NOTCH1 Mutation and Treatment Outcome In CLL Patients Treated With Chlorambucil (Chl) Or Ofatumumab-Chl (O-Chl): Results From The Phase III Study Complement 1 (OMB110911)
Eugen TauschPhilipp BeckRichard F. SchlenkSabrina KlessChristina GallerPeter HillmenFritz OffnerAnn JanssensK. GovindbabuSebastian GrosickiJiří MayerPanagiotis PanagiotidisS. Danhauser-RiedlAstrid McKeownPaul D P ́B WinterIra GuptaHartmut DöhnerStephan Stilgenbauer
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Next-generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re-sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features (unmutated IGHV, n=137; IGHV3-21 subset #2, n=51) were sequenced on the HiSeq 2000 and data were analyzed using well-established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/180 (63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/177 (84%) of all mutations. We selected 155 mutations for Sanger validation (variant allele frequency, 10–99%) and 93% (144/155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11–27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/82 (94%) mutations. In summary, this study demonstrates that targeted next-generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand-alone test without the need for confirmation by Sanger sequencing.
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Fludarabine (F) and cyclophosphamide (C) remain backbones of up-front chemotherapy regimens for chronic lymphocytic leukemia (CLL). We report long-term follow-up of a randomized F vs. FC trial in untreated CLL (#) . With median follow-up of 88 months, estimated median progression-free survival (PFS) was 19.3 vs. 48.1 months for F (n = 109) and FC (n = 118), respectively (p < 0.0001), and median overall survival (OS) was 88.0 vs. 79.1 months (p = 0.96). In multivariable analyses, variables associated with inferior PFS and OS respectively were age (p = 0.002, p < 0.001), Rai stage (p = 0.006, p = 0.02) and sex (p = 0.03, PFS only). Del(17)(p13.1) predicted shorter PFS and OS (p < 0.0001 for each), as did del(11q)(22.3) (p < 0.0001, p = 0.005, respectively), trisomy 12 with mutated Notch1 (p = 0.003, p = 0.03, respectively) and unmutated IGHV (p = 0.009, p = 0.002, respectively), all relative to patients without these features. These data confirm results from shorter follow-up and further justify targeted therapies for CLL.
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Weiss–Kruszka syndrome (WSKA) is a rare disease most often caused by mutations in the ZNF462 gene. To screen for hereditary diseases, exons from the patient’s genome were sequenced. Genomic PCR experiments followed by Sanger sequencing were used to confirm the mutated genomic regions in the patient and his parents. We report a new mutation site, a heterozygous mutation (NM_021224.6:c.6311dup) in ZNF462 in a male patient of 8 years old. The mutation in the ZNF462 gene caused WSKA. This patient is the first case with WSKA characterized by attention-deficit hyperactivity disorder and complete growth hormone deficiency without pituitary lesions. Our results suggest that the heterozygous mutation in ZNF462 is the direct cause of WSKA in this patient. Mutations in other genes interacting with ZNF462 result in similar symptoms of WSKA. Furthermore, ZNF462 and its interacting proteins ASXL2 and VPS13B may form a protein complex that is important for normal development but awaits more studies to reveal its detailed functions.
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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, <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 < 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 < 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 < 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
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Background: The immunoglobulin heavy chain gene (IgHV) mutation is one of the most reliable predictive biomarkers in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy. The incidence of CLL in Asia is about 10 times lower, than in Western countries and there are potential different IgHV genes mutations and stereotypy between Eastern and Western CLL. For instance, Chinese patients with CLL have higher percentage of mutated subgroup compared with European patients (55%) and different clinical presentation (Yi Xia et al., 2013; Marilisa Marinelli et al., 2016). The incidence of CLL in Kazakhstan is similar to that of the Western world, but to date nothing is known on the IgHV gene representation and mutations. Aims: The aim of the present study was to evaluate IgHV Repertoire Features In Kazakhstan Chronic Lymphocytic Leukemia Patients. Methods: The study included samples and medical records from 35 patients (19 male and 16 female) from the different regions of Kazakhstan. Participants’ ages ranged from 35 to 84 years (Median = 61). According to the European Research Initiative on CLL recommendations, IgHV mutational status was determined by amplifying the IgHV transcript by PCR, sequencing the gene through Sanger sequencing, and comparing the transcript to known germline genes available in immunoglobulin databases. We use the 2% cut-off to discriminate between mutated and unmutated rearrangements. Descriptive statistics for discrete variables included counts and frequency distributions. Results: Patients were divided into the four prognostic groups proposed by the CLL-IPI as follows: 6 (17.2%) at low risk, 16 (45.7%) at intermediate risk, 10 (28.6%) at high risk, and 3 (8.6%) at very high risk. The median observation time was 2.5 years. Eleven patients (31.4%) have no indications for treatment. 21 (60%) patients were treated with chemoimmunotherapy as first line and only 3 (12.5%) patients with 17p deletion received Ibrutinib. 20 (57.1%) had unmutated IGHV genes (U-CLL), 13 (37,1%) patients had mutated genes (M-CLL) and 2 (5.7%) cases carried double in-frame rearrangements. Prevalence of U-CLL which is consistent with their poor clinical outcome was close to Italian population (51%) and almost two times higher than among the Chinese patients (34%). The most of patients in this study were Caucasian (29; 83%) and only 6 patients (17%) were identified as Asians. In U-CLL group the most common IgHV genes were IGHV1-69 (8; 40%), IgHV1-2 (5; 25%), IgHV3-15 and IgHV4 (2; 10%). IgHV1-69 gene in the vast majority of cases was expressed in an unmutated state. In M-CLL most common IgHV genes were IGHV4, IgHV3 and IgHV2 without predominance of one of the subgroups and there were not any cases with IgHV1-69. Gender ratio in this study was 1:1 among patients with IgHV1-69 and all of them were Caucasian. During the study 4 participants with U-CLL died and two of them had IgHV 1-69 rearrangement. Summary/Conclusion: Hence, we conclude that Kazakhstan CLL patients show unique IgHV repertoire which is close to patients from western countries. Remarkably, IgHV1-69 was the most prevalent IgHV subgroup in Kazakhstan U-CLL patients (40%). IgHV1-69 gene is more associated with ethnicity, but not a gender.
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Follicular lymphoma (FL) is a heterogeneous disease with some patients developing progressively or transformed disease early, whereas others follow an indolent clinical course. We evaluated the prognostic value of immunoglobulin heavy chain variable (IGHV) gene usage and mutational status in FL patients. One hundred and four IGH sequences were obtained in tumour samples from 99 patients. The IGHV3 subgroup had the highest usage frequency (57.7%) with IGHV3-23 being the most common sequence. Patients with the IGHV5 subgroup or IGHV sequences from more than one subgroup had significantly less favourable prognosis with an estimated 5-year survival of 62.5 and 50.0%, respectively, as compared with a 5-year survival of 95.1% for patients with other IGHV subgroups (P=0.013 and P<0.001, log-rank). The poor survival associated with IGHV5 or >1 IGHV subgroup usage was an independent prognostic factor in Cox multivariate analysis (P=0.005). IGHV genes were unmutated showing >98% homology in 15.2% of cases. Contrasting the situation in chronic lymphocytic leukaemia (CLL), the presence of unmutated sequences did not yield prognostic information, although unmutated sequences were associated with age at diagnosis >60 years (P=0.022, Fisher's exact). In conclusion, our results indicate that analysis of IGHV gene usage might aid in predicting prognosis for FL patients.
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Follicular lymphoma
Immunoglobulin heavy chain
Subgroup analysis
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