ATM Deletion Research Articles

Compound heterozygous variants including a novel copy number variation in a child with atypical ataxia-telangiectasia: a case report

BackgroundAtaxia-telangiectasia is a rare autosomal recessive, neurodegenerative disorder caused by alterations in the ATM gene. The majority of ATM pathogenic variants are frameshift or nonsense variants which are predicted to truncate the whole ATM protein. Herein, we report on an ataxia telangiectasia child with atypical phenotype who was identified as compound heterozygous for two ATM variants involving a previously described pathogenic single nucleotide variation (SNV) and a novel copy number variation (CNV).Case presentationA 6-year-old boy presented with delayed development and oculomotor apraxia. Brain magnetic resonance imaging showed interval development of mild atrophy in the cerebellum. Serum alpha fetoprotein level was in normal range. Next-generation sequencing and single-nucleotide polymorphism array tests were performed. Next-generation sequencing revealed a heterozygous nonsense pathogenic variant in ATM, c.742C > T (p.Arg248Ter) inherited from the father. Single-nucleotide polymorphism array revealed a compound heterozygous CNV, arr[GRCh37] 11q22.3(10851766–108183226) × 1, 31460 bp (exons 24–40 deletion of ATM) inherited from the mother, which was validated by reverse transcription-polymerase chain reaction analysis (RT-PCR). We demonstrated that this variant (NM_000051.4:c.3403_6006del) generated a product of in-frame deletion of exon 24–40 of ATM (p.Ser1135_Gln2002del).ConclusionsThe compound heterozygosity for ATM variants involving a previously described pathogenic SNV and a novel CNV may be associated with the atypical clinical manifestations. This clinical report extends the genetic and phenotypic spectrum of ATM pathogenic variants in atypical ataxia-telangiectasia, thus making implementation of advanced analysis beyond the routine next-generation sequencing an important consideration in diagnosis and rehabilitation services for children with ataxia-telangiectasia.

Clinical analysis of fludarabine and cyclophosphamide combined with rituximab in the first-line treatment of 43 cases of chronic lymphoblastic leukemia

目的探讨FCR方案（氟达拉滨+环磷酰胺+利妥昔单抗）一线治疗慢性淋巴细胞白血病（CLL）的疗效。方法回顾性分析2004年5月至2017年12月一线应用FCR方案治疗的43例CLL患者的临床资料。结果①43例CLL患者中，男31例，女12例，接受FCR方案治疗时中位年龄58（36～72）岁；8例患者伴B症状，外周血中位淋巴细胞计数26（3～550）×109/L，IGHV基因未突变62.1％（18/29），P53基因缺失14.0％（6/43），RB1基因缺失18.6％（8/43），12号染色体三体占25.6％（11/33），ATM基因缺失16.7％（7/42）。全部患者FCR方案中位疗程数为4（2～6）个。②全部43例患者的总体反应率（ORR）为88.4％（38/43），完全缓解（CR）20例（46.5％），部分缓解（PR）18例（41.9％），疾病稳定（SD）4例（9.3％），疾病进展（PD）1例（2.3％）；7例（16.3％）患者获得微小残留病（MRD）阴性。③中位随访51（6～167）个月，中位无进展生存（PFS）时间为67（29～105）个月，中位总生存（OS）时间未达到，5年PFS率为（62.1±8.6）％，10年PFS率为（31.0±14.3）％，5年OS率为（70.5±8.3）％，10年OS率为（51.3±13.8）％。疗程数<4为影响OS的不良预后因素，P53基因缺失、疗程数<4为影响PFS的不良预后因素（P<0.001），且在多因素分析中仍具有预后意义［P53基因缺失：HR＝7.65（95％CI 1.74～33.60），P＝0.007；疗程数<4：HR＝3.75（95％CI 1.19～11.80），P＝0.025］。④18例（41.9％）患者于化疗后发生2～3级感染，19例（44.2％）发生3～4级血液学不良反应，1例（2.3％）患者发生肿瘤溶解综合征，所有不良反应经对症处理均恢复。结论FCR方案一线治疗CLL的治疗反应及远期生存较理想，不良反应可接受。

Disease Progression on Ibrutinib Therapy Is Associated with a Poor Clinical Outcome in Chronic Lymphocytic Leukemia (CLL) Patients Managed in Standard Clinical Practice

Introduction: Ibrutinib is an irreversible Bruton Tyrosine Kinase (BTK) inhibitor that has shown efficacy in the treatment of CLL as a front-line therapy or in patients with relapse/refractory (RR) disease. Anecdotally, accelerated disease progression and refractoriness to subsequent therapies in CLL or other B-cell malignancies patients treated with ibrutinib has been observed. In our present contribution, we studied in a real-world patient population the clinical outcome of CLL patients treated with ibrutinib who discontinued therapy. Methods: We conducted a comprehensive retrospective analysis on CLL patients receiving ibrutinib at our Institute. Demographic characteristics, response to subsequent therapies, and results of FISH analysis were obtained. Correlation between clinical outcomes and status on ibrutinib therapy (responding, progressing, or discontinuing due to toxicity) was performed. Statistical analysis was carried out in SPSS v20. Chi Square, Fisher's exact test were used to compare categorical data, Kaplan Meier and Log rank tests for time to event analysis and Cox regression for univariate analysis. Results: A total of 70 CLL patients treated with ibrutinib were followed for a median period of 19.4 mo [ range 0.3 - 41.3]. Sixty-three patients had RR CLL and 7 received ibrutinib as frontline therapy. Our patients had a median age of 67.9 years (46 - 92), a majority had advanced stage disease (Rai stage 3 - 31.4%, Rai stage 4 - 41.4%). Median number of prior treatments was 2 (range 0-7). Overall response rate (ORR) to ibrutinib was 52.8% (complete response [CR] - 11.4%, partial response [PR] - 41.4%]. Additionally, stable disease [SD] was present in 25.7% leading to a Clinical Benefit Rate (CBR) of 78.5%. A total of 25 (38%) patients discontinued therapy. Reasons for discontinuation included progressive disease (PD) in 13 (52%) (5 with Richter's transformation [RT]), drug toxicity (DT) in 7 (28%), and other reasons in 5 (20%) patients. DTs included atrial fibrillation (n=2), sepsis (n=2), pneumonia (n=1), congestive heart failure (n=1) and fatigue (n=1). Other reasons for discontinuation were other terminal cancer (n=2), patient preference, alcoholism and insurance issues. Univariate analysis showed an increased risk of discontinuation associated with del(13q) (HR 1.56, 95%CI 0.7-3.7, p=0 .32), ATM deletion (HR 1.32, 95%CI 0.5-3.4, p=0 .57), Unmutated IgH (HR 3.5, 0.5-27.3, p=0 .23) though none reached statistical significance. We did not observe an increased risk of discontinuation in patients with del(17p) (HR 0.80, 95%CI 0.3-2.7, p=0 .72). Cytogenetic data after ibrutinib discontinuation was available in 12 patients and showed new onset del(13q) (n= 3), del(17p) (n= 4) and ATM deletion (n= 1) including 1 patient who had complex cytogenetics (≥3 cytogenetic abnormalities). In patients who discontinued therapy, we observed a mortality rate of 72% (18/25) with a median overall survival (OS) of 17.8 mo (95% CI, 7.4-28.2); in comparison, median OS was not reached in patients who remained on therapy (p <0.05). A lower median OS was observed in patients who discontinued due to DT (5.9 mo, 95% CI, 3.9-8), compared to those with RT (11.5 mo, 95% CI 3.1-20), PD (18.2 mo, 95%CI 3.8-32.6) though this was not statistically significant (p= 0.117). Despite trends towards lower median OS in patients who discontinue ibrutinib, univariate analysis did not show a statistically significant association with del(13q) (HR 1.8, 95%CI 0.7-5, p=0 .248), ATM deletion (HR 1.32, 95%CI 0.5-3.9, p=0 .61), Unmutated IGH (HR 1.08, 95%CI 0.13-9, p=0 .94), and ZAP70+ (HR 1.15, 95%CI 0.36-3.64, p=0 .81). Two patients had complex cytogenetics with a median OS of 1.5 mo (HR 4.5, 95%CI 0 .9-22.4, p <0.05). Thirteen patients received subsequent therapy after ibrutinib discontinuation with idelalisib (n=3), lenalidomide (n=3), rituximab with bendamustine (n=2), R-CHOP (n=2), or other treatments (n=3). ORR to post ibrutinib therapy was 23.1% with CR in 3/13 patients (reason for discontinuation- RT - 1, other reasons- 2). Two patients had SD (both discontinued Ibrutinib for PD) leading to a CBR of 38.5%. Eight of 13 patients had PD and died during follow up period. Conclusion: Ibrutinib has good efficacy in the treatment of CLL as front-line therapy and in relapse refractory disease in a real-world setting. However, patients who discontinue therapy have poor survival outcomes and poor response to salvage therapy. DisclosuresNo relevant conflicts of interest to declare.

Gender, Age and TP53 Aberrations in Context of Complex Karyotypes in Chronic Lymphocytic Leukemia Patients in the Era of Chemo +/- Immunotherapy

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia characterized by a heterogeneous course of the disease. Determination of prognosis of patients (pts) is based on clinical and laboratory parameters, where cytogenetic examination plays a significant role. According to recent studies the complex karyotype (CK) shows to have adverse prognostic value and occurrence of CK is reported in 15-20% of untreated pts. Multiple diverse chromosomal translocations, deletions, duplications, numerical changes or dicentric chromosomes arise within complex karyotypes.Our aims were to determine the CK frequency in our cohort of untreated CLL pts, to verify its prognostic significance and to validate whether TP53 disruptions or ATM deletions involved in CK affect survival of pts. Moreover we aimed to investigate types of chromosomal aberrations implied within the CK in detail and to identify their possible adverse prognostic impact.A total of 644 untreated CLL pts (diagnosed according to iwCLL criteria) were examined at Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Olomouc, Czech Republic (in years 1996-2017). Peripheral blood or bone marrow samples were examined by classical and molecular cytogenetic and molecular genetic methods (G-banding, FISH, mFISH, arrayCGH, PCR, sequencing, NGS - Illumina, MiSeq) using standard protocols.CK was detected in 79 pts (12.3%, 57 men and 22 women), median of age at diagnosis was 64 years (34-87 years). The most frequently detected recurrent changes in pts with CK were deletions of 13q14 (68%), 11q22 (43%), 17p13 (33%) and duplication of 8q24 (27%). Chromosomes mostly included in CK were chromosomes 13, 11, 17, 2, 8 and 6, respectively. Sixty pts (76%) had unmutated IGHV status and 6 pts (8%) mutated IGHV3-21 . TP53 gene mutations were detected in 25 pts out of 71 examined ptswith CK (35%), 19 of pts (27%) had both deletion and TP53 mutation.Statistical analysis has shown a lower overall survival (OS) rate of CK pts compared to pts without CK (77 months vs. 115 months, p<0.0001). We also observed influence of gender and age on the OS of CK pts. More adverse outcome has been seen in women with CK compared to women without CK (37 months vs. 154 months, p<0.0001; men with CK vs. men without CK 84 months vs. 106 months, p = 0.0049) and also in pts with CK with age over 60 years (pts with CK over 60 vs. pts with CK under 60: 68 months vs. 123 months, p=0.0075). The effect of presence of translocations, numerical changes and dicentric chromosomes as part of the CK on patient´s survival has not been demonstrated (p=0.5; p=0.35; p=0.62). The deletions of TP53 and ATM genes have not occurred simultaneously in our CK cohort and the survival of these pts has not been worse. Nevertheless we could see a trend to worse survival in pts with CK and TP53 deletion/mutation (p=0.076). However, pts without CK with the occurrence of ATM or TP5 3 deletions have had a comparable negative prognostic impact just as the pts with CK (p=0.37/p=0.78).In accordance with other authors we proved that pts with CK occurring before treatment had significantly shorter survival compared to pts without CK. Importantly, the occurrence of CK in CLL has the worst impact on OS of pts older than 60 years. Moreover OS has shortened the most significantly in women with CK compared to women without CK in our single center experience. To our best knowledge these findings was not reported by other authors so far. To explain this phenomenon it would be necessary to extend studied cohort of pts with CK. Using of standard cytogenetic methods completed with modern molecular genetic analyses remains essential for risk stratification of CLL pts. Finally, not only pts with TP53 aberrations, but also pts with CK could be possible candidates for new treatment approaches.This work was supported by grants: VES16-32339A, IGA_LF_2017_007. DisclosuresPapajik:Abbvie: Consultancy, Research Funding; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Research Funding; Roche: Other: Travel/accomodation expenses, Speakers Bureau.

Tumor genotype dictates radiosensitization after Atm deletion in primary brainstem glioma models.

Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, palliative radiotherapy remains the standard treatment. Here, we utilized Cre/loxP technology to show that deleting Ataxia telangiectasia mutated (Atm) in primary mouse models of DIPG can enhance tumor radiosensitivity. Genetic deletion of Atm improved survival of mice with p53-deficient but not p53 wild-type gliomas after radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. Primary p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, NAD(P)H quinone dehydrogenase 1 (Nqo1). Tumors lacking p53 and Ink4a/Arf expressed the highest level of Nqo1 and were most resistant to radiation, but deletion of Atm enhanced the radiation response. These results suggest that tumor genotype may determine whether inhibition of ATM during radiotherapy will be an effective clinical approach to treat DIPGs.

Advanced Prostate Cancer with ATM Loss: PARP and ATR Inhibitors

BackgroundDeleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. ObjectiveTo characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. Design, setting, and participantsWe studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. Outcome measurements and statistical analysisATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. Results and limitationsOverall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. ConclusionsATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. Patient summaryOf aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR.

Ibrutinib Plus Rituximab (IR) Followed By Short Course R-Hypercvad/MTX in Patients (age ≤ 65 years) with Previously Untreated Mantle Cell Lymphoma - Phase-II Window-1 Clinical Trial

Ibrutinib Plus Rituximab (IR) Followed By Short Course R-Hypercvad/MTX in Patients (age ≤ 65 years) with Previously Untreated Mantle Cell Lymphoma - Phase-II Window-1 Clinical Trial

Anti-CD19 Chimeric Antigen Receptor T Cell Treatment of Relapsed /Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Resistant to Bruton's Tyrosine Kinase (BTK) Inhibitor

CASE PRESENTATION: A 68-year-old male was diagnosed with CLL/SLL in November 2007. Bone marrow asp/bx: 36.5% lymphocytes, 78% CD19, 65% ATM (11q22 deleted) positive cells, 13.5% D13S25 (13q14.3 deleted). On December 10, 2009, the patient took FCR scheme for five cycles, followed by FR scheme for one cycle, and then a month of Chlorambucil. On September 5, 2013, the patient took BR scheme for four cycles with no effect. From March 2015 to Feb 2016, 420 mg of Ibrutinib was administered daily. On January 15, 2016, the patient developed swollen lymph nodes in his right neck with intermittent lumps, fever and nausea. He was admitted into the hospital at Feb 2, 2016. Test results: multiple swollen superficial lymph nodes over the body, with the biggest measuring 60×30mm on the right neck, with no tenderness. Supplementary tests: peripheral white blood cells (WBC) 11.94×10E9/L, lymphocyte 7.5×10E9/L, CD19 cells 6.73×10E9/L, bone marrow lymphocyte 62%, peripheral blood lymphocyte 52%. Immunophenotype: CD5, CD19, CD20dim, CD23, CD11b dim, HLA-DR expression, visible CD5+CD19+ cell clusters, and visible immunoglobulin cKappa with restricted expression. On March 10, 2016, peripheral blood platelet 60 × 10E9/L, CD19 cells 1.94×10E9/L, lactate dehydrogenase 460U/L, FER 115.6ng/ml, hepatitis B virus carrier. Diagnosis: CLL/SLL IV stage, ATM (11q22) deletion, D13S25 (13q14. 3) positive, CD19 positive. Relapse of CLL/SLL occurred again after four months and at this stage the patient was considered for therapy in a clinical trial of CD19-specific chimeric antigen receptor (CAR-) T cell therapy. Ethical approval and informed consent were obtained for anti-CD19 CAR T Cell treatment of ibrutinib resistance in relapsed/refractory CLL/SLL. We infused autologous T cells transduced with a CAR T 19 retroviral vector with CLL/SLL at doses of 3.3 × 10E8 CART19 cells on Mar. 16 2016. Patients were monitored for responses, toxic effects, and the expansion and persistence of circulating CART19 cells. After CART19 cells were infused, the patient experienced chills, fever, headache, weak, anorexia, nausea, shortness of breath, chest tightness, heart palpitation, hypotension and shock for 9 days. The serum levels of IFN-Υ were at their highest at day 7 after CAR T cells infusion. Serum interleukin 6 (IL-6) was at 680pg/ml and CD3+ cells were 97.5%, CD8+ cells 72.8% (18.7-32.8%), FER was 1529.5ng/ml (Normal No. 22-322ng/ml) 14 days after CAR-T cell infusion. The serum levels of IL-6 were at their highest at day14. The patient was diagnosed as having cytokine release syndrome. After the patient took the anti-IL-6R antibody and anti-TNF antibody, he began to recover gradually. Enlarge lymph nodes shrunk after being infused with CART19 cells for 7 days. The peripheral blood CD19 B lymphocytes were 0 on day 14 after infused with CAR T19 cells. Q-PCR was used to detect the amount of the peripheral blood CART19 cells, which stood at 5485 copies/μl, 924 copies/μl, 191 copies/μl respectively 2 weeks, 6 weeks and 3 months after infusing with CART19 cells. The peripheral blood CART 19 cells were not detectable 4 months after infusing with CART19 cells until present. The lymphadenopathy was decreased gradually after 14 days of infusion. The MRI test showed that lymphadenopathy reduced markedly or disappeared after 6 months of infusion. ATM (11q22 deleted) negative, D13S25 (13q14.3 deleted) negative. After treatment with CAR T 19 cell therapy for 53 months, the patient remained disease-free, the patient's lymph nodes, lymphocytes and I mmunoglobulins were normal. CONCLUSIONS : Cancer immunotherapy as a method of cancer treatment is the most effective after conventional treatments such as radiotherapy, chemotherapy, and surgery. For BTK Inhibitor resistance in relapsed and refractory CD19+ CLL/SLL, CD19 is a favorable target, because the expression of CD19 is limited to B cells and not present in other tissues or cells. Currently, the efficacy of this treatment in treating CLL/SLL remains to be seen. The effects of chemotherapy on the patient's B cell lymphoma are negligible, due to the fact that his CLL/SLL have become relapsed and refractory. As a result we chose the CAR T19 cell therapy genetic engineering technique as a method of treatment, to which the patient has responded well. Therefor, CAR T cell technology overcome the limitations of existing cancer therapies and has great potential for development and application. Disclosures No relevant conflicts of interest to declare.

BRD4 Inhibitors Enhance the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia

Background: The development of drugs targeting BTK and BCL2 has dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents has been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulates transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of two BRD4i, AZD5153 and PLX51107, and their cooperative role with the BCL2 inhibitor venetoclax and BTK inhibitor ibrutinib in CLL. Methods: Primary tumor cells were isolated from 36 CLL patients. Peripheral blood mononuclear cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults under RPCCC protocol CIC-01-16. B-cells were then isolated from enriched lymphocytes by MACS separation using a human B-cell Isolation Kit II. CLL cells were seeded at 3 x106/mL in a 384 well tissue culture plates and exposed to different doses of AZD5153 (1-20uM) or PLX51107 (1-20uM) as a single agent or in combination with venetoclax (1-100nM) or ibrutinib (50-1000nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. In addition, CLL cells were plated in 6 well plates and exposed to AZD5153 or PLX51107 alone or in combination with either venetoclax or ibrutinib for 24 hrs. Induction of apoptosis and changes in B-cell receptor signaling, BCL2 and NF-kB pathway related proteins were evaluated by flow cytometry or Western blotting respectively. Results: The study included 33 treatment naïve and 3 relapsed/refractory CLL patients. Deletion 11q was detected in 6 patients and del 17p was seen in 1 patient. Both AZD5153 and PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells. Activity was observed in both treatment naïve and relapsed/refractory samples. Moreover, activity was observed in del 17p or ATM deletion samples. Synergistic activity was observed between BRD4is and venetoclax. PLX51107 exhibited synergy with venetoclax in 83% of the samples tested. Similarly, AZD5153 exhibited synergistic effects when combined with venetoclax in 61% of the samples. To a lesser degree, BRD4is exhibited synergistic activity when combined with ibrutinib, (44% with PLX51107 and 39% with AZD5153). Induction of apoptosis was observed in CLL cells exposed to either BRD4i. Morevoer, an increase in the percentage of apoptosis was observed when BRD4i was combined with venetoclax. Western blotting showed a accumulation of p21, reduced levels of C-MYC, as well as modulation of BCL-2 family member proteins such as BCL2, MCL-1 following BRD4i ex vivo exposure. Conclusions: Our data indicate that BRD4 inhibition results in apoptosis of primary tumor cells isolated from patients with various subtypes of CLL. PLX51107 and AZD5153 exhibit synergistic effects when combined with venetoclax and to a lesser degree with ibrutinib. Modulation of BCL-2 family members following BRD4 inibition could play a role in the synergistic activity observed in our studies. A better understanding of the key signaling pathways involved in proliferation and survival of CLL cells impacted by BRD4i in combination with venetoclax and ibrutinib would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Disclosures Hernandez-Ilizaliturri: Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; Astra Zeneca: Consultancy; Epyzome: Consultancy.

A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

Fluorescence in Situ Hybridization for Detecting Molecular Cytogenetic Abnormalities of Chronic Lymphocytic Leukemia

To investigate the value of fluorescence in situ hybridization (FISH) in the diagnosis and prognosis evaluation of patients with chronic lymphocytic leukemia (CLL). Ninty-three patients with newly diagnosed CLL were tested by five probes including RB1 (13q14.1), D13S25 (13q14.3), p53(17p13.1), ATM( 11q22.3) and CSP12, while conventional cytogenetics (CC) was used for karyotype analysis. Then the correlation of the molecular cytogenetic abnormalities with the clinical Binet stages, Rai stages and the other related laboratory examinations was analyzed. The detection rate of chromosome abnormality in 93 patients was 79.6%, out of which detection rate of 13q (13q- was the highest and accounted for 45.2%), followed by trisomy 12 (+12) 26.9%, p53 deletion (17p-) 19.4% and ATM deletion (11p-) 17.2%. There were 27 cases (29.0%) with 2 or more abnormalities, including 13 cases with 13q-/17q-, 5 with 13q-/11q-, and 4 with 13q-/+12. Compared with CC test results, the positive rate of FISH detection was significantly higher (χ2=32.127, P＜0.01). There was no significant correlation between FISH results and Rai stages (P＞0.05), meanwhile 17p- highly correlated with later stage of the Binet stages (P=0.012). The molecular cytogenetic abnormalities significantly correlated with age, absolute value of peripheral lymphocyte count and CD38 expression level (P＞0.05). The incidence of 13q- in female (65.4%) was statistically significantly higher than that in male (37.3%) (P=0.015). The unmutated IGHV rate of CLL patients with a 17p- was significantly higher than that in patients without this genetic abnormality (P=0.013). The expression of CD38 was detected among 29.0% of the patients, which significantly correlated with Binet stages (P=0.027) and unmutated IGHV (P=0.006). FISH can greatly increase the detection rate of molecular cytogenetic abnormalities in CLL patients, which, as a powerful supplement to the conventional cytogenetics, can be applied for the clinical staging and prognosis evaluation of CLL patients.

Germline variants of Brazilian women with breast cancer and detection of a novel pathogenic ATM deletion in early-onset breast cancer.

It is estimated that 5-10% of breast cancer cases are hereditary. The identification of pathogenic germline variants allows individualized preventive health care, improvement of clinical management and genetic counseling. Studies in ethnically admixed Latin American populations have identified regions with increased frequency of deleterious variants in breast cancer predisposing genes. In this context, the Brazilian population exhibits great genetic heterogeneity, and is not well represented in international databases, which makes it difficult to interpret the clinical relevance of germline variants. We evaluated the frequency of pathogenic/likely pathogenic (P/LP) germline variants in up to 37 breast cancer predisposing genes, in a cohort of 105 breast and/or ovarian cancer Brazilian women referred to two research centers between 2014 and2019. A total of 22 patients (21%) were found to carry P/LP variants, and 16 VUS were detected in 15 patients (14.3%). Additionally, a novel pathogenic ATM intragenic deletion was identified in an early-onset breast cancer. We also detected a BRCA1 pathogenic variant (c.5074+2T>C) in higher frequency (10×) than in other studies with similar cohorts. Our findings contribute to the characterization of the genetic background of breast cancer predisposition in the Brazilian population as a useful resource to discriminate between deleterious variants and VUS, thus enabling improvement in the preventive health care and clinical management of carriers.

Leukemic phase of Richter transformation: A mimic of acute myeloid leukemia that responded to Ibrutinib monotherapy.

Here we report an unusual case of RT without any clinical, laboratory, or radiologic evidence suggestive of transformation other than cytopenia. Without any significant extramedullary involvement, RT presented in a leukemic phase that mimicked acute myeloid leukemia (AML). Importantly, despite the presence of a number of risk factors associated with a poor outcome, such as clonal relatedness, complex karyotype, expression of CD38 and ZAP70, thrombocytopenia, and multiple prior therapies, the patient responded well to ibrutinib monotherapy. This article is protected by copyright. All rights reserved.

Expression and Prognostic Value of MiR-155 in Patients with Chronic Lymphocytic Leukemia

To explore the expression of miR-155 in patients with chronic lymphocytic leukemia (CLL) and its correlation with the clinical and biological features in CLL. The expression of miR-155 was detected by quantitative real time polymerase chain reaction (qRT-PCR) in the peripheral mononuclear cells collected from 73 CLL patients and CD19 positive B cells collected from 60 healthy controls, respectively. The expression of miR-155 in CLL patients was compared with the healthy controls, and the correlation of miR-155 expression with the clinical characteristics of CLL patients such as age, sex, Binet stage, cytogenetic and molecular genetic, as well as the relationship of miR-155 expression level with time to treatment (TTT) and overall survival (OS) was further analysed. The expression of miR-155 was significantly elevated in CLL patients compared with the healthy controls. Further analysis showed that miR-155 expression decreased in the patients with the mutated immunoglobulin heavy chian variable region (IGHV) as compared with the patients unmutated (P＜0.05), and its expression was significant higher in the IGHV-39 family (P＜0.05). Fluorescence in situ hybridization (FISH) showed that the expression of miR-155 increased in patients with P53 mmutation/deletion and ATM deletion (P＜0.05). However, OS and TTT were not different between the patients with high and low expression of miR-155. MiR-155 is increasingly expresses in CLL patients and correlates with poor prognosis, suggesting its important role in the genesis and progress of CLL.

Expression and Prognostic Value of MiR-155 in Patients with Chronic Lymphocytic Leukemia

To explore the expression of miR-155 in patients with chronic lymphocytic leukemia (CLL) and its correlation with the clinical and biological features in CLL. The expression of miR-155 was detected by quantitative real time polymerase chain reaction (qRT-PCR) in the peripheral mononuclear cells collected from 73 CLL patients and CD19 positive B cells collected from 60 healthy controls, respectively. The expression of miR-155 in CLL patients was compared with the healthy controls, and the correlation of miR-155 expression with the clinical characteristics of CLL patients such as age, sex, Binet stage, cytogenetic and molecular genetic, as well as the relationship of miR-155 expression level with time to treatment (TTT) and overall survival (OS) was further analysed. The expression of miR-155 was significantly elevated in CLL patients compared with the healthy controls. Further analysis showed that miR-155 expression decreased in the patients with the mutated immunoglobulin heavy chian variable region (IGHV) as compared with the patients unmutated (P＜0.05), and its expression was significant higher in the IGHV-39 family (P＜0.05). Fluorescence in situ hybridization (FISH) showed that the expression of miR-155 increased in patients with P53 mmutation/deletion and ATM deletion (P＜0.05). However, OS and TTT were not different between the patients with high and low expression of miR-155. MiR-155 is increasingly expresses in CLL patients and correlates with poor prognosis, suggesting its important role in the genesis and progress of CLL.

The importance of phenotypic information to guide genetic testing

For suspected monogenic disorders, patient phenotype is used by clinicians to guide genetic testing and by laboratory professionals to help decide what findings should be reported. We present a 23-month-old boy with developmental delay, dystonia and sialorrhoea. Fragile X testing and microarray analysis were the first-tier tests, with normal results for the former. An intragenic ATM deletion was observed but not reported as it was heterozygous and not considered a strong phenotypic match. The proband’s 4-year-old sibling presented with the same clinical features. Both had biochemical results suggestive of ataxia telangiectasia, so the clinician requested ATM analysed further as mutations in this gene cause autosomal recessive ataxia telangiectasia. The heterozygous chromosome 11 deletion, corresponding to exons 6 to 37 of ATM, was identified in both siblings. Subsequent Sanger sequencing of ATM identified a second pathogenic variant [c.9091del; p.(Gln3031Lysfs*2)]. The diagnosis was also supported by abnormal chromosome breakage studies. Clinical laboratories should have clear and consistent reporting policies when determining the significance of deletions involving genes associated with autosomal recessive conditions. This case demonstrates how phenotypic information is critical in guiding what findings should be reported and what follow up testing should be considered if a diagnosis has not been established.

Composite Copy Number Variation in CDK4, CDKN2A and RB1 Predisposes Mantle Cell Lymphoma to Expansion of PD1+ Tumor Cells and Resistance to CDK4/6 Inhibitor Therapy as Revealed by Integrative Longitudinal scRNA-seq

Composite Copy Number Variation in CDK4, CDKN2A and RB1 Predisposes Mantle Cell Lymphoma to Expansion of PD1+ Tumor Cells and Resistance to CDK4/6 Inhibitor Therapy as Revealed by Integrative Longitudinal scRNA-seq

ATM whole gene deletion in an Italian family with hereditary pancreatic cancer: Challenges to cancer risk prediction associated with an 11q22.3 microdeletion

Hereditary pancreatic cancer has been attributed to variants of several cancer predisposition genes including ATM. While heterozygous pathogenic variants in the ATM gene are implicated as a cause of familial breast and pancreatic cancers to our knowledge ATM whole gene deletions have not been previously reported. We describe a contiguous gene deletion of the ATM locus in a multi-generation family of Italian descent with a strong family history of pancreatic cancer. A deletion of one copy of the entire ATM gene was identified by routine panel testing and further characterized by chromosomal microarray analysis. An 11q22.3 microdeletion of approximately 960 kb was identified that is predicted to result in loss of 10 genes including ATM. The deletion was identified in two additional family members including a presymptomatic daughter and an affected sibling. A normal disomic complement of the 11q22.3 region was detected in a third family member with a history of prostate and pancreatic cancer. Additional family members were not available for testing. Given available evidence that ATM haploinsufficiency can increase cancer risk, we predict that the observed copy number loss has likely contributed to hereditary cancer in this family. However, absence of the familial microdeletion in at least one affected family member suggests that ATM deletions are unlikely the sole contributing factor influencing tumor development in affected individuals. This case highlights 11q22.3 microdeletions of the ATM gene region as a possible risk factor for hereditary cancer, including pancreatic cancer. The same case provides a further cautionary tale for over interpretation of cancer risk associated tumor suppressor microdeletions and suggests that the variant may not be sufficient for tumor development or may modify the cancer risks associated with other, yet unidentified hereditary cancer genes.

Diagnostic and prognostic value of CD160 antigen in patients with chronic lymphocytic leukemia

Objective To analyze the expression of CD160 antigen in patients with chronic lymphocytic leukemia (CLL), and to explore its clinical diagnostic value as well as the correlation of CD160 with genetic abnormalities. Methods A retrospective study was conducted from January 2015 to December 2017. Clinical data of 336 patients in the First Affiliated Hospital with Nanjing Medical University (Jiangsu Province) were collected. Among them, 200 patients were diagnosed with CLL according to WHO Classification Tumors of Hematopoietic and Lymphoid Tissues (the 4th edition of 2008), including 122 patients with typical CLL and 78 with atypical CLL based on Royal Marsden Hospital Immunomarker Integral System. Besides, there were 49 patients diagnosed with MCL and 87 patients with CD5-small B cell lymphoma (SBL). All patients′ tumor cells were detected for CD160 expression and its mean fluorescence intensity (MFI) by flow cytometry. At the same time, fluorescence in situ hybridization (FISH) was used to detect P53 deletion, 13q14 deletion, ATM deletion, 6q23 deletion,+12 and IGH rearrangement in CLL cases. Molecular characteristics and genetic abnormalities were compared between CD160+ and CD160-CLL patients. Results The CD160 positive rate in typical CLL patients and atypical CLL patients was 59.8% (73/122) and 64.1% (50/78), with MFI ranging from 14.9 to 173.9, and 29.6 to 193.7, respectively; while the CD160 positive rate in patients with CD5-SBL was 1.1% (1/87) and all the MCL patients were CD160 negative. The CD160 positive rate was significantly higher in typical and atypical CLL patients than that in MCL patients or patients with CD5-SBL (P<0.01). The rearrangement rate of IGH was significantly higher in CD160+ CLL patients than that in CD160-CLL patients (62.1% vs 31.6%, P<0.05). Conclusion CD160 has significant value for auxiliary diagnosis of CLL, especially can provide a reliable evidence for the diagnosis and differential diagnosis among atypical CLL, MCL, and SBL. Key words: Leukemia, lymphocytic, chronic, B-cell; Lymphoma, mantle-cell; Receptors, immunologic; Antigens, CD; GPI-linked proteins; Diagnosis, differential; Flow cytometry

Cutting Edge: ATM Influences Germinal Center Integrity.

The DNA damage response protein ATM has long been known to influence class switch recombination in ex vivo-cultured B cells. However, an assessment of B cell-intrinsic requirement of ATM in humoral responses in vivo was confounded by the fact that its germline deletion affects T cell function, and B:T cell interactions are critical for in vivo immune responses. In this study, we demonstrate that B cell-specific deletion of ATM in mice leads to reduction in germinal center (GC) frequency and size in response to immunization. We find that loss of ATM induces apoptosis of GC B cells, likely due to unresolved DNA lesions in cells attempting to undergo class-switch recombination. Accordingly, suboptimal GC responses in ATM-deficient animals are characterized by decreased titers of class-switched Abs and decreased rates of somatic hypermutation. These results unmask the critical B cell-intrinsic role of ATM in maintaining an optimal GC response following immunization.