Caterina Ilari

Comparative analysis between RQ-PCR and digital-droplet-PCR of immunoglobulin/T-cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia

Real‐time quantitative polymerase chain reaction (RQ‐PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow‐up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ‐PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases.

Inter- and intra-patient clonal and subclonal heterogeneity of chronic lymphocytic leukaemia: evidences from circulating and lymph nodal compartments.

Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non‐silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB‐specific and 7 (10·9%) were LN‐specific. Most of the LN‐ or PB‐specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5–5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN‐ or PB‐specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN‐specific lesions, and Case 100, with 6 LN‐specific and 8 PB‐specific lesions, showed, in the PB, the clonal expansion of LN‐derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3‐p11·1), del9(p24·3‐p13·1) and gain 2(p25·3‐p14). CLL shows an intra‐patient clonal heterogeneity according to the disease compartment, with both LN and PB‐specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN‐derived mutations/CNAs can appear in the PB at relapse.

Stereotyped subset #1 chronic lymphocytic leukemia: a direct link between B-cell receptor structure, function, and patients' prognosis

Chronic lymphocytic leukemia (CLL) with stereotyped B‐cell receptor (BCR) belonging to subset #1 (IGHV1‐5‐7/IGKV1‐39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non‐subset #1 CLL). There were no distinctive features regarding CD38, ZAP‐70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non‐stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti‐IgM revealed a significant higher proliferation of subset #1 versus unmutated non‐subset #1 CLL, both at baseline and after 24–48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis. Am. J. Hematol. 89:74–82, 2014.

Immunoglobulin gene rearrangements in Chinese and Italian patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western world, whereas in Asia the incidence is about 10 times lower. The basis for this ethnic and geographic variation is currently unknown. The aim of this study was to characterize IGHVDJ rearrangements and stereotype of the HCDR3 region in a series of 623 Chinese CLL, in order to identify possible differences in immunoglobulin gene usage and their potential pathogenetic implications. Chinese CLL were compared to 789 Italian CLL. Chinese patients showed a higher proportion of mutated IGHV and a more frequent usage of IGHV3-7, IGHV3-74, IGHV4-39 and IGHV4-59 genes. A significantly lower usage of IGHV1-69 and IGHV1-2 was documented, with comparable IGHV3-21 frequency (3% Chinese vs 3.8% Italian CLL). The proportion of known stereotyped receptors was significantly lower in Chinese (19.7%) than in Italian CLL (25.8%), despite a significantly higher frequency of subset #8 (p= 0.0001). Moreover, new paired clusters were identified among Chinese cases. Overall, these data support a potential different antigenic exposure between Eastern and Western CLL.

A case of concomitant chronic lymphocytic leukaemia and hairy cell leukaemia evaluated for IGHV-D-J rearrangements and BRAF-V600E mutation: lack of evidence for a common origin.

Keywords: chronic lymphocytic leukaemia ; hairy cell leukaemia; IGH ; BRAF V600E; quantitative PCR

Clinicopathological features and outcome of chronic lymphocytic leukaemia in Chinese patients

Chronic lymphocytic leukaemia (CLL) is uncommon in Chinese population and its biology, genetics and treatment outcome in Chinese patients have not been comprehensively investigated. In this study, we studied the clinicopathological features and outcome of 212 Chinese patients with newly diagnosed CLL in Hong Kong and Singapore. The median age at diagnosis was 64 years. The majority of patients presented with early-stage disease (Binet stage A, 56.1%). Del(13)(q14) was the most frequent abnormality (41.7%) detected by fluorescence in situ hybridization (FISH) analysis. Del(17p) and TP53 gene mutations were detected in 7.8% and 8.2% of patients, respectively. MYD88 mutations were found at a higher frequency (11.5%) than expected. CLL with unmutated variable region of the immunoglobulin heavy chain genes (IGHV) occurred in only 31.2% of cases, and was associated with advanced-stage disease (p <0.01) and adverse FISH abnormalities (p<0.01). With a median follow-up of 39 months, the median overall survival (OS) was 108 months. The presence of del(17p) or TP53 mutations was associated with a significantly shorter time to first treatment and an inferior OS (p <0.01). Unmutated IGHV was also associated with a significantly shorter time to treatment (p <0.01). Among patients who required treatment, the median OS and progression-free survival (PFS) were 107 and 23 months, respectively. The presence of del(17p) was associated with a significantly inferior OS and PFS (p <0.01). In summary, Chinese CLL patients had similar genetic aberrations at diagnosis compared with those of Western populations. FISH abnormalities are major factors affecting outcome.

Whole-genome amplification for the detection of molecular targets and minimal residual disease monitoring in acute lymphoblastic leukaemia

Accurate genomic characterization requires sufficient amounts of optimal quality DNA. An approach for increasing the DNA amount is the whole‐genome amplification (WGA) method. We applied WGA to the molecular quantification and minimal residual disease (MRD) evaluation of acute lymphoblastic leukaemia (ALL), aiming to compare the results obtained from genomic DNA and amplified DNA with WGA, and to evaluate the applicability and the reliability of WGA‐DNA. Twenty paired samples from adult ALL patients were sequenced to identify the functional germline V‐D‐J segment at diagnosis; real‐time quantitative polymerase chain reaction (RQ‐PCR) quantitative analysis was performed both at diagnosis and follow‐up. Genomic DNA and WGA‐DNA screening identified equivalent 87 rearrangements. At diagnosis, the quantitative evaluation of genomic DNA samples showed 1 logarithm difference to WGA‐DNA samples; these levels are comparable, being within the degree of acceptability and confidence. In the follow‐up samples, RQ‐PCR analysis on genomic DNA and WGA showed concordant MRD results in 16/18 samples, while 2/18 were MRD‐positive outside the quantitative range by RQ‐PCR (i.e. <5 × 10−5) on genomic DNA and MRD‐negative on WGA‐DNA. WGA‐DNA enables: (i) the design of accurate targets for MRD evaluation in ALL patients, (ii) accurate disease quantification at diagnosis, (iii) MRD quantification comparable to genomic DNA.

Refined karyotype-based prognostic stratification of chronic lymphocytic leukemia with a low- and very-low-risk genetic profile

Refined karyotype-based prognostic stratification of chronic lymphocytic leukemia with a low- and very-low-risk genetic profile

Unravelling the suboptimal response of TP53-mutated chronic lymphocytic leukaemia to ibrutinib

TP53‐disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long‐term response to ibrutinib. We hereby report that ibrutinib‐induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53‐mutated (TP53‐M) CLL cells compared to TP53 wild‐type cells. Contrariwise, venetoclax effectively killed TP53‐M cells. Gene expression profile analysis of TP53‐M cells revealed a downmodulation of B‐cell receptor (BCR)‐related genes and an upmodulation of genes with anti‐apoptotic/pro‐survival activity, suggesting that the survival and proliferation of TP53‐M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53‐M CLL patients.

Biallelic BIRC3 inactivation in chronic lymphocytic leukaemia patients with 11q deletion identifies a subgroup with very aggressive disease

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