The morphology of CLL revisited: the clinical significance of prolymphocytes and correlations with prognostic/molecular markers in the LRF CLL4 trial.

Historically, an increase in the percentage and number of circulating prolymphocytes in chronic lymphocytic leukaemia (CLL) has been associated with strong expression of surface immunoglobulin, trisomy 12 and a poor outcome. This study re-examines the biological and clinical significance of increased peripheral blood prolymphocytes in 508 patients at entry into the randomized UK Leukaemia Research Fund CLL4 trial. It also investigates the associations between increased prolymphocytes and a comprehensive array of biomarkers. 270 patients (53%) had <5% prolymphocytes, 167 (33%) had 5-9%, 60 (12%) had 10-14% and 11 (2%) had ≥15% prolymphocytes. We show that a higher proportion of prolymphocytes (≥10%) was independently associated with NOTCH1 mutations (P = 0·006), absence of 13q deletion (P = 0·001), high CD38 expression (P = 0·02) and unmutated IGHV genes (P = 0·01). Deaths due to Richter syndrome were significantly more common amongst patients who had ≥10% vs <10% prolymphocytes (13% vs 2%) respectively (P < 0·0001). ≥10% prolymphocytes was also associated with a shorter progression-free survival (Hazard ratio [HR] 1·50 [95% confidence interval [CI]: 1·16-1·93], P = 0·002) and overall survival (HR 1·99 [95% CI: 1·53-2·59], P < 0·0001). Our data support the routine examination of blood films in CLL and suggest that a finding of an increased proportion of prolymphocytes may be a trigger for further evaluation of clinical and laboratory features of progressive disease.

Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification.

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission.

Phase 1, dose-escalation study of guadecitabine (SGI-110) in combination with pembrolizumab in patients with solid tumors.

BACKGROUND: Data suggest that immunomodulation induced by DNA hypomethylating agents can sensitize tumors to immune checkpoint inhibitors. We conducted a phase 1 dose-escalation trial (NCT02998567) of guadecitabine and pembrolizumab in patients with advanced solid tumors. We hypothesized that guadecitabine will overcome pembrolizumab resistance. METHODS: Patients received guadecitabine (45 mg/m2 or 30 mg/m2, administered subcutaneously on days 1-4), with pembrolizumab (200 mg administered intravenously starting from cycle 2 onwards) every 3 weeks. Primary endpoints were safety, tolerability and maximum tolerated dose; secondary and exploratory endpoints included objective response rate (ORR), changes in methylome, transcriptome, immune contextures in pre-treatment and on-treatment tumor biopsies. RESULTS: Between January 2017 and January 2020, 34 patients were enrolled. The recommended phase II dose was guadecitabine 30 mg/m2, days 1-4, and pembrolizumab 200 mg on day 1 every 3 weeks. Two dose-limiting toxicities (neutropenia, febrile neutropenia) were reported at guadecitabine 45 mg/m2 with none reported at guadecitabine 30 mg/m2. The most common treatment-related adverse events (TRAEs) were neutropenia (58.8%), fatigue (17.6%), febrile neutropenia (11.8%) and nausea (11.8%). Common, grade 3+ TRAEs were neutropaenia (38.2%) and febrile neutropaenia (11.8%). There were no treatment-related deaths. Overall, 30 patients were evaluable for antitumor activity; ORR was 7% with 37% achieving disease control (progression-free survival) for ≥24 weeks. Of 12 evaluable patients with non-small cell lung cancer, 10 had been previously treated with immune checkpoint inhibitors with 5 (42%) having disease control ≥24 weeks (clinical benefit). Reduction in LINE-1 DNA methylation following treatment in blood (peripheral blood mononuclear cells) and tissue samples was demonstrated and methylation at transcriptional start site and 5' untranslated region gene regions showed enriched negative correlation with gene expression. Increases in intra-tumoural effector T-cells were seen in some responding patients. Patients having clinical benefit had high baseline inflammatory signature on RNAseq analyses. CONCLUSIONS: Guadecitabine in combination with pembrolizumab is tolerable with biological and anticancer activity. Reversal of previous resistance to immune checkpoint inhibitors is demonstrated.

Immunohistochemical distinction of haematogones from B lymphoblastic leukaemia/lymphoma or B-cell acute lymphoblastic leukaemia (B-ALL) on bone marrow trephine biopsies: a study on 62 patients.

Haematogones are normal, maturing B-cell precursors. They can be confused with neoplastic immature lymphoid cells of B lymphoblastic leukaemia/lymphoma or B-cell acute lymphoblastic leukaemia (B-ALL). Though multi-colour flow-cytometry strategies for distinguishing haematogones from cells of B-ALL are well-described, similar strategies have not been determined for bone marrow trephine biopsies (BMTB). We revisited the morphological and immunohistochemical features (CD20, CD34, TdT and PAX5 expression) in 69 BMTB from 62 patients - 27 with excess haematogones; seven with residual B-ALL after therapy; 18 with no reported excess of haematogones or residual acute leukaemia on BMTB; and 17 diagnostic samples of B-ALL. The distinctive immunophenotypic pattern of BMTB with excess haematogones was of CD34, TdT, CD20 and PAX5 accounting for increasing proportions of cells in the order mentioned, whereas among B-ALL, the immunohistochemical pattern was of CD20, PAX5 and TdT accounting for an equal proportion of cells. Furthermore, among haematogones, the intensity of CD20 expression was extremely heterogeneous as compared to the neoplastic cells in CD20-positive B-ALL. The TdT-positive haematogones were generally small and uniform, while a certain degree of heterogeneity was noticed among neoplastic B-ALL cells. This study provides a practical strategy to distinguish haematogones from B-ALL cells in BMTB.

Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia: data from the randomised UK LRF CLL4 trial.

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders.

The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Inter-laboratory validation of a harmonized PNH flow cytometry assay.

BACKGROUND: A network comprising 11 laboratories aimed to consolidate PNH testing by developing and validating an assay guided by previous guidelines and studies. Network analyses of >20 native samples yielded key findings that were used to create and reshape the final protocol. METHODS: Twenty-seven native samples were distributed to all participating laboratories for blind testing, local analysis, and subsequent re-analysis by a central laboratory. Inter-laboratory clone size precision (coefficient of variation [CV]) was monitored for each sample, and the findings used to refine the test protocol. Linearity and precision tests were performed, assay limits of blank and detection were calculated, and limits of quantification were determined. RESULTS: When using the final protocol, enumeration of cells with a PNH phenotype by all participating laboratories was comparable, with no clinically significant discrepancies or false-positive or false-negative results reported. Of note, the biological characteristics of the sample affected precision. For example, CVs were higher when PNH and normal cells showed contiguous expression of GPI-linked antigens. A red cell gating strategy that eliminated non-specific type II PNH phenotypic events was devised, enabling reliable reporting of events in the type II red cell gate. The final protocol provided an assay with a Limit of Quantification of 0.01% for neutrophils and red cells, and 0.1% for monocytes. CONCLUSIONS: We describe a robust network-validated PNH assay that may aid other laboratories to set up and validate high resolution PNH analysis. © 2018 International Clinical Cytometry Society.

Case 35: an unusual haematological neoplasm characterized by cells with cytoplasmic tails.

A middle aged Northern European man presented with a submental lymph node and was found on imaging to have generalized lymphadenopathy. There was bone marrow infiltration by small apparently lymphoid cells, many with cytoplasmic tails; these cells were CD56(+) and weakly CD4(+). A provisional diagnosis was confirmed by further immunophenotyping.

The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells.

The nature of hemopoietic progenitors subject to leukemic transformation in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and downstream flanking regions of CD2 were found to be hypersensitive to DNase I in primary APL blasts, with an identical pattern of hypersensitive sites to those detected in cells of T-lineage. All of the sites were confirmed to be inaccessible to DNase I in B-lineage leukemia cells. The demonstration of T-cell-associated chromatin features in primary APL blasts has implications for the origin of APL that may arise in more primitive progenitors than previously considered to be the case.

CD34 and CD117 are overexpressed in AML and may be valuable to detect minimal residual disease.

We estimated by quantitative flow cytometry (FC) the expression of CD13, CD33, CD34 and CD117 antigens in cells from 64 patients with acute myeloid leukaemia (AML) and 22 normal bone marrows (BMs). The method converts fluorescence intensity into number of antigen molecules per cell, measured by antibody binding capacity (ABC). The number of molecules per cell in normal BM was 9.5+/-5.7 for CD13, 7+/-2.3 for CD33, 6+/-0.7 for CD34, and 6.3+/-1.5x10(3) for CD117. AML blasts expressed 11.4+/-12.4 molecules per cell for CD13, 9.5+/-9.7 for CD33, 74+/-2328.5 for CD34 and 12.5+/-33 x 10(3) for CD117. The number of CD34 and CD117 molecules were significantly higher in AML than in normals (P<0.0001 and P<0.05, respectively) while only in a few cases, CD13 and CD33 were abnormally expressed in myeloblasts. Our results indicate that quantitative analysis of CD34 and CD117 may be useful to detect minimal residual disease (MRD) and could be tested in a future to monitor therapy in AML.

The diagnostic value of CD123 in B-cell disorders with hairy or villous lymphocytes.

BACKGROUND AND OBJECTIVES: CD123 is an antibody that identifies the a chain of the human interleukin-3 receptor and is expressed in a variety of normal hematopoietic cells, acute leukemia and hairy cell leukemia (HCL). The aim of the study was to investigate the diagnostic value of CD123 expression in B-cell disorders with circulating hairy and villous lymphocytes. DESIGN AND METHODS: We investigated the diagnostic value of CD123 expression in neoplastic cells from 59 patients with B-cell disorders with circulating hairy or villous lymphocytes: HCL (n=24), the variant form of HCL (n=11) and splenic lymphoma with villous lymphocytes (SLVL) (n=24). Cells from 12 patients with chronic lymphocytic leukemia were used as controls. Immunophenotypic analysis was performed by flow cytometry on 77 samples from peripheral blood (n=48), bone marrow (n=25) and spleen cell suspensions (n=4). RESULTS: Our findings show that cells from 95% of typical HCL express CD123 with strong to moderate intensity while this molecule is absent in circulating cells from most cases of HCL-variant (91%) and SLVL (97%). INTERPRETATION AND CONCLUSIONS: We conclude that CD123 is a useful new marker for distinguishing B-cell disorders with circulating villous lymphocytes as its expression is characteristic of typical HCL with high sensitivity and specificity. However CD123 does not allow the distinction between HCL-variant and SLVL, as both are CD123 negative.

CD38 expression as a prognostic indicator in chronic lymphocytic leukaemia.

Staging systems and laboratory features help predict survival in chronic lymphocytic leukaemia but they do not distinguish patients who will progress from those whose disease will remain indolent. CD38 expression has emerged as an independent prognostic factor, yet there is debate as to what level of CD38 affects prognosis. We plotted the hazard ratios for the treatment-free interval (TFI) between the higher and lower groups defined by CD38 cut-offs from 0 to 100%. The maximum hazard ratio was achieved for a cut-off of 7%. We examined by triple colour analysis the values for CD38 in 289 untreated patients using both >or=30 and >or=7% as thresholds for prognosis. Using a >or=7% threshold (but not >or=30%), we showed a significant correlation with advanced stage and male sex. The interval from diagnosis to first therapy or TFI was longer (median 36 months) in patients with <7% CD38 positive cells than those with >or= 30% (8.7 months) or with intermediate values between 7 and 29% (P<0.00005). The <7% threshold also identified patients in stage A with a long TFI (P=0.0001). Multivariate analysis showed that CD38 has independent prognostic value for TFI in all patients. In 135 patients tested for deletions of p53, 13q14 and 11q23 and for trisomy 12, we showed a correlation between 13q14 deletion and <30%/<7% CD38 positive cells and a tendency for trisomy 12 to be associated with >or=30%/>or=7% CD38 positive cells. We conclude that 7% may be a more useful threshold for disease progression than higher values of CD38.

Characterisation of TP53 abnormalities in chronic lymphocytic leukaemia.

Abnormalities of TP53 in chronic lymphocytic leukaemia (CLL) correlate with aggressive disease and transformation. We studied 115 patients with CLL including 90 untreated, 25 with heavily pretreated/refractory CLL using fluorescent in situ hybridisation (FISH) to detect allelic loss at chromosome 17p and flow cytometry (FC) to test p53 protein overexpression. A total of 17 cases were identified with TP53 deletion and/or protein expression. Both tests correlated in 10 of 17 patients; in six, one or the other abnormality was detected and in one case, with a deletion, flow cytometry failed. Material for direct DNA sequencing was available in 14 of 17 cases. Mutations were found in seven cases. Five of 14 patients with allelic loss and seven of 13 expressing p53 protein had a mutation. These were single-base substitutions and were located in exons 5, 7 or 8. Mutations were not found in 13 of 14 other cases without deletions by FISH or protein expression. The incidence of p53 abnormalities in this series was 15%, with a significant difference between untreated patients (7%) and the pretreated/refractory group (50%; P<0.01). Abnormal p53 was predicted for shorter survival, regardless of the method used. We confirm that p53 abnormalities are more common in refractory CLL and that mutations occur at the known hot spots. Testing for TP53 deletions by FISH and protein expression by FC is an effective and simple way of screening patients who are likely to have aggressive disease. DNA sequencing adds little to these methods in identifying the population at risk.

Diagnostic significance of CD20 and FMC7 expression in B-cell disorders.

We analyzed by flow cytometry the expression of CD20 and FMC7 in cell suspensions from 932 patients, including 630 cases of chronic lymphocytic leukemia (CLL), 23 cases of other B-cell leukemias, and 279 cases of B-cell non-Hodgkin lymphoma (B-cell NHL). CD20 was positive in 94.5% of cases; FMC7 was positive in 35.7%. There was a correlation between CD20 and FMC7 expression in patients with B-cell NHL (P < .001) but not CLL (P = .1). We also tested a scoring system in which FMC7 was replaced by CD20 and compared it with our current scoring system for CLL. With this modification, the accuracy of the scoring system for differentiating CLL from other non-CLL disorders fell from 94.4% to 81.5%. In CD20+ CLL, the intensity of CD20 expression correlated with FMC7 and low scores (P < .001 for both comparisons). We suggest that the particular conformation of CD20 recognized by FMC7 is manifested only in cells with strong CD20 expression, which is not the case for CLL. FMC7 is of greater diagnostic value than CD20 for distinguishing CLL from other B-cell disorders; we recommend its continued use for this purpose.

Isolated bone marrow involvement in diffuse large B cell lymphoma: a report of three cases with review of morphological, immunophenotypic and cytogenetic findings.

Diffuse large B cell lymphoma (DLBL) comprises a heterogenous entity characterized by the presence of large cells, exhibiting a mature B cell phenotype. The high proliferation rate and aggressive disease remain a therapeutic challenge, but the apparent biological diversity permits a risk-stratification model for prognostic grouping through the International Prognostic Index (IPI). Empirical to this approach is the consideration of cytogenetic data, offering an insight into the pathogenetic events which may underlie neoplastic clonal evolution and disease progression. We describe three cases of DLBL presenting with isolated marrow disease, a rare primary finding in this lymphoma. All three cases showed involvement of blood and bone marrow without evidence of splenic or lymph node involvement on imaging studies. Histological and immunophenotypic findings were similar in all three cases, outlining the phenotypic maturity of this disease. Cytogenetic analysis revealed complex karyotypes in the two cases examined. M-FISH (multicolour fluorescent in situ hybridization) performed on bone marrow from case 1 showed several cryptic translocations not evident on G-banding, including a novel translocation between 2p and 9p, and an unbalanced translocation between 14q and 11q. Cytogenetic analysis in case 2 showed abnormalities involving 7q, 9p at the site of the INK4a gene, and the bcl-2 locus, findings confirmed by M-FISH. These cases serve to highlight the biological and cytogenetic heterogenity of DLBL and emphasize the need for complementary investigations in the characterization of this entity.