbbVie Company: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Niesvizky:Amgen Inc.: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Ritchie:Bristol-Myers Squibb: Research Funding; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Speakers Bureau; ARIAD Pharmaceuticals: Speakers Bureau.

A Descriptive Analysis of Obinutuzumab Usage in a Single Canadian Centre

Background:Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in North America with the majority of patients being over the age of 70 (Goede et al, 2014). CLL is a heterogeneous disease with some patients undergoing treatment at time of diagnosis, while others follow along a more indolent, asymptomatic course. When indicated, the choice of treatment is based on the patient's functional status, renal function and other comorbidities (Shanafelt, 2013). Previous studies have shown that unfit patients treated with Obinutuzumab plus Chlorambucil have shown prolongation of progression free survival and higher rates of complete response compared to other monoclonal based regimens (Goede et al, 2014). The aim of this study is to investigate the clinical use and uptake of Obinutuzumab in combination with Chlorambucil in a Canadian based population.Methods:All patients receiving Obinutuzumab were identified using the CLL CAISIS database. Then a retrospective chart review was conducted for patients approved for first-line antibody treatment with Obinutuzumab from January 1, 2014 to December 31, 2017. Data including patient characteristics, patterns of treatment, reported toxicities, response rates and survival data were collected and analyzed. This data was used to determine the overall safety and efficacy of this treatment regimen.Results: There were a total of 66 patients that met the inclusion criteria for this study. This cohort consisted of 54.5% males and 45.5% females, with a median age at diagnosis of 68 years (range: 46-94). Within this population 47 patients underwent florescence in situ hybridization testing, which identified 32 patients with chromosomal abnormalities. The immunoglobulin heavy chain variable region gene mutation was also tested in 41 patients, and identified 22 patients that were unmutated. At the time of treatment the median age was 73 (range: 55-98) with a median Cumulative Illness Rating Scale (CIRS) score of 8 (range: 3-15). Of the 66 patients who started treatment with Obinutuzumab, 50 patients (76%) achieved a partial response and 5 patients (8%) had no response. Of note, 21 patients (31.8%) discontinued treatment prior to completion of all six cycles due to cytopenias (4), other comorbidities (4), progression of CLL (2), decline in functional/mental status (2), patient request (2) and other (7). In regards to their treatment there was a high incidence of infusion related reactions (IRRs) on the first day of treatment, with 30 patients (45.5%) requiring intervention. There was a subset of 15 patients that had received low dose Chlorambucil prior to treatment with Obinutuzumab which resulted in a lower average lymphocyte count (38.1) and lower rate of IRRs (33%) compared to those with no prior Chlorambucil (average lymphocyte count: 66.5, rate of IRR: 42%). Due to toxicity and tolerability of Chlorambucil, dose reduction was observed in 37 patients (56%) throughout all six cycles of treatment. In the end, only 18 patients (27.3%) received all doses of Obinutuzumab and Chlorambucil while 37 patients (56%) received all doses of Obinutuzumab. Within the entire cohort, 12 patients have relapsed and 9 of these patients have required additional therapy. This relapse rate can be attributed to lack of response to Obinutuzumab (3), a 17p del (1), poor prognostic markers (5), and inadequate Obinutuzumab (1).Conclusion:In this current study, our results demonstrate that the majority of patients in this cohort were able to complete treatment with Obinutuzumab. Although our demographics were similar to previous studies, we found higher partial response rates (76%) likely due to differences in standards of practice, such as imaging studies after treatment. DisclosuresWhiteside:Roche: Membership on an entity's Board of Directors or advisory committees, Other: teaching sessions. Dawe:AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Johnston:Roche: Other: unrestricted grant received in the past; Abbvie: Other: unrestricted grant received in the past; Teva: Other: unrestricted grant received in the past; Gilead: Other: unrestricted grant received in the past; Janssen: Other: unrestricted grant received in the past. Banerji:Roche: Other: Unrestricted grant received in the past; Janssen: Other: Unrestricted grant received in the past; Abbvie: Other: Unrestricted grant received in the past; Gilead: Other: Unrestricted grant received in the past; Teva: Other: Unrestricted grant received in the past.

Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era.Aim. To apply a NGS approach to investigate ID in CLL.Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B).Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F).Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. DisclosuresZaja:Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

Evaluation of the CLL-IPI in relapsed and refractory chronic lymphocytic leukemia in idelalisib phase-3 trials

The CLL-IPI is a risk-weighted prognostic model for previously untreated patients with chronic lymphocytic leukemia (CLL), but has not been evaluated in patients with relapsed CLL or on novel therapies. We evaluated the CLL-IPI in 897 patients with relapsed/refractory CLL in 3 randomized trials testing idelalisib (PI3Kδ inhibitor). The CLL-IPI identified patients as low (2.2%), intermediate (12.8%), high (48.7%), and very high (36.2%) risk and was prognostic for survival (log-rank p < .0001; C-statistic 0.706). Of CLL-IPI factors, age >65, β2-microglobulin >3.5mg/L, unmutated immunoglobulin heavy chain variable region gene, and deletion 17p/TP53 mutation were independently prognostic, but Rai I-IV or Binet B/C was not. The CLL-IPI is prognostic for survival in relapsed CLL and with idelalisib therapy. However, low/intermediate risk is uncommon, and regression parameters of individual factors in this risk-weighted model appear different in relapsed CLL. Reassessment of the weighting of the individual variables might optimize the model in this setting.

NDRG2 mRNA levels and miR-28-5p and miR-650 activity in chronic lymphocytic leukemia

BackgroundNDRG2 is identified as a tumor suppressor gene in many tumors, and functions in cell proliferation, differentiation and apoptosis. Recent data indicate that NDRG2 expression is up-regulated by TP53. Moreover, proposed mechanisms of NDRG2 inactivation include epigenetic silencing of the NDRG2 promoter and down-regulation by microRNAs (miRNAs). However, few studies have ever been done on the role of NDRG2 and the NDRG2-regulating miRNAs interference in chronic lymphocytic leukemia (CLL).MethodsNDRG2 and microRNAs mRNA levels in CLL subjects were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The dual-luciferase reporter assay was performed to determine NDRG2-related miRNAs. Low expression of mature exogenous miRNAs in CLL cells was established by transient transfection. NDRG2 protein levels in CLL cells were detected by western blot. In addition, flow cytometry was conducted to examine the apoptosis of CLL cells.ResultsLower expression of NDRG2 was found in the B-cells from 102 CLL patients compared the 40 normal subjects (P < 0.001). Patients with advanced Binet stage (P = 0.001), high lactate dehydrogenase (LDH) level (P = 0.036), un-mutated immunoglobulin heavy chain variable region gene (IGHV) (P = 0.004) and those with p53 aberrations (P < 0.001) had a markedly lower levels of NDRG2 mRNA. This decrease was associated with briefer time-to-treatment (P = 0.001) and poorer survival (P < 0.001). High expression of miR-28-5p and miR-650 was associated with Binet B/C stage (P = 0.044) and IGHV un-mutated (P = 0.011), as well as Binet B/C stage (P = 0.013) and p53 aberrations (P = 0.037), respectively. Inhibition of miR-28-5p or miR-650 could induce more apoptosis in CLL cells with germline TP53.ConclusionsNDRG2 mRNA levels might be a useful prognostic variable for patients of CLL and up-regulating NDRG2 transcription may be a therapy approach in CLL without p53 aberrations.

Real-world testing and treatment patterns in chronic lymphocytic leukemia: A SEER patterns of care analysis.

Laboratory testing and treatments for chronic lymphocytic leukemia (CLL) have changed dramatically within the last decade. The authors evaluated changes in patterns of real-world testing and treatment over time by comparing 2 population-based cohorts. The National Cancer Institute-sponsored Patterns of Care study was conducted among patients with CLL who were sampled from 14 Surveillance, Epidemiology, and End Results (SEER) program registries. Demographics, testing, and treatment data were abstracted from medical records within 24 months of diagnosis. A total of 1008 patients diagnosed in 2008 and 1367 patients diagnosed in 2014 were included. There was a significant increase in fluorescence in situ hybridization (FISH) testing, immunoglobulin heavy-chain variable region gene (IgVH ) mutation analyses, and lymph node biopsies between 2008 and 2014. FISH testing was performed in the majority of, but not all, treated patients (53% in 2008, which increased to 62% in 2014). Some differences in the receipt of FISH testing by age and insurance status were observed over time (older patients and Medicare patients without private insurance were less likely to be tested in 2014). There were contrasting testing patterns noted by practice type and year, with nonteaching hospitals more likely to perform bone marrow biopsies in 2008, and teaching hospitals more likely to perform FISH and IgVH testing in 2014. There also were differences in treatments over time, with the use of bendamustine and rituximab being more common in 2014, at the expense of fludarabine, cyclophosphamide, and rituximab. There have been rapidly changing practices in the testing and treatment patterns of patients with CLL within the last decade.

Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) were mutated in 12 patients (2.6%), and genes downstream of BRAF (MAPK2K1, MAPK2K2, and MAPK1) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P=0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.

Identification of prognostic parameters in CLL with no abnormalities detected by chromosome banding and FISH analyses.

Chronic Lymphocytic Leukaemia (CLL) is a heterogeneous disease with a clinical course dependent on cytogenetic features. However, in 15-20% of cases both chromosome banding and fluorescence insitu hybridisation analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we performed a comprehensive analysis on 171 patients including genomic arrays (comparative genomic hybridisation and single nucleotide polymorphism), immunoglobulin heavy chain variable region genes (IGHV) status, flow cytometry and targeted sequencing. Genomic arrays detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV status was unmutated in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (15/171; 9%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of these, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, by integrating genomic and molecular data, we identified patients at higher risk for treatment need.

Trisomy 12 chronic lymphocytic leukemia expresses a unique set of activated and targetable pathways.

Although trisomy 12 (+12) chronic lymphocytic leukemia (CLL) comprises about 20% of cases, relatively little is known about its pathophysiology. These cases often demonstrate atypical morphological and immunophenotypic features, high proliferative rates, unmutated immunoglobulin heavy chain variable region genes, and a high frequency of NOTCH1 mutation. Patients with +12 CLL have an intermediate prognosis, and show higher incidences of thrombocytopenia, Richter transformation, and other secondary cancers. Despite these important differences, relatively few transcriptional profiling studies have focused on identifying dysregulated pathways that characterize +12 CLL, and most have used a hierarchical cytogenetic classification in which cases with more than one recurrent abnormality are categorized according to the abnormality with the poorest prognosis. In this study, we sought to identify protein-coding genes whose expression contributes to the unique pathophysiology of +12 CLL. To exclude the likely confounding effects of multiple cytogenetic abnormalities on gene expression, our +12 patient cohort had +12 as the sole abnormality. We profiled samples obtained from 147 treatment-naïve patients. We compared cases with +12 as the only cytogenetic abnormality to cases with only del(13q), del(11q), or diploid cytogenetics using independent discovery (n=97) and validation (n=50) sets. We demonstrate that CLL cases with +12 as the sole abnormality express a unique set of activated pathways compared to other cytogenetic subtypes. Among these pathways, we identify the NFAT signaling pathway and the immune checkpoint molecule, NT5E (CD73), which may represent new therapeutic targets.