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Νext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, "(S+Rc) to Rnc IGHV mutation ratio". When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

Summary: Large‐scale next‐generation sequencing (NGS) studies revealed extensive genetic heterogeneity, driving a highly variable clinical course of chronic lymphocytic leukaemia (CLL). The evolution of subclonal populations contributes to diverse therapy responses and disease refractoriness. Besides, the dynamics and impact of subpopulations before therapy initiation are not well understood. We examined changes in genomic defects in serial samples of 100 untreated CLL patients, spanning from indolent to aggressive disease. A comprehensive NGS panel LYNX, which provides targeted mutational analysis and genome‐wide chromosomal defect assessment, was employed. We observed dynamic changes in the composition and/or proportion of genomic aberrations in most patients (62%). Clonal evolution of gene variants prevailed over the chromosomal alterations. Unsupervised clustering based on aberration dynamics revealed four groups of patients with different clinical behaviour. An adverse cluster was associated with fast progression and early therapy need, characterized by the expansion of TP53 defects, ATM mutations, and 18p− alongside dynamic SF3B1 mutations. Our results show that clonal evolution is active even without therapy pressure and that repeated genetic testing can be clinically relevant during long‐term patient monitoring. Moreover, integrative NGS testing contributes to the consolidated evaluation of results and accurate assessment of individual patient prognosis. [ABSTRACT FROM AUTHOR]

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutatedIGHV(U-CLL) usually progress rapidly, whereas patients with mutatedIGHV(M-CLL) have a more indolent disease. The expression of several genes correlates closely with theIGHVmutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance ofCOBLL1,LPL,andZAP70gene expression, which correlated withIGHVmutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR  = 2.300/5.970, 95% CI: 1.587–3.450/4.621–15.86). Our approach provides a reliable alternative method to prognosis assessment viaIGHVmutational status analysis. [ABSTRACT FROM PUBLISHER]

The canonical Wnt pathway, dependent on β-catenin-controlled transcription, is the most explored Wnt pathway, known to drive the malignant transformation of multiple cell types. Several reports have suggested that this pathway also participates in chronic lymphocytic leukaemia ( CLL) pathogenesis. To get a better insight into the role of the Wnt/β-catenin pathway in CLL we analysed in detail the expression of the most overexpressed Wnt ligand, encoded by the WNT3 gene, in a well-defined cohort of 137 CLL patients. Our analysis demonstrated that (i) untreated patients with more aggressive disease (with a notable exception of patients with 11q deletion) express less WNT3, (ii) WNT3 declines with disease progression in a significant proportion of patients and (iii) low WNT3 was identified as a strong independent marker indicating shorter treatment-free survival in CLL patients with IGHV mutation. Interestingly, CLL-related lymphoid cell lines, but not stromal cells, failed to respond to the ligand-induced activation of the Wnt/β-catenin pathway. This opens the possibility that CLL cells use Wnt-3 to communicate with the cells in the microenvironment. We thus propose that the Wnt/β-catenin pathway plays a more complex role in CLL pathogenesis than previously anticipated. [ABSTRACT FROM AUTHOR]

The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in chronic lymphocytic leukemia, as indicated by the adverse prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM) detected by fluorescence in situ hybridization (FISH) analysis. In addition to deletions, mutations in these respective genes are also associated with chemoresistance, and add to the prognostic information provided by FISH. In order to explore the possibility that dysfunction of the ATM-p53 pathway might also result from mechanisms other than ATM/ TP53 deletion/mutation, assays have been developed that probe the functional integrity of the ATM-p53 pathway. Currently, four different p53 function assays have been developed that are based on the measurement of p53 and p53-dependent genes at the RNA (real-time polymerase chain reaction [RT-PCR] p21; RT-PCR miR34a; reverse transcription-multiplex ligation-dependent probe amplification assay [RT-MLPA] p21, bax, puma and CD95) or protein (fluorescence activated cell sorting [FACS]p53-p21) level in untreated cells or following irradiation or drug treatment. Here we provide an overview of these assays based on the available literature. [ABSTRACT FROM AUTHOR]

Follicular lymphoma (FL) is characterized by an indolent and relapsing course. Recently, the clinical outcome of FL has been distinguished by immune microenvironment-associated gene signatures. In our study, gene expression profiling (GEP) was performed in 31 non-selected patients with follicular lymphoma (FL), 12 of whom were in relapse and the remaining 19 newly diagnosed. A custom oligonucleotide microarray (Agilent 8 × 15K) was used which contained probes for about 3500 genes, including those that had been previously published as demonstrating significant prognostic value. An unsupervised approach was not able to recognize clinically different FLs. As the previously published prognostically relevant gene signatures could not be properly verified, probably due to microarray platform differences, template matching was therefore used in order to define two gene sets with differential gene expression among our samples. These gene sets shared an overrepresentation of genes with similar biological functions and were termed 'T-CELL' and 'PROLIFERATION' profiles. The 'poor profile' was then defined by a high PROLIFERATION score (upper tertile) and/or low T-CELL score (lower tertile). The 'poor profile' cohort contained a significantly higher proportion of relapsed cases ( p < 0.05, Fisher's exact test). Additionally, a comparison of samples from initial diagnosis and from relapse showed significant differences mainly in the T-CELL profile ( p = 0.036; χ2). This supports the hypothesis that the number of T-cells and their expression pattern play a major role in FL development. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article about the prognostic relevance of MYD88 mutations in chronic lymphocytic leukemia (CLL) in the previous issue.