A Clinically Applicable Approach to the Classification of B-Cell Non-Hodgkin Lymphomas with Flow Cytometry and Machine Learning.

The immunophenotype is a key element to classify B-cell Non-Hodgkin Lymphomas (B-NHL); while it is routinely obtained through immunohistochemistry, the use of flow cytometry (FC) could bear several advantages. However, few FC laboratories can rely on a long-standing practical experience, and the literature in support is still limited; as a result, the use of FC is generally restricted to the analysis of lymphomas with bone marrow or peripheral blood involvement. In this work, we applied machine learning to our database of 1465 B-NHL samples from different sources, building four artificial predictive systems which could classify B-NHL in up to nine of the most common clinico-pathological entities. Our best model shows an overall accuracy of 92.68%, a mean sensitivity of 88.54% and a mean specificity of 98.77%. Beyond the clinical applicability, our models demonstrate (i) the strong discriminatory power of MIB1 and Bcl2, whose integration in the predictive model significantly increased the performance of the algorithm; (ii) the potential usefulness of some non-canonical markers in categorizing B-NHL; and (iii) that FC markers should not be described as strictly positive or negative according to fixed thresholds, but they rather correlate with different B-NHL depending on their level of expression.

Prognostic relevance of cytometric quantitative assessment in patients with myelodysplastic syndromes.

OBJECTIVES: Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. METHODS: Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (<3% vs. ≥3%), CD117, and CD11b(-) /CD66b(-) (<5% vs. ≥5%); myeloid maturation was analyzed by the expression of CD11b(+) /CD66b(++) (<15% vs. ≥15%) and CD11b(+) /CD66b(+) (<25% vs. ≥25%). RESULTS: In univariate analysis, the expression of immaturity markers (CD34(+) , CD117(+) , and CD11b(-) /CD66b(-) ) was associated with shorter LFS and OS (P < 0.0001); higher expression of differentiation markers (CD11b(+) /CD66b(++) and CD11b(+) /CD66b(+) ) was associated with longer LFS (P < 0.0001 and P = 0.0002, respectively) and OS (P < 0.0001). In multivariate analysis, expression of CD34(+) (P = 0.007), CD117(+) (P = 0.013), and CD11b(+) /CD66b(++) (P = 0.023) retained independent prognostic value for OS, while only the expression of CD34(+) was a prognostic factor for LFS (P = 0.0003). Two different risk groups were defined according to the presence of 0-1 or ≥2 of these factors with significant different LFS and OS (P < 0.0001). This score showed prognostic value in predicting survival even in subanalysis according to IPSS and WHO subgroups. CONCLUSIONS: Flow cytometric analysis in MDS may provide meaningful prognostic information. Blast percentage expressed as CD117(+) or CD34(+) cells and the quantitative assessment of myeloid maturation showed prognostic value for survival.

Cluster analysis of immunophenotypic data: the example of chronic lymphocytic leukemia.

Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have previously proposed an original method to identify the immunophenotypic signature of chronic lymphocytic leukemia (CLL) subsets with different prognosis, named surface-antigen expression profiling. According to this method, expression data for surface markers can be successfully analyzed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim to identify the immunophenotypic signature of CLL subsets with different prognosis. By employing an identical approach for investigating the reactivity of a wide panel of monoclonal antibodies provided by the "Ninth International Workshop on Leukocyte Differentiation Antigens", we were able to identify some of them (i.e. TCL1, CCR7, FCRL2, FCRL3, and CD150) as additional potential markers with prognostic relevance in CLL. These suggestions need to be confirmed: (i) in a new set of clinically characterized CLL cases; (ii) in combination with other prognostic markers in the context of comprehensive scoring systems for clinical outcome prediction.

Clonal CD8+ TCR-Vbeta expanded populations with effector memory phenotype in Churg Strauss syndrome.

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

Age-dependent accumulation of monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly.

Multicolour flow cytometric analysis enabled the identification of monoclonal B-cell lymphocytosis (MBL), frequently resembling chronic lymphocytic leukaemia, at a rather high frequency in peripheral blood (PB) samples from an elderly population. PB T lymphocytes from 103 otherwise healthy subjects >65 years of age and 51 younger donors (<65 years) were analysed. Besides CD4(+) and CD8(+) single positive (SP) cells, CD4(+)CD8(+) double positive (DP) mature T lymphocytes were present in both series and could be further distinguished into CD4(high)CD8(low) and CD4(low)CD8(high) subsets. An age-dependent increase of both DP T-cell subsets was observed, while SP T cells remained stable throughout life. Flow cytometry and polymerase chain reaction analysis of the TRBV expression profiles showed the presence of a TRBV restriction within CD4(+)CD8(+) DP cells in more than half (53/103; 55.3%) of the individuals >65 years of age, regardless the actual number of DP T cells observed. Clonal expansions were more prominent within the CD4(high)CD8(low) subset, accounting for most circulating DP clones (47/103; 45.6%). A few cases showed more than one (up to three) monoclonal expansion. Clonal CD4(low)CD8(high) DP T-lymphocyte expansions were detected in only 10/103 samples (9.7%) and showed a close phenotypic similarity to the rare T-cell large granular lymphocyte leukaemias. The similarities between DP clones and MBL in the elderly may help to better understand the mechanisms of immunosenescence and their relationships with the development of lymphoproliferative disorders.

Alternative BCR/ABL splice variants in Philadelphia chromosome-positive leukemias result in novel tumor-specific fusion proteins that may represent potential targets for immunotherapy approaches.

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

Monoclonal CD5+ and CD5- B-lymphocyte expansions are frequent in the peripheral blood of the elderly.

The responsiveness and diversity of peripheral B-cell repertoire decreases with age, possibly because of B-cell clonal expansions, as suggested by the incidence of serum monoclonal immunoglobulins and of monoclonal chronic lymphocytic leukemia (CLL)-like B lymphocytes in clinically silent adults. We phenotyped peripheral blood cells from 500 healthy subjects older than 65 years with no history or suspicion of malignancies and no evidence of lymphocytosis. In 19 cases (3.8%) a kappa/lambda ratio of more than 3:1 or less than 1:3 was found: 9 were CD5+, CD19+, CD23+, CD20low, CD79blow, sIglow (classic CLL-like phenotype); 3 were CD5+, CD19+, CD23+, CD20high, CD79blow, sIglow (atypical CLL-like), and 7 were CD5-, CD19+, CD20high, CD23-, CD79bbright, FMC7+, sIgbright (non-CLL-like). In 2 subjects, 2 phenotypically distinct unrelated clones were concomitantly evident. No cases were CD10+. Polymerase chain reaction (PCR) analysis demonstrated a monoclonal rearrangement of IgH genes in 15 of 19 cases. No bcl-1 or bcl-2 rearrangements were detected. Using a gating strategy based on CD20/CD5/CD79 expression, 13 additional CLL-like B-cell clones were identified (cumulative frequency of classic CLL-like: 5.5%). Thus, phenotypically heterogeneous monoclonal B-lymphocyte expansions are common among healthy elderly individuals and are not limited to classic CLL-like clones but may have the phenotypic features of different chronic lymphoproliferative disorders, involving also CD5- B cells.