Melissa Dabusti

CD34+ cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation

OBJECTIVE The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT). MATERIALS AND METHODS Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay. RESULTS Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused. CONCLUSIONS These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity.

Clonal evolution including 14q32/IGH translocations in chronic lymphocytic leukemia: analysis of clinicobiologic correlations in 105 patients

Abstract To better define the significance of clonal evolution (CE) including 14q32 translocations involving the immunoglobulin heavy chain gene (IGH) in chronic lymphocytic leukemia (CLL), 105 patients were analyzed sequentially by fluorescence in situ hybridization (FISH) with the following panel of probes: 13q14/D13S25, 11q22/ATM, 17p13/TP53, #12-centromere and 14q32/IGH break-apart probe. CE was observed in 15/105 patients after 24–170 months (median 64). Recurring aberrations at CE were 14q32/IGH translocation in seven patients; other aberrations were 17p −, 11q −, biallelic 13q − and 14q32 deletion. CE was detected in 15/58 pre-treated patients; in contrast, none of 47 untreated patients developed CE (p < 0.0001). In two cases the appearance of 14q32/IGH translocation was first detected in the bone marrow (BM) or in the lymph node (LN) and 13–58 months later in the peripheral blood (PB). ZAP70 + and high-risk cytogenetics predicted for the occurrence of CE with borderline statistical significance (p = 0.055 and 0.07, respectively). Shorter time to first treatment (TTT) and time to chemorefractoriness (TTCR) were noted in 15 patients with CE when compared to patients without CE (TTT: 35 vs. 71 months, p = 0.0033 and TTCR: 34 vs. 86 months, p = 0.0046, respectively). Survival after the development of CE was 32 months (standard error 8.5). We arrived at the following conclusions: (i) 14q32/IGH translocation may represent one of the most frequent aberrations acquired during the natural history of CLL and (ii) it may be detected earlier in BM or LN samples; (iii) CE including 14q32/IGH translocation occurs in pre-treated patients with short TTT and TTCR; (iii) survival after CE is relatively short.

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: Correlation with colony assay

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.

Therapy-related adult acute lymphoblastic leukemia with t(4;11)(q21; q23): MLL rearrangement, p53 mutation and multilineage involvement

A diagnosis of pro-B acute lymphoblastic leukemia (ALL) with CD15+ was made in a 42-year-old woman, 12 months after the treatment of uterine adenocarcinoma by carboplatinum, anthracyclines, etoposide and radiotherapy. Molecular cytogenetic studies revealed a karyotype with multiple chromosome changes, including the t(4;11)(q21;q23) and a 17p-chromosome, with MLL disruption and 17p13/p53 gene deletion in 86% of the cells. A p53 exon 6 mutation was documented, resulting in p53 protein stabilization, with 20% of the cells reacting with the 1801 anti-p53 monoclonal antibody. Dual-color FISH using MLL and p53 probes was performed on peripheral blood smears, providing direct evidence of the involvement of the blast cells and of the granulocytic lineage. Only a partial, short-lasting response was obtained by induction treatment, confirming that a poor prognosis is associated with therapy-related ALL with the 4;11 translocation.

Expression of the immunoglobulin superfamily cell membrane adhesion molecule Cd146 in acute leukemia

The expression of the immunoglobulin superfamily cell membrane adhesion molecule CD146 has been reported on several normal and pathological cell types in human. The aim of this study was to investigate CD146 expression in acute leukemia using a multiparametric cytofluorimetric approach.

Diagnostic work-up for clinical and prognostic assessment of acute leukaemia

SummaryAcute leukaemia is a neoplastic disorder of haematopoietic precursors characterized by a rapid clinical course and a relatively high early death rate in spite of recent therapeutic progress. Prompt and reliable diagnostic and prognostic assessment has a favourable impact on patient outcome. Diagnostic suspicion relies on signs and symptoms of bone marrow failure or quali-/quantitative abnormalities of blood cells, which are accurately detected by modern automated counters. Cytomorphological examination of a stained blood and bone marrow film plays a key role in the diagnostic process, with relevant additional information coming from immunophenotyping of blast cells. Cytogenetic and molecular genetic data are the basis of prognostic stratification. In this context, a coordinated intervention of specialized laboratories combining solid expertise in each field involved in the diagnostic work-up is essential; at the same time, the availability of all the required technologies at the same hospital structure would probably lack efficiency due to the low number of tests performed. The organization of laboratory networks, either at regional or national level, especially in the context of clinical trials, may offer a great opportunity for centralization of more sophisticated technologies in reference laboratories, each highly specialized in a particular field of investigation and all interconnected. The challenge for laboratory haematology is the reorganization of clinical and scientific activity according to this model, without loosing educational potential in favour of new generations of medical doctors, haematologists, biologists and laboratory technicians.RiassuntoLa leucemia acuta è una malattia neoplastica dei precursori emopoietici caratterizzata da una rapida evolutività clinica, con un tasso di mortalità precoce che si mantiene elevato nonostante i continui progressi terapeutici. La tempestività di un corretto inquadramento diagnostico secondo criteri classificativi internazionali e di una precisa stratificazione prognostica ha un impatto clinico dimostrato in termini di outcome. Il sospetto diagnostico può essere formulato sulla base di sintomi o segni, in genere associati alla soppressione della normale emopoiesi, oppure sulla base di alterazioni emocromocitometriche, quantitative o qualitative, sensibilmente rilevate dai moderni contatori automatici. La valutazione morfologica dello striscio di sangue periferico e della citologia midollare rappresenta un momento fondamentale nell’iter diagnostico e può ricevere contributi di rilievo dall’indagine immunofenotipica. La stratificazione prognostica si fonda su dati citogenetici e molecolari sempre più specifici e dettagliati. In questo contesto, l’integrazione tra le diverse competenze cliniche e laboratoristiche coinvolte nel processo diagnostico acquista grande importanza; allo stesso tempo però la disponibilità presso ogni centro delle risorse strumentali necessarie e di personale altamente qualificato nei diversi settori si scontra con esigenze di efficienza economica. Lo sviluppo, a livello locale e nazionale, di reti di laboratori a elevata specializzazione in ognuno dei differenti ambiti diagnostici e di ricerca, consente una proficua ottimizzazione delle risorse. La sfida che si propone quindi all’Ematologia di laboratorio è quella di una riorganizzazione dell’attività clinica e scientifica secondo un modello che soddisfi elevati standard di accuratezza diagnostica ed efficienza economica, senza sacrificare le esigenze formative delle nuove generazioni di medici, specialisti ematologi, biologi e tecnici di laboratorio.

Biological significance and prognostic/predictive impact of complex karyotype in chronic lymphocytic leukemia

The complex karyotype (CK) is an established negative prognostic marker in a number of haematological malignancies. After the introduction of effective mitogens, a growing body of evidence has suggested that the presence of 3 or more aberrations by conventional banding analysis (CBA) is associated with an unfavorable outcome in chronic lymphocytic leukemia (CLL). Thus, the importance of CBA was recognized by the 2018 guidelines of the International Workshop on CLL, which proposed the introduction of CBA in clinical trials to validate the value of karyotype aberrations. Indeed, a number of observational studies showed that cytogenetic aberrations and, particularly, the CK may have a negative independent impact on objective outcome measures (i.e. time to first treatment, progression free survival, time to chemorefractoriness and overall survival) both in patients treated with chemoimmunotherapy and, possibly, in patients receiving novel mechanism-based treatment. Here, we set out to present the scientific evidence supporting the significance of CK as a prognostic marker in CLL and to discuss the biological basis showing that the CK is a consequence of genomic instability.