Technical Note Cell Dysplasia - Cell Dysplastic Features (A Morphological Note).

Cell dysplasia is a currently used term describing various cellular developmental abnormalities visible by microscopy. However, detailed description of these developmental abnormalities might provide useful information not only on the cell state but also on the abnormal developmental steps of cell lineages, tissues and organs. The frequently noted visualized cell dysplastic features reflect nuclear- or nucleolar-cytoplasmic anarchy (asynchrony), premature heterochromatin condensation state, marked aneuploidy, abnormal nucleus-cytoplasm ratio, abnormality of cell organelles including mitochondria, abnormal presence or absence of cell lineage-specific granules, and formation of peripheral buds or blebbing on the cell surface. The description of these frequently occurring cell dysplastic features might also be helpful in recognizing and studying defined specific disorders of the "whole macro-body" expressed as a disease.

Polatuzumab vedotin plus bendamustine and rituximab in patients with relapsed/refractory diffuse large B-cell lymphoma in the real world.

OBJECTIVES: Polatuzumab vedotin with bendamustine and rituximab (Pola-BR) was approved for treatment of transplant-ineligible patients with relapsed/refractory DLBCL (R/R DLBCL). However, the number of patients treated in the GO29365 trial including the extension cohort was limited, and more data evaluating the efficacy of this treatment regimen is needed. METHODS: We analyzed 21 patients with R/R DLBCL to determine real-life efficacy and safety of Pola-BR regimen. Data of all patients entered the database of the NiHiL project (NCT03199066). RESULTS: Median overall survival was 8.7 months, and progression-free survival 3.8 months. The overall response rate was 33%. Grade 3-4 neutropenia was detected in 29%, thrombocytopenia in 38%, anemia in 19%, infections in 24% cases, and peripheral neuropathy in 5%. Discontinuation of treatment was caused by progression in 50%, adverse events in 31%, and intended bridging to CAR-T therapy in 19%. CONCLUSION: Although the outcome of patients is worse than in GO29365 trial, the use of Pola-BR regimen in the real world demonstrates tolerable toxicity profile and efficacy in transplant-ineligible patients with R/R DLBCL. Moreover, this regimen might represent a perspective option as a bridge to CAR-T therapy.

CD31/PECAM-1 impacts engraftment, growth and spread of mantle cell lymphoma cells and positively correlates with extramedullary involvement.

Platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31) is an immunoglobulin superfamily member expressed on the surface of platelets, leukocytes and endothelial cells. The role of CD31 in biology of lymphomas has not yet been systemically studied. Expression of cell surface CD31 was analyzed by flow cytometry on primary MCL cells isolated from peripheral blood, bone marrow or malignant effusions obtained from 29 newly diagnosed MCL patients. CD31 was significantly more expressed in patients with documented extranodal involvement. Knock-down of CD31 expression in JEKO1 and MINO MCL cell lines hampered their subcutaneous engraftment in immunodeficient mice and prolonged overall survival of intravenously-xenografted animals. In contrast, transgenic overexpression of CD31 accelerated growth of subcutaneous JEKO1 and MINO tumors, shortened overall survival of intravenously-xenografted mice, and resulted in significantly increased frequency of extramedullary murine tissue infiltration Our observations suggest that CD31 facilitate survival and regulate extranodal spread of MCL cells.

Prospective observational study in comorbid patients with chronic lymphocytic leukemia receiving first-line bendamustine with rituximab.

Chemoimmunotherapy with bendamustine and rituximab is an alternative treatment for elderly patients with CLL. The aim of this observational multicenter study was to prospectively assess efficacy and safety of bendamustine and rituximab in front-line therapy in patients with CLL and significant comorbidities in real hematological practice. Eighty-three consecutive patients with cumulative illness rating scale (CIRS) >6 who received at least one cycle of BR as first-line treatment were included in the study. The median age was 71 years (range, 53-83), the median CIRS was 8 (range, 7-17), and 60.2% of patients had a creatinine clearance ≤70 mL/min. FISH analysis, available for 78 cases, showed a del(17p) in 11.5% and del(11q) in 20.5% of patients. Overall response rate was 88.0% with a complete response rate of 20.5%. With median follow-up time of 22 months, the estimated median progression free survival was 35.9 months. Progression free survival and overall survival rates at 2 years were 69.9% and 96.2%, respectively. Grade 3 or 4 neutropenia, thrombocytopenia, and anemia were documented in 40 (48.2%), 14 (16.9%), and 8 (9.6%) patients, respectively. Grade 3 or 4 infections occurred in 14.5% of patients. Chemoimmunotherapy with BR is an effective therapeutic option with manageable toxicity for the initial treatment of CLL patients with significant comorbidities. ClinicalTrials.gov Identifier: NCT02381899.

Oncogenic microRNA-155 and its target PU.1: an integrative gene expression study in six of the most prevalent lymphomas.

The transcription factor PU.1 and its inhibitory microRNA-155 (miR-155) are important regulators of B-cell differentiation. PU.1 downregulation coupled with oncogenic miR-155 upregulation has been reported in lymphoid malignancies; however, these data have not been studied across different subtypes in relation to clinical outcomes. We studied expression of miR-155 and PU.1 in the six most prevalent human B-cell lymphomas (n = 131) including aggressive (DLBCL, HL, MCL) and indolent (B-CLL/SLL, MZL, FL) types. Levels of miR-155 and PU.1 inversely correlated in DLBCL, B-CLL/SLL, and FL tumor tissues. In HL tissues, an exceptionally high level of miR-155 was found in patients with unfavorable responses to first-line therapy and those who had shorter survival times. PU.1 downregulation was noted in B-CLL/SLL samples positive for the adverse prognostic markers CD38 and ZAP-70. Upregulation of miR-155 and downregulation of PU.1 expression are integral aspects of lymphoma biology that could mark aggressive behavior of some, but not all, lymphoma types.

Ofatumumab added to dexamethasone in patients with relapsed or refractory chronic lymphocytic leukemia: Results from a phase II study.

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high-dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab-dexamethasone (O-Dex) combination in relapsed or refractory CLL. The trial was an open-label, multicenter, nonrandomized, Phase II study. The O-Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2-6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1-4 and 15-18; Cycles 1-6). The O-Dex regimen was given until best response, or a maximum of six cycles. Thirty-three patients (pts) were recruited. Twenty-four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression-free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3-5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O-Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101).

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia.

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

Endothelial cells (EC) and endothelial precursor cells (EPC) kinetics in hematological patients undergoing chemotherapy or autologous stem cell transplantation (ASCT).

Our objective was to study the kinetics of circulating endothelial cells (EC) and endothelial precursor cells (EPC) in hematological patients during chemotherapy and autologous stem cell transplantion (ASCT). Eighteen newly diagnosed patients and 17 patients undergoing ASCT were studied and compared to healthy controls. ECs were evaluated as CD146+CD31+Lin- cells, while EPCs were evaluated as CD34+CD133+Lin-, or CD34+VEGFR2+Lin- cells, or CFU-En colony forming units. Numbers of these cells were evaluated before and after treatment, and, in patients treated with ASCT, during mobilization of hematopoietic progenitors. Both newly diagnosed patients and patients before ASCT had significantly higher number of CD146+CD31+Lin- cells and significantly lower number of CFU-En colonies than healthy controls. These parameters did not return to normal for at least 3 months after chemotherapy or ASCT. Numbers of CFU-En did not correlate either with numbers of CD34+CD133+Lin- cells or with numbers of CD34+VEGFR2+Lin- cells but they did correlate with numbers of CD4+ lymphocytes and NK cells. In conclusion, we have found that hematological patients have higher number of EC and lower numbers of CFU-En than healthy controls and that these parameters do not return to normal after short-term follow-up. Furthermore, our observations support emerging data that CFU-En represent cell population different from flowcytometrically defined EC and endothelial precursors and that their development requires cooperation of monocytes and CD4+ lymphocytes. However, cells forming CFU-En express endothelial surface markers and can contribute to proper endothelial function by NO production.

Incidence of chromosomal anomalies detected with FISH and their clinical correlations in B-chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia. Molecular genetic characterization of B-CLL has made significant progress and typical chromosomal anomalies have been assessed. The most frequent chromosomal abnormalities are deletions at 13q14, 17p13, and 11q22 approximately q23 and trisomy 12. The aim of this study was to establish incidence of chromosomal changes in bone marrow or peripheral blood cells (or both) of B-CLL patients using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH), and to evaluate the prognostic implications. We performed I-FISH on bone marrow and blood smears from 217 B-CLL patients (124 male, 93 female). Trisomy 12 was found in 35 of the 217 (16%); deletion 13q14 was analyzed in 207 patients and found in 112 (54%). Deletion 17p13 was found in 34 (16%) out of 206 examined. Deletion of 11q23 was analyzed in 56 patients and was present in 7 (12%). Statistical analyses were performed to correlate the molecular-cytogenetic findings with disease status (stable versus progressive), Rai stage, CD38/CD19 antigen coexpression, immunoglobulin variable heavy chain (IgV(H)) mutational pattern, and other clinical and laboratory parameters. No apparent differences in distribution were noted for anomalies +12, del(13)(q14), or del(17)(p13) among patients with stable and progressive disease, and no consistent pattern in the distribution of type of genomic changes were found among various Rai stages and in CD38/CD19-positive or -negative patients. Patients without IgV(H) mutation had a worse prognosis; however, distribution of chromosomal abnormalities identified with FISH was the same for patients with and without IgV(H) mutations.

Biallelic IgH rearrangements in patients with indolent lymphoproliferative disorders: molecular and practical implications.

We report a group of patients (pts) with indolent lymphoproliferative disorder who had both alleles for the immunoglobulin heavy chain genes rearranged (biIgH). This group of 17 pts consisted of 9 small lymphocytic lymphomas (SLL) and 8 chronic lymphocytic leukemia (CLL). The polymerase chain reaction (PCR) amplification of clonal immunoglobulin heavy (IgH) rearrangement using the complementarity determining region III (CDRIII) constantly retrieved two distinct bands in all PCR informative samples of those pts. To rule out biclonality, we evaluated samples by fluorescein activated cell sorting (FACS) analysis and sequenced the PCR products. We were able to obtain both IgH sequences from 12 patients. FACS suggested biclonality in one case, which also correlated with sequencing results as both IgH rearrangements were in-frame. Recently, we reported a patient who sustained transformation into an aggressive disease after biIgH was detected in the setting of monoclonal disease (Cerny et al., 2003b, Haematologica 88(05):ECR15 B.). We decided to compare clinical characteristics and prognosis of 17 pts with biIgH and 37 pts with monoIgH rearrangements. Although we found some minor differences in disease characteristics between both groups, these did not translate into a significantly different overall survival. Our findings suggest that true biclonal cases of CLL are rare.

Familial chronic lymphocytic leukemia.

Familial aggregation of chronic lymphocytic leukemia (CLL) has been observed more frequently than familial aggregation of any other type of oncohematologic disorder. The presence of cells with a CLL-like immunophenotype (CLL-like cells) was recently documented in 13.5% healthy first-degree relatives of CLL patients. We present a family with CLL in which 2 brothers, a sister and their mother were affected.

Unusual sequence of VDJ rearrangement revealed by molecular analysis in a patient with indolent lymphoma.

We report a unique case of indolent lymphoma with an unusual VDJ rearrangement. Polymerase chain reaction (PCR) analysis of bone marrow at the time of diagnosis was positive for both BCL-2/JH and CDRIII rearrangements. After treatment, the patient achieved complete remission (CR) with slow disappearance of both rearrangements (CDRIII and then BCL-2/JH). Subsequently, two new CDRIII rearrangements were detected in bone marrow, peripheral blood, and lymph node tissue. After this conversion, fluorescent activated cell sorting (FACS) analysis demonstrated monoclonal disease, suggesting that both CDRIII rearrangements originated from one cell. Histological evidence of a B-cell small lymphocytic lymphoma (B-SLL) infiltrate in the bone marrow became evident approximately 1 year after the two CDRIII rearrangements appeared. Direct sequencing revealed that one of the CDRIII sequences consisted of a VDVDJ rearrangement. This is the first report of such a rearrangement in a case of indolent lymphoma. This type of rearrangement has been described to result from a secondary VDJ recombination in childhood acute lymphoblastic leukemia (ALL) leading towards oligoclonality and poorer prognosis. Our observations suggest that such a finding in an indolent lymphoma patient may precede transformation into an aggressive disease. Early detection by PCR could have substantial impact on the prognosis of such patients.