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PubMed1993

[Immunophenotypic study of acute myeloid leukemia (AML): biological significance and prognostic implications].

The immunophenotype of 61 de novo AML referring to the Institute of Hematology of Ferrara between January 1988 and September 1992 was studied in order to evaluate the prognostic impact and the biological implications of this approach. In this study we confirm the possible aberrant expression of some antigens and the lack of a strict correlation between FAB classification and immunophenotype. The expression of the CD14 and CD34 was correlated with a worse prognosis, while the CD34+ patients further showed a lower probability to achieve a complete remission.

Aged↗
PubMed1993

[Preliminary study of immunophenotype of multiple myeloma cells].

Using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, plasma cells from multiple myeloma (MM, 23 cases), plasma cell leukemia (PCL, 2 cases) and reactive plasmacytosis (RP, 13 cases) were immunophenotyped with a panel of monoclonal antibodies (McAb). The results showed that McAbCD38 was strongly positive in high percentage of MM and RP cases and the CD9 was the next. 9/23 MM expressed CD10. Our results might indirectly support that CD10 is a malignant marker of MM with poor prognosis, a concept proposed by Durie. The results were (1) all RP but 1 acute monocytic leukemia related to RP were CD10 negative. (2) In our series 2 cases of plasma cell leukemia (PCL) expressed CD10; (3) 4 MM cases survived more than 2 years were CD10 negative. A few MM cases also expressed other surface markers of pre-B and B lymphocyte, such as CD19, CD20, CD22, HLA-DR, cytoplasmic mu chain. CD20 was positive in 4/21 MM and negative in all RP cases. 7/22 MM expressed HLA-DR, and 1/13 RP did so, among them there was a significant difference. HLA-DR seems to be another malignant marker of plasma cells. 1 MM expressed CD8, and 1 PCL highly expressed CD4 indicating PCL might be heterogeneous. Lymphoid stem cells may be involved in MM and PLC. We conclude that multiple myeloma cells have different immunophenotypes and CD10, CD20 and HLA-DR may help to differentiate MM from RP.

Antigens, CD↗
PubMed1993

Acute myeloid leukemia: correlation between purine metabolism enzyme activities and membrane immunophenotype.

A total of 34 AML patients with heterogenous age distribution (from 2 years up to 82 years) were observed. Purine metabolism enzyme activities were compared and correlated with membrane immunophenotype. Analysis of bone marrow and peripheral blood samples based on FAB criteria and immunologic phenotyping of acute myeloid leukemia (AML) provided useful--either confirmatory or contradictory-information on the distribution of M1-M6 patients demonstrating a predominance of M1+M2 and M4 groups (44% and 32.4%, respectively). In contrast, it was demonstrated that less frequent subtypes were M3 and M6 (5.9% and 2.9%, respectively). AML subtypes were correlated with expression of surface antigens detected by the following monoclonal antibodies: CD13, CD33, CDw65, CD11b, CD15, CD14, HLA-DR and CD34. On the basis of immunophenotyping we found the following characteristic markers: M1, M2-CD34, HLA-DR, CD13, CD33, CDw65; M3-CD13, CD33, HLA-DR (negative); M4, M5-CDw65, CD14, CD13, CD33 and HLA-DR. CD14 was confirmed to be a typical marker for discriminating myeloid from monocytoid FAB AML subtypes. Analysis of purine metabolism enzyme activities showed that there is a correlation between the values of adenosine deaminase and purine nucleoside phosphorylase and various immunotypes of AML. High ADA/PNP ratio (> 1.0) was found in M1, M2, M3 subtypes. It was due to the increased level of ADA activity (> 100 pkat.10(-6) cells), though these activities overlapped to a certain extent. It was shown that PNP activity simultaneously decreased. With maturation of cells within AML lineage ADA activity decreased and PNP activity increased. This corresponded with ADA/PNP ratio that was < 1.0 in cells of more mature AML subtypes. We found that the enzymatic values were characteristic mainly in cells of M5 (monocytic) AML subtype and were characterized by decreased values of ADA activity with a simultaneous increase in PNP activity. It follows from our results that ADA/PNP ratio enables to discriminate between myeloid and monocytoid subtypes of AML.

Adenosine Deaminase↗
PubMed1993

Malignant thymoma associated with T-cell lymphocytosis. A case report with immunophenotypic and gene rearrangement analysis.

Patients with malignant thymoma rarely may also have a peripheral T-cell lymphocytosis. "Lymphocyte spillover" from the thymus into the peripheral blood as well as a second, associated neoplasm (ie, T-cell chronic lymphocytic leukemia) are two hypotheses that have been proposed to explain this clinical phenomenon. We describe another patient with a lymphocyte-rich malignant thymoma associated with peripheral T-cell lymphocytosis. At the time of initial diagnosis, the patient's complete blood cell count was unremarkable. However, subsequent to the development of pulmonary metastases, the patient developed persistent lymphocytosis. The total leukocyte count ranged from 20 to 30 x 10(9)/L, 80% of these cells being lymphocytes. Immunophenotypic analysis of peripheral blood specimens from this patient proved that the circulating cells were mature, polyclonal T cells. The cells expressed the alpha/beta T-cell receptor and the pan-T-cell antigens CD2, CD3, CD5, and CD7, and were negative for both terminal deoxynucleotidyl transferase (TdT) and the CD1 antigen. A mixture of T-helper (CD4+) and T-suppressor (CD8+) cells were present in a ratio of 1:1.6. Gene rearrangement studies revealed that the T-cell receptor beta chain and the immunoglobulin heavy-chain genes were in the germline configuration. Serum samples from this patient were also analyzed for thymic hormones; the level of thymosin alpha 1 was markedly elevated, while that of thymosin beta 4 was decreased. These results effectively exclude the hypothesis that the lymphocytosis represents a second, associated neoplasm. The lymphocyte spillover hypothesis also seems unlikely (although not excluded), since the lymphocytes in lymphocyte-rich thymomas usually have an immature thymic cortical immunophenotype. Furthermore, one might expect nonspecific elevation of all thymic hormone levels with lymphocyte spillover. Thus, we suggest that the lymphocytosis results from a poorly defined immunoregulatory disorder, related to the presence of thymoma, and perhaps secondary thymic hormone imbalance.

Aged↗
PubMed1993

Hodgkin's disease following non-Hodgkin's lymphoma. A clinicopathologic and immunophenotypic study of nine cases.

We describe nine patients who initially developed non-Hodgkin's lymphoma and subsequently developed Hodgkin's disease. The median interval from the diagnosis of non-Hodgkin's lymphoma (NHL) to the diagnosis of Hodgkin's disease (HD) was 5 years (range, 2-12 years). The median age of the patients at time of diagnosis of NHL was 54 years (range, 27-81 years). Seven of nine cases (78%) of NHL were primarily nodal. According to the Working Formulation, seven NHL were follicular (two small cleaved cell, three mixed small and large cell, two large cell), one was diffuse large cell, and one was large cell immunoblastic. All NHL had histologic or immunophenotypic findings indicative of B-cell lineage. Seven of the nine patients were treated in a nonuniform manner: four with chemotherapy and three with chemotherapy and radiation therapy. At the time of HD, the median age of the patients was 59 years (range, 35-85 years). Lymph nodes were involved in all patients. Six HD biopsies were subclassified as nodular sclerosis, one as mixed cellularity, and two cases were not further subclassified. Immunophenotypic studies revealed that the Reed-Sternberg and Hodgkin cells were LeuM1 or BerH2 positive and LCA negative in eight of nine biopsies, supporting the histologic diagnosis. These results further demonstrate that patients with NHL may subsequently develop HD. The NHLs are usually of B-cell lineage. The results also emphasize the need for rebiopsy in patients with NHL who experience an apparent clinical relapse.

Adult↗
PubMed1993

Cellular drug resistance profiles that might explain the prognostic value of immunophenotype and age in childhood acute lymphoblastic leukemia.

Immunophenotype and age have prognostic value in childhood acute lymphoblastic leukemia (ALL) but how this operates is not understood. In 84 children with ALL at initial diagnosis we studied the correlation between these factors and the in vitro resistance to eight drugs, determined with the 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay. B-lineage ALL samples were classified into four differentiation stages: the CD10- proB ALL; cALL; preB ALL with cytoplasmic mu positive ALL cells; and B-ALL with surface immunoglobulin-positive (Ig+) cells. cALL and preB ALL cases have the best prognosis; proB and T-ALL cases show a worse prognosis and B-ALL the poorest prognosis. Patients aged < 18 months and > 10 years have a poor prognosis compared to patients in the intermediate age group. Our results show that cALL and preB ALL cells were the most drug-sensitive cells compared to the other phenotypes. No differences were found between cALL and preB ALL cases with the exception that preB cells were more sensitive to mustine and mafosfamide (Maf). Compared to cALL and preB ALL cases, T-ALL cases were significantly more resistant to prednisolone (Pred), daunorubicin (DNR), L-asparaginase (L-Asp), cytosine arabinoside (AraC), and Maf; proB ALL cases were more resistant to Pred, DNR, L-Asp, and 6-thioguanine. The three B-ALL cases were resistant to vincristine and DNR. Two out of three B-ALL were resistant to Pred. Compared to cells from patients aged 18 months to 10 years, cells from children < 18 months were more resistant to Pred and DNR; cells from children > 10 years were more resistant to Pred. We conclude that cellular drug-resistance patterns might at least partly explain the prognostic value of immunophenotype and age in childhood ALL.

Aging↗
PubMed1993

Expression of unusual immunophenotype combinations in acute myelogenous leukemia.

Immunophenotypes for 272 patients with acute myelogenous leukemia (AML) were analyzed using a panel of 22 antibodies. Numerical evidence for unusual coexpressions (present in normal marrow at < or = 0.1%) of surface markers on > or = 10% of the blast cells was found in 85% of all cases. Asynchronous expression of myeloid differentiation antigens occurred in 70% of the cases. Unusual coexpression of T-lymphoid, B-lymphoid, or natural killer (NK) markers with myeloid markers occurred in 38%, 13%, and 21%, respectively, of all AML cases. Two- and three-color analyses confirmed coexpression in 15 of 15 cases, and indicated that these percentages are an underestimate, because coexpression can be demonstrated in cases without numerical overlap. These data indicate that the unusual coexpression of normal differentiation antigens is a common occurrence in AML. Markers in 12 of 13 patients were similar between presentation and relapse, and in two patients, unusual phenotypes detected at first relapse were shown at second relapse, indicating these immunophenotypes are stable in the majority of AML patients. Significant correlations were found between t(8;21) cytogenetics and coexpression of CD19 with CD15 or CD34, t(9;22) and coexpression of CD19 and CD34, and t(15;17) and coexpression of CD2 and myeloid antigens. Multiparameter fluorescence analysis allows detection of unusual phenotypes when the blast counts are < 5% (classical remission). Analysis of 16 patients in remission indicated the presence of presentation phenotypes in 0.2% to 7.9% of the lymphocyte + blast light scatter region, representing 0.03% to 1.4% of the total nucleated marrow cells. Of the 16 patients with > or = 4 months follow-up after detection of these cells, 6 of 6 patients with > or = 0.2% unusual presentation phenotypic marrow cells have relapsed, while 9 of 10 patients with < 0.2% remain in remission. The detection of cells with the unusual presentation phenotype may reflect residual AML cells, and their increase may predict relapse.

Adult↗
PubMed1995

Cutaneous lymphoma-simulating Merkel cell carcinoma-molecular genetic demonstration of a clonal disease with divergent immunophenotypes.

Merkel cell carcinoma and malignant lymphoma are important differential diagnoses for undifferentiated cutaneous round cell tumors and immunohistochemistry is instrumental in their evaluation. We describe a case of a 73-year-old man who had cutaneous large cell lymphoma in the right leg (immunophenotype CD45+, CD19+, CD20+ CD22+, lambda clonal, cytokeratin-, NSE-) and lymphoma in left leg simulating Merkel cell carcinoma showing absence of leukocyte antigens (CD45-, CD20-, no light chains) and focal expression of keratin and NSE. However, analysis of polymerase chain reaction amplification products of DNA extracted from both lesions showed two amplifiable sharp bands indicating clonal rearrangements of both alleles of the immunoglobulin heavy chain. Cloning and sequencing of the products from left and right leg lesions showed either 100% homology (one band), or close similarity (the other band), indicating that both tumors were derived from the same B-cell lymphoma clone. This case shows the value of polymerase chain reaction and sequencing in analyzing the ultimate nature of lymphoproliferations and illustrates the potential limitations of immunophenotyping.

Aged↗
PubMed1994

Quality control in immunophenotyping: U.S. efforts to establish common methodology and their impact. American Society for Histocompatibility and Immunogenetics.

Several organizations in the United States have recently been involved in the quality assurance of immunophenotyping using flow cytometry. These activities include publication of guidelines for immunophenotyping, particularly in human immunodeficiency virus infection, performance evaluation and proficiency testing programs, and training and continuing education programs. Through questionnaires and results from performance evaluation programs, variability in test results has been decreasing the last few years, and more laboratories are beginning to use the same specimen processing and data analysis procedures.

Flow Cytometry↗
PubMed1996

Characterization of postcardiac transplant lymphomas. Histology, immunophenotyping, immunohistochemistry, and gene rearrangement.

OBJECTIVE: Between 2% and 9% of cardiac transplant recipients develop posttransplant lymphoproliferative disease, which includes lymphomas. These are usually aggressive Epstein-Barr virus-associated B-cell proliferations similar to those seen in other immunodeficiency states. A retrospective pathologic study of the tumor tissue from 21 cardiac transplant recipients with posttransplant lymphoproliferative disease was undertaken. DESIGN: Tumor histology, immunohistochemistry, immunophenotyping, and DNA analysis for clonal gene rearrangement and the presence of Epstein-Barr virus DNA were performed. PATIENTS: The mean patient age was 53.4 +/- 10.2 years (range 33-67 years); 33% of the patients were alive at the time of study. RESULTS: Histologically, the samples comprised one Burkitt's lymphoma, three diffuse mixed lymphomas, eight diffuse large-cell lymphomas, and nine immunoblastic lymphomas. Thirteen (93%) of 14 samples were infiltrated by small reactive T cells; five of the lymphomas qualified as T-cell rich. Of 14 cases studied, 12 had clonal immunoglobulin gene rearrangements, 1 had oligoclonal bands, and 1 exhibited only a germline pattern. The B cells were CD10+, CD19+, and CD20+, and the reactive T cells were CD2+, CD3+, CD5+, CD7+, CD8+, and CD57+ by immunophenotyping. CONCLUSIONS: In this patient series, morphologically aggressive lymphomas and disseminated disease occurred early as well as late after transplantation. Most of the tumors showed a reactive T-cell component, which may represent a host attempt at controlling the B-cell proliferation.

Adult↗
PubMed1996

[The diagnostic significance of blast immunophenotype assay in patients with acute leukemia].

A detailed analysis of immunophenotypes of 120 adult newly diagnosed patients with acute leukaemias was performed. Using the immunopheno-typing, it was possible to defined 96,7% of leukemia cases. The proportion of leukemia subtypes was: AML in 62,5%, ALL in 32,5%, acute biphenotypic leukaemia 1,7% and acute undifferentiated leukaemia (AUL) in 3,3%. The diagnosis initially made according FAB criterias in 12,5% cases after the immunophenotyping was verified. Above analyses showed the existence of the atypical blasts phenotypes in 31%: co-expression of CD 19, CD2 and CD7 markers in AML, co-expression of CD 33 marker in ALL, co-expression of CD 19 marker in T cell ALL and biphenotypic (mixed-lineage) leukaemia.

Adolescent↗
PubMed1996

Immunophenotype, mRNA expression, and gene structure of p53 in Wilms' tumors.

The anaplastic variant of Wilms' tumor is regarded as the result of tumor progression of the more common classic Wilms' tumor. Anaplasia is rare and occurs in only 4.5% of tumors. Three anaplastic Wilms' tumors in our collection were examined in comparison with 10 classic Wilms' tumors for p53 expression by immunohistochemical techniques and Northern blot analysis, and their p53 gene structure was determined by single-stranded conformation polymorphism and sequence analysis. All classic tumors contained a wild-type p53 gene and expressed marginal levels of protein as expected for normal p53. In contrast, three out of three anaplastic tumors demonstrated evidence of p53 alterations consistent with a role of p53 in tumor progression. One of the anaplastic mutants (W4) did not express protein or p53 mRNA. Its apparently normal immunophenotype would have disguised the mutated nature of p53, which was detected only by mRNA and sequence analysis. The second anaplastic mutant (W16) contained normal levels of p53 mRNA, but overexpressed the protein in a fashion typical of mutated p53. The same immunophenotype was displayed by fixed primary tissue of the third anaplastic tumor (W17), but p53 mutation could not be confirmed for lack of frozen primary material. The present study emphasizes the importance of p53 function in the anaplastic progression of Wilms' tumor and the risk of error in assessing normal p53 function using a single methodology.

Adolescent↗
PubMed1996

Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery.

We prospectively studied immune reconstitution in 102 children who underwent T-lymphocyte depleted bone marrow transplants using either closely matched unrelated donors or partially matched familial donors by assaying total lymphocyte counts (TLC), T-cell subsets, B cells, and natural killer cells. TLC, CD3+, and CD4+ T-cell counts remained depressed until 2 to 3 years posttransplant, whereas CD8+ T-cell counts normalized by 18 months, resulting in an inverted CD4:CD8 ratio until 12 months posttransplant. Although the percentage of NK cells was elevated early posttransplant, their absolute numbers remained normal. CD20+ B cells were depressed until 12 to 18 months posttransplant. Factors affecting immunophenotypic recovery were analyzed by nonparametric statistics. Younger patients tended to have higher TLC posttransplant. Higher marrow cell doses were not associated with hastened immunophenotypic recovery. Graft-versus-host disease (GVHD) and/or its treatment significantly delayed the immune reconstitution of CD3+, CD4+, and CD20+ cells. The presence of cytomegalovirus was associated with increased CD8+ counts and a decrease in the percentages of CD4+ and CD20+ cells.

Adolescent↗
PubMed1996

Role of immunophenotyping in characterisation of blast crisis of chronic myeloid leukemia--a study of 25 cases.

The blast cell populations of 25 patients of chronic myeloid leukemia in blast crisis (CML-BC) were studied for morphological, cytochemical and immunophenotypic features. The patients were divided into 6 broad groups based upon the pattern of surface marker positivity-myeloblastic, mixed myeloblastic, megakaryoblastic, mixed lineage, lymphoid and undifferentiated blast crisis. Myeloperoxidase (MPO), Sudan Black B (SBB) and Chloroacetate esterase (CAE) stains showed 100% specificity for the myelomonocytic lineage but the sensitivity was low. Periodic acid Schiff (PAS) stain was neither specific nor sensitive for the lymphoid lineage. Immunophenotyping as compared to morphologic and cytochemical assessment, was seen to be most useful for assigning a lineage to leukemic cells in CML-BC.

Adult↗
PubMed1996

Transferrin receptor expression by blast cells in acute lymphoblastic leukemia correlates with white cell count & immunophenotype.

Transferrin receptor (TR) expression by blast cells in 127 cases of acute lymphoblastic leukemia (ALL) at presentation and 19 cases at relapse was examined using three anti-TR monoclonal antibodies to find its correlation with prognostic features such as the total leucocyte count (TLC), the morphology of blast cells and their cytochemical and immunophenotypic properties, as well as age and sex of the patients. Blasts in 62 per cent of thymic (T) ALL cases at presentation showed significant TR expression as compared to only 10.9 per cent in common ALL (CALL) (P < 0.001). This differential expression of TR was also observed among cases with > 50 x 10(9)/l TLC, while in cases with < 50 x 10(9)/ l TLC no such pattern was observed (30% TR positivity in T-ALL vs 20% TR positivity in non-T-ALL). Furthermore, the percentage of TR positive blasts was significantly higher (P < 0.005) in cases with > 50 x 10(9)/l TLC as compared to those with < 50 x 10(9)/l (48.3-54.4% vs 24.9-28.8%). In contrast to CALL cases at presentation, those at relapse showed a very high TR positivity (54-66%), similar to the T-ALL cases (53-84%). This suggests a high proliferative rate of blast cells in ALL at relapse, irrespective of its immunophenotype. There was no correlation of TR expression with blast cell morphology (FAB L1 vs L2), their cytochemical properties and sex of the patients. However, a significantly higher incidence of TR positivity was observed in patients above 10 yr of age compared to those below 10 yr (47% vs 15%; P < 0.001). The incidence of T-ALL was also significantly higher in the former group (56%) compared to the latter (33%) (P < 0.005). Our data suggest that by virtue of its association with features of poor prognosis, e.g., age above 10 yr, expression of thymic markers, high leucocyte count and disease relapse, TR expression by blast cells in ALL could serve as a biological marker of poor prognosis.

Humans↗
PubMed1996

Refractory T cell acute lymphoblastic leukemia with unusual karyotype and interesting immunophenotype.

Karyotype, immunophenotype, and molecular studies are important in the evaluation of Acute Lymphocytic Leukemia as these data provide diagnostic as well as prognostic information. We present a case of acute lymphoblastic leukemia with unusual cytogenetics, 45,XY,i(7q),der(9)t(3;9)(q12;p22),del(12)(p12), :der(18)t(3;18)(p14;q22),-3. This karyotype is hypodiploid, showing loss of chromosome 3, a very rare occurrence. Hypodiploidy and translocations are suggestive of a poor clinical outcome. Cytogenetics also showed a chromosome 12p deletion which has been implicated in the oncogenesis of some acute leukemias. Immunophenotype by flow cytometry was positive for CD7 and CD10, T, and precursor B cell markers respectively. Given the specificity of CD7 for T cell processes, it was felt that the flow cytometry was more suggestive of a T cell process. Gene rearrangement studies showing a T cell receptor rearrangement helped confirm the T cell lineage of this malignancy. Hypodiploidy and T cell phenotype are indicators of poor prognosis. Interestingly this patient was refractory to two conventional chemotherapeutic protocols and finally responded to an unconventional protocol of high dose Ara C, etopside, and L asparaginase.

Antigens, CD↗
PubMed1996

The role of cytology, cytochemistry, immunophenotyping and cytogenetic analysis in the diagnosis of haematological neoplasms. General Haematology Task Force of the BCSH.

Cytology, cytochemistry, immunophenotyping and cytogenetic analysis have specific roles in the diagnosis and management of various haematological neoplasms. Careful examination of Romanowsky-stained films of blood and bone marrow is fundamental in all haematological diagnosis and, when considered together with clinical and haematological features, indicates which of the more specialized techniques are most likely to be useful. The major role of cytochemistry is in the diagnosis of acute myeloid leukaemia and the myelodysplastic syndromes. The major role of immunophenotyping is in the diagnosis of the chronic lymphoproliferative disorders and of acute leukaemia which is not obviously myeloid. Cytogenetic analysis has a role in confirming the diagnosis of chronic granulocytic leukaemia and gives important supplementary information in the acute leukaemias and the myelodysplastic syndromes.

Acute Disease↗