Belinda Austen

Mutation Status of the Residual ATM Allele Is an Important Determinant of the Cellular Response to Chemotherapy and Survival in Patients With Chronic Lymphocytic Leukemia Containing an 11q Deletion

PURPOSE The ataxia telangiectasia mutated (ATM) gene is located on chromosome 11q and loss of this region is common in B-cell chronic lymphocytic leukemia (CLL). Our aim was to determine if CLL tumors with a chromosome 11q deletion might be divided into two subgroups based on the status of the remaining ATM allele. METHODS The sequence of the residual ATM allele was determined in 72 CLLs with an 11q deletion. This was related to the cellular response to irradiation or cytotoxic drug exposure in vitro and clinical outcome. RESULTS We show that the residual ATM allele is mutated in 36% of CLLs with an 11q deletion and that these leukemias demonstrate an impaired cellular response to irradiation or cytotoxic drug exposure in vitro. Inactivation of the second ATM allele was associated with a reduction in patient survival beyond that already dictated by the presence of an 11q deletion (P = .0283). Furthermore, we demonstrate that ATM mutations may arise during the evolution of an 11q deleted subclone and are associated with its expansion. CONCLUSION CLL with 11q deletion can be divided into two subgroups based on the integrity of the residual ATM allele. Patients with complete loss of ATM function, due to biallelic ATM defects, have defective responses to cytotoxic chemotherapeutics in vitro and a poorer clinical outcome. ATM mutant subclones can develop during an individuals disease course and give rise to additional expansion of the 11q deleted subclone.

Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers

Bcl-2 family proteins play a critical role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their association with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clinical outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), V(H) gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addition, Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, respectively). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, respectively) and in stage A patients only (P = .002 and P = .001, respectively). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and V(H) gene mutation status, CD38 expression, and ZAP-70 expression offers a biologic explanation for their association with adverse prognosis.

A novel functional assay using etoposide plus nutlin-3a detects and distinguishes between ATM and TP53 mutations in CLL

A novel functional assay using etoposide plus nutlin-3a detects and distinguishes between ATM and TP53 mutations in CLL

Frequent epigenetic inactivation of the SLIT2 gene in chronic and acute lymphocytic leukemia

Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all 10 leukemia cell lines analyzed (8 completely and 2 partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5’ CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.

ATM germline heterozygosity does not play a role in chronic lymphocytic leukemia initiation but influences rapid disease progression through loss of the remaining ATM allele

Ataxia telangiectasia patients, with constitutional bi-allelic ATM mutations, have a marked risk of lymphoid tumors and ATM mutation carriers have a smaller risk of cancer. Sporadic ATM mutations occur in 10–20% of chronic lymphocytic leukemia and are often associated with chromosome 11q deletions which cause loss of an ATM allele. The role of constitutional ATM mutations in the pathogenesis of chronic lymphocytic leukemia is unknown. Here we investigated the frequency of constitutional ATM mutations in either of two chronic lymphocytic leukemia cohorts, those with and without a chromosome 11q deletion. We found that in comparison to controls, constitutional pathogenic ATM mutations were increased in patients with chromosome 11q deletions (6 of 140 vs. 0 of 281, P=0.001) but not in those without 11q deletions (2 of 178 vs. 0 of 281, P=0.15). These results suggest that ATM germline heterozygosity does not play a role in chronic lymphocytic leukemia initiation but rather influences rapid disease progression through ATM loss.

South Asian chronic lymphocytic leukaemia patients have more rapid disease progression in comparison to White patients

Ethnicity has a major impact on the prevalence of chronic lymphocytic leukaemia (CLL) and may also influence disease phenotype. We compared the clinical features of Southern Asian and White CLL patients managed within one UK region. Asians required treatment almost twice as often as Whites (HR, 1·94) and had a shorter time to first treatment (P = 0·0063). This difference remained significant after adjusting for stage, lymphocyte doubling time and IGHV status (P = 0·026). CLL was diagnosed at younger ages in Asians and racial‐specific variations in IGHV usage were demonstrated. Our findings indicate that Southern Asian race has a negative impact on CLL phenotype.

Pathogenic ATM mutations occur rarely in a subset of multiple myeloma patients.

Ataxia Telangiectasia (A‐T) patients have biallelic inactivation of the ATM gene and exhibit a 200‐fold‐increased frequency of lymphoid tumours. ATM mutations have been found in a number of adult lymphoid malignancies but there is no data on the occurrence of ATM mutations in multiple myeloma tumours. The purpose of our work was to investigate the occurrence of ATM mutations in multiple myeloma and to this end we screened 45 sporadic cases for ATM mutations using denaturing high‐performance liquid chromatography analysis and DNA sequencing. Pathogenic ATM mutations were identified in 2/45 of the myelomas compared with a published estimate of ATM mutant allele frequency in the UK population of 2/521 (P = 0·033). One was the missense mutation 7181C>T which was then modelled in an expression system and the S2394L protein shown to have no ATM kinase activity. The second myeloma had the pathogenic ATM splice site mutation IVS40‐1G>C leading to loss of exon 41. We also report a 48‐year‐old ataxia telangiectasia patient who developed multiple myeloma. Taken together our study suggests that ATM mutation may play a role in the pathogenesis of a subset of multiple myelomas.

Calreticulin mutations and their importance in splanchnic vein thrombosis

We have read the recent publications on Calreticulin (CALR) gene mutated splanchnic vein thrombosis (SVT) with great interest (Castro et al, 2015; Haslam & Langabeer, 2015; Roques et al, 2015). Between 25% and 40% of patients with SVT have overt or occult myeloproliferative neoplasms (MPNs), which are usually associated with JAK2 mutations (Smalberg et al, 2012; Sekhar et al, 2013). CALR mutated MPNs are typically associated with a lower incidence of thrombosis than their JAK2 mutated counterparts (Andrikovics et al, 2014). We have summarized the data from five recent reports that have been published regarding this group of patients (Table I). We wish to highlight data on our patients with CALR related SVT. Between 2006 and 2014, 24 of 83 patients (29%) with SVT in whom local pathology had been ruled outwere found to have a JAK2 V617F mutation and two patients (2 4%) were found to have CALR mutations (Table II). Both of these patients had significant disease burden related to SVT, ascites and portal hypertension (PHT). Given previous reports of therapy with alpha interferon in CALR mutated essential thrombocythaemia (Cassinat et al, 2014) and with a view to improving the underlying myeloproliferative state, splenomegaly and PHT, both patients were treated with Ruxolitinib. Ruxolitinib treatment at doses of 20–40 mg/d resulted in improvement in some but not all disease characteristics (Table II). We agree with the conclusion reached by some of authors (Plompen et al, 2015; Roques et al, 2015; Turon et al, 2015) that the diagnostic algorithm for SVT should include CALR mutation analysis when looking for an underlying myeloproliferative disorder, as this can be done when screening for the presence of JAK2 mutation. CALR mutations have been reported in 0–2% of MPN-SVT patients; our cohort of 83 patients confirm this, as 2 4% of our patients had CALR mutations. However, the burden of disease is significant in all of the affected individuals listed in Table I, with four patients requiring TIPPS procedures and two patients dying of gastrointestinal haemorrhage. In conclusion, we have presented here the CALR mutation data from our cohort of 83 SVT patients; in particular the long-term clinical follow-up of two patients with MPN-SVT in whom the data demonstrate the severity of thrombosis in the context of CALR mutated MPN. In addition, we present the long-term follow-up of treatment (12 and 9 months) with Ruxolitinib in these patients. These patients demonstrate that (i) CALR mutated MPN can be associated with