Additional lesions identified by genomic microarrays are associated with an inferior outcome in low-risk chronic lymphocytic leukaemia patients.

We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low-risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL-IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time-to-first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time-to-first treatment in the subset of patients with wild-type TP53 and mutated IGHV (p = 0.025). In CLL patients with low-risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk-adapted follow-up and for early treatment including targeted agents within clinical trials.

Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases.

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.

Redefining the prognostic likelihood of chronic lymphocytic leukaemia patients with borderline percentage of immunoglobulin variable heavy chain region mutations.

In chronic lymphocytic leukaemia (CLL), caution is warranted regarding the clinical implications of immunoglobulin variable heavy chain region (IGHV) rearrangements with a 'borderline' (BL) percentage of mutations (i.e. 97-97·9% IGHV identity). We analysed the IGHV mutational status in 759 untreated CLL patients (cohort 1). BL-CLL (n = 36, 5%) showed a time to first treatment (TFT) similar to that of M-CLL (n = 338) and significantly longer than that of UM-CLL (n = 385), despite the enrichment in subset #2 cases. In fact, CLLs belonging to subset #2 (n = 15/759, 2%) were significantly more frequent among BL-CLLs (n = 5/36, 14%), with a brief TFT. TFT of BL-CLL remained comparable to that of M-CLL also considering the 327 CLL patients evaluated at diagnosis. These findings were then validated in an independent cohort 2 of 759 newly diagnosed CLL patients (BL-CLL: n = 11, 1·4%) and in all newly diagnosed patients from cohorts 1 and 2 (n = 1 086, 84% stage A; BL-CLL: n = 47, 4·3%). BL-CLL at diagnosis showed a biological profile comparable to that of M-CLL with a low frequency of unfavourable prognostic markers, except for a significant enrichment in subset #2. Our data suggest that the prognosis of BL-CLL is good and similar to that of M-CLL, with the exception of subset #2 cases.

Minimal Residual Disease in Chronic Lymphocytic Leukemia: A New Goal?

In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with TP53 deletion/mutation or unmutated IGHV genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.

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.

In chronic lymphocytic leukaemia with complex karyotype, major structural abnormalities identify a subset of patients with inferior outcome and distinct biological characteristics.

Complex karyotype (CK) is a negative prognostic factor in chronic lymphocytic leukaemia (CLL). However, CK is a heterogeneous cytogenetic category. Unbalanced rearrangements were present in 73·3% of 90 CLL patients with CK (i.e. ≥3 chromosome aberrations in the same clone), and were associated with a shorter overall survival (P = 0·025) and a shorter time to first treatment (P = 0·043) by multivariate analysis. Patients with unbalanced rearrangements presented a distinct mRNA expression profile. In conclusion, CLL patients with unbalanced rearrangements might represent a subset of very high-risk CLL patients with distinct clinical and biological characteristics.

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.

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. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value.

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.

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.

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.

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 24­48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.