Immunity profile in breast cancer patients.

OBJECTIVES: Despite the multifactorial pathogenesis of malignant transformation, it is assumed that deficiency in some immune mechanisms plays a considerable role in its development. BACKGROUND: Chronically activated immune cells exert tumour-promoting effects directly by influencing the proliferation and survival of neoplastic cells, as well as by indirect modulation of neoplastic microenvironments in favour of tumour progression. PATIENTS AND METHODS: We refer to results of two separate investigations that aim to monitor the immune functions in patients with breast cancer. In the first investigation, we compare the picture of basic cellular immunity profile of patients in early stage of breast cancer with those suffering from advanced disease; in the second one, we compare the production of Th1-cytokines in patients in different stages of breast cancer and atopic healthy controls. RESULTS: We recognized that the totals of T-lymphocytes and T-helpers were lower and the expression of HLADR on T-lymphocytes were higher in patients with advanced disease; the expression of IL-2 and LFN-gamma by T-lymphocytes was decreased in metastatic breast cancer patients, however IL-2 production was increased in patients in early stage of disease. CONCLUSION: We conclude that the role of immune system in cancer development is ambivalent as it may be not only protective, but also harmful (Tab. 1, Fig. 3, Ref. 22). Full Text (Free, PDF) www.bmj.sk.

Normal maturation sequence of immunoglobulin light and heavy chains in hematogone stages 1, 2 and 3 in acute leukemia after treatment.

The cellular diversity of bone marrow samples was studied by using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on acute leukemia blast cells and regeneration of normal B-cells, hematogones. This approach should enhance the ability to study normal hematopoiesis, and to identify and monitor hematopoietic disorders. The study was performed on a homogeneous group of patients (mainly children), all of them after finishing complete therapy for AL, mostly B-ALL. In all of these patients complete pattern of all three individual Hg stages was present. Maturation spectra of surface immunoglobulin kappa (sIgkappa) and lambda (sIglambda) light chains and IgM, IgA heavy chains in all three stages of Hgs are presented as reliable reports on sIgs as their incidence on Hgs are scarse and even contradictory. The Ig expression paralles CD20 expression. SIg of light (kappa,lambda) and heavy (IgM, IgA) chains were completally absent in stage 1 Hgs and their expression increased through stage 2 to 3; IgM was expressed similarly. Light Ig chains kappa/lambda were expressed in a polytypic way. The results completed information on normal maturation sequence of bone marrow stage 1, 2 and 3 hematogone regeneration in treated acute leukemia patients.

Increased myeloid precursors in regenerating bone marrow; implications for detection of minimal residual disease in acute myeloid leukemia.

Presented study is focused on exact immunophenotypic definition of myeloid precursors and their following stages in regenerating bone marrow during treatment of ALL/AML for correct interpretation of the immunophenotype results and proper distinction from minimal residual disease (MRD) by multiparameter flow cytometry. This study includes bone marrow samples from 36 controls, 27 patients with AML, 39 patients with B-ALL undergoing therapy who remained in complete remission after treatment and also 30 B-ALL patients one year after the end of therapy. We observed substantial expansion of immature bone marrow populations in the regenerating bone marrows, which were identified by expression of CD34 and/or CD117 markers by 4-color flow cytometry. Myeloid precursors were significantly increased after cessation of induction therapy cycle of B-ALL (1.27+/-2.04%, p=0.0064) and also AML patients (0.87+/-0.77%, p=0.001), but also during follow-up of B-ALL patients (1.42+/-2.36%, p=0.0001) when compared with non-treated controls (0.38+/-0.29%). Some cases where their frequencies achieved up to 12% reflect the massive regeneration of myeloid lineage in bone marrow after chemotherapy cycles. Especially in these cases accurate interpretation of such a high frequency of immature myeloid cells as myeloid precursors was very important to exclude incoming relapse or secondary leukemia. The myeloid precursors represented by CD34+ in regenerating bone marrow expressed CD45 (94.8+/-5.5%), CD117 (38.3+/-26.2%), CD38 (91.4+/-5.7%), HLA-DR (90.6+/-7.6%), CD13 (73.0+/-20.8%) and CD33 (85.2+/-15.6%), while CD90 (2.7+/-2.5%), CD133 (10.0+/-8.2%) and T or B lymphocyte markers were negative. Comparing immunophenotypes with control bone marrows, only difference in expression of CD33 marker was found (85.2+/-15.6% versus 63.0+/-17.4% p=0.024). In addition, according to expression of these markers three different subsets of myeloid precursor cells were identified in regenerating bone marrow samples: CD34+ CD117- HLA-DR+, CD34+ CD117+ HLA-DR+ and CD34- CD117+ HLA-DR-/+ without aberrant marker expression. In conclusion for the correct discrimination of MRD in acute leukemia it is indispensable to define the range of normality in myeloid differentiation by extensive studies of bone marrows not only from healthy donors but also from regenerating bone marrow of patients undergoing therapy.

The impact of cell heterogeneity and immunophenotypic changes on monitoring minimal residual disease in acute myeloid leukemia.

Monitoring of minimal residual disease (MRD) becomes increasingly important for the more accurate stratification of the therapy in acute leukemia. The purpose of this study was to characterize in detail the phenotypes of heterogeneous population in various AML subtypes and to identify the leukemia associated aberrant phenotype (LAP) in individual patients with AML for precise investigation of MRD. The impact of heterogeneity of pathological populations, the effectiveness of location AML blasts on CD45/SSC dot plots in AML patients during follow-up and phenotype changes on MRD monitoring were evaluated in the second step. Bone marrow samples from 63 patients with AML were analyzed at diagnosis, 33 were selected for monitoring of MRD during follow-up and 13 analyzed at relapse using a wide antibody panel in quadruple combinations by multiparameter flow cytometry. In 88% of AML patients at least one LAP was defined at diagnosis, two or more aberrancies coexisted in 60% of them. The total number of LAPs identified by application of various combinations of antibodies was 112 (mean = 2.04 LAP/patient) and the median percentage of blasts carrying the LAP was 53.57%. In half of the patients, we were able to detect the presence of at least two subpopulations, which not always shared the same aberrancy. Although AML cells often have light scattering properties similar to those of normal (myeloid and B-lymphoid precursors, basophiles etc.) in a fraction of cases we found also very useful the location on CD45/SSC dot plots for MRD discrimination. In 13 patients relapse occurred and although we found in 69% changes of phenotype when comparing diagnosis and the first relapse, at least one LAP was constant in 92%. According to our observations, in majority of patients with AML monitoring of MRD by multiparameter flow cytometry is feasible although in some cases could present some specific difficulties owing to their immunophenotypic heterogeneity, similarity with other cell subpopulations or shifts at relapse. In conclusion, investigation of MRD should be based on the phenotypic characteristics of each subpopulation even if it is present in low frequencies.

The knowledge on the 3rd type hematogones could contribute to more precise detection of small numbers of precursor B-acute lymphoblastic leukemia.

Bone marrow hematogones (benign B-lymphocyte precursors) may cause diagnostic problems due to their morphologic and immunophenotypic similarities with neoplastic lymphoblasts. Hematogone populations in presented study containing 358 bone marrow specimens of 251 individuals always exhibited a continuous and complete maturation spectrum of antigen expression typical for normal evolution of B-lineage precursors; lacking aberrant or asynchronous antigen expression. In contrast lymphoblasts of 19 bone marrows of precursors B-ALL patients showed maturation arrest and exhibited several immunophenotypic aberrancies. Hematogones were identified by 4-color flow cytometry using optimal antibody combinations in many bone marrow samples. They were more commonly found in higher numbers in children, and there was found a general decline in hematogones with increasing age. Bone marrow hematogones were separately assessed as hematogones 1 population of early stage and hematogones 2 of mid-stage precursor B-cells, respectively. In some (about 30%) of hematogones a third type hematogones could be assessed in bone marrow samples. This small B-cell subpopulation was defined by CD10-positivity, coexpressing more mature markers CD19,CD20,CD22 and CD45bright. These cells obviously blended with those of mature B-lymphocytes (CD10-negative) on CD45/SSC, and could be better recognized on CD10-gating. Quantitative immunophenotyping of this study completed the percent antigen expression data in two main hematogone subtypes and lymphocytes in 16 bone marrow specimens and precursor B-ALL lymphoblasts in some samples. Increased information on benign B-lymphocyte precursors, especially that of existence of the 3rd type hematogones could provide a basis for better discrimination of B-leukemia cells even in a very small amounts.

The value of dot plot patterns and leukemia-associated phenotypes in AML diagnosis by multiparameter flow cytometry.

The immunophenotypic features in patients with acute myeloid leukemia (AML) were investigated at diagnosis using a wide antibody panel including progenitor-associated, myeloid and lymphoid markers in quadruple combinations. Analyzed were bone marrow samples from 37 adult and pediatric patients for exact identification of AML blasts according their localization on CD45/SSC dot plots and aberrant immunophenotypes in various subtype of AML. We found the localization of AML blasts on CD45/SSC dot plots, which in combination with immunophenotype profile of blasts allow discrimination of several AML subtypes (M0-M2, M3, M4/M5 and other types). In 27/37 AML patients (73%) at least one leukemia-associated phenotype (LAP) was found, two or more aberrancies coexisted in more than a half of them (78%). Asynchronous expression was the most frequent type of LAP (77.8%, 21/27) followed by coexpression of lymphoid-associated antigens, which occurred in 18/27 (66.7%) patients. Presented study showed that leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on typical light scatter profiles and aberrant antigen expression that could further be used for detection of minimal residual disease.

The value of multiparameter flow cytometry of cerebrospinal fluid involved by leukemia/lymphoma cells.

The usefulness of multiparameter flow cytometric (FC) analysis of cerebrospinal fluid (CSF) was evaluated in leukemia/lymphoma patients having central nervous system (CNS) involvement of the disease. In 12 specimens of 8 patients with different types of leukemia/lymphoma (one case of T-ALL, 3 cases of early B-cell ALL, one case of AML, and 3 proven or suspicious NHL cases) the presence of pathological clone in CSF has been confirmed or excluded. The phenotypic patterns of CSF cells were defined according to those of bone marrow (BM)/peripheral blood (PB) at diagnosis or during follow-up of the same patients. Furthermore, in one case of suspicious CNS infiltration of NHL, the pathological clone was characterized as a highly suspicious of solid tumor and was proved to be a lung cancer metastasis. The definition was made on the basis of CD45 (common leukocyte antigen) and other studied CD markers negativity. The exact comparison of immunophenotypic profiles of specimens from different sites (CSF, BM, PB) of the same patient has been performed and no phenotypic changes were found. In some CSF specimens, where no cells of suspicious pathological clone were detected, in 4-color analysis only normal lymphocyte population was found even in small cell samples (even if the cellularity was < than 0.3x10-6). In these populations the high values of T-cells (CD3+) predominated and the high prevalence of CD4+ over CD8+ cells, and an almost total lack of B-lymphocytes was found. Our results suggest that positive CSF immunology is a useful indicator of malignancy and reflects leptomeningeal involvement. Simultaneously we demonstrated that FC analysis of CSF in the aim to detect possible CSF seeding of leukemia/lymphoma is a reliable and quick technique.