Variability in CD39 and CD73 protein levels in uveal melanoma patients.

Extracellular adenosine is produced from ATP by CD39 and CD73, and can modulate tumor development by acting on cancer cells or immune cells. Adenosine metabolism has been poorly studied in uveal melanoma. We studied the protein levels of CD39 and CD73 in a small, well described cohort of patients with uveal melanoma. Our results show a high variability in the levels of the two proteins, both in positivity and in intensity. Our results suggest that similar studies on larger cohorts could determine the clinical value and the druggability of these enzymes in the given clinical setting.Supplemental data for this article is available online at http://dx.doi.org/10.1080/15257770.2022.2032738.

Single-agent ibrutinib in RESONATE-2™ and RESONATE™ versus treatments in the real-world PHEDRA databases for patients with chronic lymphocytic leukemia.

After analyzing treatment patterns in chronic lymphocytic leukemia (CLL) (objective 1), we investigated the relative effectiveness of ibrutinib versus other commonly used treatments (objective 2) in patients with treatment-naïve and relapsed/refractory CLL, comparing patient-level data from two randomized registration trials with two real-world databases. Hazard ratios (HR) and 95% confidence intervals (CIs) were estimated using a multivariate Cox proportional hazards model, adjusted for differences in baseline characteristics. Rituximab-containing regimens were often prescribed in clinical practice. The most frequently prescribed regimens were fludarabine + cyclophosphamide + rituximab (FCR, 29.3%), bendamustine + rituximab (BR, 17.7%), and other rituximab-containing regimens (22.0%) in the treatment-naïve setting (n = 604), other non-FCR/BR rituximab-containing regimens (38.7%) and non-rituximab-containing regimens (28.5%) in the relapsed/refractory setting (n = 945). Adjusted HRs (95% CI) for progression-free survival (PFS) and overall survival (OS), respectively, with ibrutinib versus real-world regimens were 0.23 (0.14-0.37; p < 0.0001) and 0.40 (0.22-0.76; p = 0.0048) in the treatment-naïve setting, and 0.21 (0.16-0.27; p < 0.0001) and 0.29 (0.21-0.41; p < 0.0001) in the relapsed/refractory setting. When comparing real-world use of ibrutinib (n = 53) versus other real-world regimens in relapsed/refractory CLL (objective 3), adjusted HRs (95% CI) were 0.37 (0.22-0.63; p = 0.0003) for PFS and 0.53 (0.27-1.03; p < 0.0624) for OS. This adjusted analysis, based on nonrandomized patient data, suggests ibrutinib to be more effective than other commonly used regimens for CLL.

Unclassifiable Isolated Monoclonal Lymphocytosis: Comprehensive Description of a Retrospective Cohort.

According to the World Health Organization (WHO) classification, the nosology of B-cell neoplasms integrates clinical, morphological, phenotypic, and genetic data. In this retrospective analysis, we identified 18 patients with isolated neoplastic lymphocytosis that could not be accurately classified within the WHO classification. Most of them were asymptomatic at the time of diagnosis and the evolution was relatively indolent, as only five patients required treatment after a median follow-up of 48 months. The neoplastic B-cells expressed CD5 in most cases, but the Royal Marsden Hospital score was strictly below 3. Trisomy 12 was the most frequent cytogenetic abnormality. High-throughput sequencing highlighted mutations found in both chronic lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL). Similarly, the immunoglobulin heavy chain variable region repertoire was distinct from those reported in CLL or MZL. However, as treatment choice is dependent on the correct classification of the lymphoproliferative disorder, a histological diagnosis should be performed in case patients need to be treated.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53.

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

Toll-like receptor expression and function differ between splenic marginal zone B cell lymphoma and splenic diffuse red pulp B cell lymphoma.

In splenic marginal zone lymphoma (SMZL), specific and functional Toll-like Receptor (TLR) patterns have been recently described, suggesting their involvement in tumoral proliferation. Splenic diffuse red pulp lymphoma with villous lymphocytes (SDRPL) is close to but distinct from SMZL, justifying here the comparison of TLR patterns and functionality in both entities. Distinct TLR profiles were observed in both lymphoma subtypes. SDRPL B cells showed higher expression of TLR7 and to a lesser degree TLR9, in comparison to SMZL B cells. In both entities, TLR7 and TLR9 pathways appeared functional, as shown by IL-6 production upon TLR7 and TLR9 agonists stimulations. Interestingly, circulating SDRPL, but not SMZL B cells, constitutively expressed CD86. In addition, stimulation with both TLR7 and TLR9 agonists significantly increased CD80 expression in circulating SDRPL but not SMZL B cells. Finally, TLR7 and TLR9 stimulations had no impact on proliferation and apoptosis of SMZL or SDRPL B cells. In conclusion, SMZL and SDRPL may derive from different splenic memory B cells with specific immunological features that can be used as diagnosis markers in the peripheral blood.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2, TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

Advances in the role of cytogenetic analysis in the molecular diagnosis of B-cell lymphomas.

INTRODUCTION: Cytogenetic abnormalities represent essential determinants of diagnosis and prognosis in B-cell lymphomas. Their theranostic value is increasingly significant with the development of targeted therapies, in order to adapt the treatment at diagnosis as well as when relapse occurs. Areas covered: As the significance of these biomarkers is influenced by the technology used to detect them, an overview describing the strength and weakness of conventional and emerging technologies is provided. This review also updates the diverse cytogenetic abnormalities found in B-cell lymphomas, emphasizing their value in treatment decision. Expert commentary: Cytogenetics remains an essential analysis for the diagnostic work-up of lymphomas. As whole genome sequencing becomes more and more affordable routinely, the next challenge will be to recover all the information conveyed by conventional karyotype, including the analysis of the clonal architecture at the single cell level, in whole genome data.

Splenic diffuse red pulp lymphoma has a distinct pattern of somatic mutations amongst B-cell malignancies.

Splenic Diffuse Red Pulp Lymphoma (SDRPL) has been recently introduced as a provisional entity but differential diagnosis with other splenic lymphomas is needed to be clarified since the therapeutic approaches are distinct. Recently described recurrent mutations or CD180 expression appear useful for differential diagnosis. We completed our previous description in a larger cohort including 53 patients selected on the presence of characteristic villous cells in peripheral blood (PB) and a specific immunophenotype. Immunoglobulin heavy variable (IGHV), BRAF, MYD88, and NOTCH2 mutations were determined and CD180 and BRAF expressions were assessed. Most cases (79%) were IGHV mutated with an overrepresentation of IGHV3-23 (19%) and IGHV4-34 (21%). MYD88 L265P and NOTCH2 mutations were observed in one case each, whereas no BRAF V600E mutation or expression was found. All cases demonstrated a high CD180 expression. Those results strengthen the concept that SDRPL does emerge as a new lymphoma entity distinct from the other splenic lymphomas with circulating lymphocytes.

Cytogenetics in the management of lymphomas and lymphoproliferative disorders in adults and children: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Non-Hodgkin's lymphomas and lymphoproliferative disorders include a high number of heterogeneous entities, described in the 2008 WHO classification. This classification reflects the crucial role of a multidisciplinary approach which integrates cytogenetic results both for the notion of clonality and for differential diagnosis between these entities. The prognostic impact of some cytogenetic abnormalities or genome complexity is also confirmed for many of these entities. Novel provisional entities have been described, such as BCLU (B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma) for which karyotype is critical to distinguish BCLU from Burkitt's lymphoma. The karyotype can be established from any tumour or liquid infiltrated by lymphoma cells. Recent adaptations of technics for cellular cultures according to the subtype of known (or suspected) lymphoma have significantly improved the percentage of informative karyotypes. Conventional karyotypes remain the best technical approach recommended for most of these subtypes. Interphase and/or metaphase FISH also represents a solid and rapid approach, because of the significant number of recurrent (sometimes specific) rearrangements of these entities. Next generation sequencing technologies contribute to enrich genomic data and substantially improve the understanding of oncogenic mechanisms underlying these lymphoid malignancies. Some molecular biomarkers are already part of the diagnostic process (for example, somatic mutation of MYD88 in Waldenström disease) thus reinforcing the essential principle of a multidisciplinary approach for the diagnosis of all the mature lymphoid malignancies.

Cytogenetics in the management of chronic lymphocytic leukemia: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Acquired recurrent cytogenetic abnormalities are frequent in chronic lymphocytic leukaemia (CLL). They can be associated with good or poor prognostic factors, and also with gene mutations. Chromosomal abnormalities could be clonal or sub-clonal. Assessing the TP53 status (deletion/mutation) is currently mandatory before treating patients. The search for 11q deletion (ATM gene) is also recommended. Finally, the prognostic value of other chromosomal abnormalities including complex karyotype is still debated.

Genetic differences between paediatric and adult Burkitt lymphomas.

Dysregulation of MYC is the genetic hallmark of Burkitt lymphoma (BL) but it is encountered in other aggressive mature B-cell lymphomas. MYC dysregulation needs other cooperating events for BL development. We aimed to characterize these events and assess the differences between adult and paediatric BLs that may explain the different outcomes in these two populations. We analysed patterns of genetic aberrations in a series of 24 BLs: 11 adults and 13 children. We looked for genomic imbalances (copy number variations), copy-neutral loss of heterozygosity (CN-LOH) and mutations in TP53, CDKN2A, ID3 (exon 1), TCF3 (exon17) and CCND3 (exon 6). Young patients displayed more frequent 13q31.3q32.1 amplification, 7q32q36 gain and 5q23.3 CN-LOH, while 17p13 and 18q21.3 CN-LOH were only detected in adult BLs. ID3 mutations were present in all adult samples, but only in 42% of childhood cases. CCND3 and ID3 double-hit mutations, as well as 18q21 CN-LOH, seemed to be associated with poorer outcome. For the first time, we report different genetic anomalies between adult and paediatric BLs, suggesting age-related heterogeneity in Burkitt lymphomagenesis. This may explain the poorer prognosis of adult BLs. Additional studies are needed to confirm these results in the setting of clinical trials.

CD180 overexpression in follicular lymphoma is restricted to the lymph node compartment.

BACKGROUND: Altered Toll-like receptor (TLR) expression levels and/or mutations in its signaling pathway (such as MyD88 mutation) contribute to the pathogenesis of lymphoproliferative disorders (LPD). CD180 is an orphan member of the TLR family that modulates the signaling of several TLRs, but only limited studies have evaluated its expression by flow cytometry (FCM) in LPD. METHODS: Using a multiparameter FCM approach, we have assessed CD180 mean fluorescence intensity (MFI) in lymph nodes (LNs) and peripheral blood (PB) samples obtained from patients with follicular lymphoma (FL; LN/PB, n = 44/n = 15), chronic lymphocytic leukemia (CLL, n = 26/n = 21), mantle cell lymphoma (MCL, n = 13/n = 17), and marginal zone lymphoma (MZL, n = 16/n = 12). Specimens from non-tumoral PB and LN (n = 8/n = 12) were used as controls. RESULTS: In the LN specimens, FL and control B-cells showed similar CD180 expression (MFI = 1,049 vs. 1,381, P > 0.05; Mann-Whitney U-test). This level was markedly lower in the other LPDs, MCL (MFI = 396, P < 0.05), or CLL (MFI = 502 P < 0.05), and similar to MZL (MFI = 858, P > 0.05). However, the CD180 expression of FL B-cells assessed in PB was dim and/or negative, in the same range as MCL and CLL (FL MFI = 453, MCL MFI = 305, CLL MFI = 420, P > 0.05) but lower than in MZL (MFI = 895, P < 0.05). Therefore, these results suggest a modulation of CD180 expression by neoplastic FL B-cells based on the anatomical compartment. CONCLUSION: These FCM data confirm the usefulness of CD180 in the accurate diagnosis of LPDs and emphasize the need to interpret this marker according to the origin of the sample. © 2015 Clinical Cytometry Society.

Characterization of a de novo Supernumerary Neocentric Ring Chromosome Derived from Chromosome 7.

Supernumerary ring chromosomes (SRC) are usually derived from regions adjacent to the centromere. Their identification may be challenging, particularly in case of low mosaicism. Here, we report on a patient who was referred for major in utero growth retardation, severe developmental delay, facial dysmorphism, cleft palate, and hypospadias. The karyotype showed a small SRC in mosaic. The combination of FISH, M-FISH and array-CGH was necessary for a complete characterization of this SRC. M-FISH revealed that the SRC originated from chromosome 7. Array-CGH performed with a 400K oligonucleotide array showed a gain in region 7q22.1q31.1 present in low mosaic. This result was confirmed by FISH using BAC probes specific for chromosome 7. The SRC was a neocentric ring derived from 7q22.1q31.1 and was found in only 8% of the cells. This is the first patient carrying a mosaic neocentric SRC derived from the long arm of chromosome 7. Our study emphasizes the need to combine different techniques and to use adapted bioinformatic tools for low-mosaicism marker identification. It also contributes to the delineation of the partial trisomy 7q phenotype.