Dissecting diffuse large B-cell lymphomas of the “not otherwise specified” type: the impact of molecular techniques [version 1; referees: 2 approved]

The updated edition of the Classification of Tumours of Haematopoietic and Lymphoid Tissues, published in September 2017 by the World Health Organization (WHO), presents many important changes to the document published in 2008. Most of these novelties are linked to the exceptional development of biomolecular techniques during the last 10 years. To illustrate how much new technologies have contributed to the better classification of single entities, as well as the discovery of new ones, would go beyond the objectives of this work. For this reason, we will take diffuse large B-cell lymphoma as an example of the cognitive improvement produced by high-yield technologies (such as the gene expression profile, the study of copy number variation, and the definition of the mutational spectrum). The acquisition of this knowledge not only has a speculative value but also represents the elements for effective application in daily practice. On the one hand, it would allow the development of personalised therapy programs, and on the other it would promote the transition from the bench of the researcher's laboratory to the patient's bedside

If it is in the marrow, is it also in the blood? An analysis of 1,000 paired samples from patients with B-cell non-Hodgkin lymphoma

<p>Abstract</p> <p>Background</p> <p>Staging of B-cell non Hodgkin's lymphoma (NHL) routinely involves bone marrow (BM) examination by trephine biopsy (BM-TB). The evidence of disease in the BM-TB results in a clinical stage IV classification affecting therapeutic strategies for NHL patients. BM immunophenotyping by flow cytometry (FC) is also used, although its clinical value is still under debate.</p> <p>Methods</p> <p>Using FC we analyzed 1,000 paired BM aspirates and peripheral blood (PB) samples from 591 NHL patients to investigate the concordance between BM and PB. B-lymphocytes were defined monoclonal when a ratio of 0.3 < κ/l > 3 was observed. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. BM-TB was also performed in 84.1% of samples (841/1000), and concordance between BM-TB and BM-FC was evaluated. Concordance was defined as the presence of a positive (in terms of disease detection) or negative result in both BM-FC and PB-FC or BM-TB and BM-FC.</p> <p>Results</p> <p>Using FC, the overall concordance between BM and PB was 95%. Among the discordant cases (ie presence of neoplastic B-lymphocyte in the BM but under the sensibility of the technique in the PB) the most frequent diagnosis was Waldenstrom's macroglobulinemia (WM, accounting for 20.8% of all discordant cases). The expression of CXCR4, a receptor involved in B-cell trafficking and homing, was found to be down regulated in WM compared to other NHL types, thus suggesting a possible role of CXCR4 in WM cell homing in the BM. WM excluded, FC investigation of BM and PB in NHL patients gives overlapping information.</p> <p>BM involvement was observed by FC in 38% of samples, and concordance between BM-FC and BM-TB was 85%.</p> <p>Conclusions</p> <p>The finding that FC data from BM and PB samples overlap in NHL might have major implications for the design of future clinical studies and for patients' follow-up.</p

Flow cytometry strategy to detect CD109+CECs and CD146+CECs.

<p>Fluorescence Minus One for each fluorescence is reported.</p

Expression of Ulex Europeaus Lectin.on CD109+CECs and CD146+CECs+ (4A–B1).

<p>Epcam staining (4C–C1) confirmed that Epithelial Cells even if present in the DNA+CD45− cell compartment, are negative for the expression of CD31 present only on endothelial cells.</p

Flow cytometry strategy to detect Ac-LDL uptake.

<p>After dublets (A) and debris (B) exclusion, Ac-LDL+ cells were detected (C). Among these cells, CD45+CD31+ and CD45−CD31+ cells were detected (D). CD45+CD31+ were also positive for CD14 (monocytes,E) and CD45−CD31+ were also positive for CD109+ (endothelial cells, F). Ac-LDL+ cells were nucleated (Syto16+, F). C1, D1, E1 and F1 are the negative controls.</p

Flow cytometry strategy to detect CD109+CEC and CD146+CEC phenotype.

<p>After exclusion of debris (A) and selection of CD45− (B), nucleated (Syto16+) and CD31+ cells (C), CEC were identified as positive for CD109 or CD146 (E). (D): negative control. CD109+ CECs and CD146+CECs were evaluated by flow cytometry for the expression of CD34, CD117, CD90 and CD13.</p

Baseline values of CD109+ CECs and CD146+ CECs.

<p>Baseline values of CD109+ CECs and CD146+ CECs.</p

Patients characteristics.

<p>Patients characteristics.</p