Cellular phenotypes of normal and leukemic hemopoietic cells determined by analysis with selected antibody combinations

Individual leukemic cells and the corresponding rare normal cell types in nonleukemic bone marrow were analyzed with various combinations of antisera (labeled with different fluorochromes: TRITC and FITC). Double staining for membrane Ia-like molecules (TRITC) and nuclear terminal transferase (FITC) was a very useful combination that distinguished common non-T, non-B ALL (Ia+,TdT+) and thymic ALL (Ia-,TdT+) from the rare cases of B ALL (Ia+,TdT-) and from AML (frequently Ia+, TdT-; in some cases Ia-, TdT-). Additional antisera (such as anti-ALL, anti- HuTLA, anti-immunoglobulin reagents, etc.) confirmed the diagnosis and further characterized the leukemic blasts. Ia+,TdT+ cells could be observed in low numbers in normal and nonleukemic regenerating marrow and were probably normal precursor cells; this reagent combinations was, therefore, not useful for monitoring residual non-T, non-B ALL blasts in treated patients. Other marker combinations detecting pre-B ALL blasts (double staining for cytoplasmic IgM and nuclear TdT) and Thy-ALL blasts (HuTLA+,TdT+) were, however, virtually leukemia specific in the bone marrow and could be used to effectively monitor residual leukemic cells throughout the disease. These combined single-cell assays are not only economical and informative but are also important for assessing the heterogeneity of leukemia and for standardizing new mouse or rat monoclonal antibodies for diagnosis.     

Translocation 4; 11 in acute lymphoblastic leukemia: clinical characteristics and prognostic significance

Banded bone marrow chromosome analyses have been done on 83 unselected patients with acute lymphoblastic leukemia (ALL). Seven patients, all with non-T, non-B ALL, had a translocation involving the long arms of chromosomes 4 and 11. Five of these patients, 4 children and 1 adult, were first studied at diagnosis, and the t(4;11) (q21;q23) was the only karyotypic abnormality. All 5 presented with a marked leukocytosis (greater than 150 X 10(9)/liter). Four of these 5 patients achieved a complete remission following the same intensive treatment regimen; however, remission duration and survival were very short (medians 2.5 and 8 mo, respectively). The fifth patient is currently receiving induction chemotherapy. The remaining 2 patients, both adults, were studied in relapse only, and had other karyotypic abnormalities in addition to the t(4;11). One of these relapse patients was a female whose clinical presentation and course were similar to those above. The last patient was a male who presented with a leukocyte count of 7 X 10(9)/liter and maintained an initial complete remission for 37 mo. Our data suggest that patients who have a t(4;11) (q21;q23) at the time of diagnosis of ALL have a poor prognosis with conventional therapy and require a new therapeutic approach.     

Change in Cellular Phenotype from Lymphoid to Erythroid in a Case of ALL

Acute leukaemia was diagnosed in a 3-month-old boy. Initial blood leucocyte count was 97 × 10⁹/1, 92% of the cells were morphologically small lymphoblasts which according to surface marker analysis were of the common ALL (non-T non-B) type. Remission was achieved with initial treatment. During relapse 5 months later a morphologically different leukaemic cell appeared in the bone marrow, blood and liquor. This cell type which dominated during the terminal stage of the disease was larger and had an abundant bosophilic cytoplasm which contained PAS-positive granulae. Sudan, myeloperoxidase and benzidine staining gave negative results. Neither T nor B lymphocyte markers were seen but a strong surface expression of glycophorin A was found by indirect immunofluorescence with specific rabbit anti-glycophorin A antiserum. The cells showed a unique surface glycoprotein pattern. Bone marrow karyotype analysis gave in about 80%: 47,XY, + 8,t(4:11) indicating that this blast cell represented a malignant clone. As glycophorin A is expressed exclusively on erythroid cells and their precursors, this finding indicates a change from a ‘lymphoid’ to an erythroid phenotype of the leukaemic cells during the course of the disease.     

Lineage specific classification of leukaemia: results of the analysis of sixty cases of childhood leukaemia

In addition to conventional morphological, histochemical and immunological marker studies, cells from 60 children with leukaemia were further analysed using the Southern blot hybridization technique to look at differences in the organization of immunoglobulin (Ig) genes. Of the 60 patients studied by conventional means, 47 were diagnosed as acute lymphocytic leukaemia (ALL) and 13 as non-lymphocytic leukaemia. Seven patients were initially classified as T ALL and 40 as non-T, non-B ALL. Further subclassification of the 40 patients with non-T, non-B ALL indicated three pre-B ALL and 29 patients diagnosed as common ALL, expressing Ia and CALLA antigens. All 29 patients with common ALL demonstrated C mu gene rearrangements with or without light chain (kappa and lambda) genes rearrangement. Based on the developmental hierarchy of Ig gene rearrangement, it was possible to further subclassify the patients with common ALL into different stages of B cell development. Eight (of the 40) patients with non-T, non-B ALL were identified as CALLA- but further analysis indicated T-lineage origin in two patients and three patients were reclassified as acute undifferentiated leukaemia (AUL). C mu gene rearrangements were detected in two patients with T ALL, two patients with AUL and one patient with acute myelogenous leukaemia (AML). In contrast to the patients with common ALL, Ig gene rearrangement observed in these non-B-lineage cells was restricted to a single C mu gene while retaining germ-line configuration of the other allele of the C mu gene and both light chain genes.     

Ph1-positive acute lymphoblastic leukemia with a 14q+ chromosome abnormality

A 13-yr-old Japanese female with acute lymphoblastic leukemia (ALL) that was associated with a Philadelphia chromosome (Ph1) as well as a 14q+ chromosome abnormality is reported. The cell surface phenotype of leukemic cells was determined to be non-T, non-B ALL on the basis of positive Ia-like antigen, terminal deoxynucleotidyl transferase activity, and lack of receptors for sheep erythrocytes, surface immunoglobulin, or intracytoplasmic mu-chain immunoglobulin. The combination of both a Ph1 and a 14q+ has not been reported previously in patients with ALL.     

Investigation of the CD10 (cALLA) negative acute lymphoblastic leukaemia: further description of a group with a poor prognosis

The absence of CD10 (cALLA) in non-T non-B acute lymphoblastic leukaemia (ALL) is usually considered to be of adverse prognostic significance. From a large multicentre series of phenotyped ALL, we have identified a group of 23 non-T non-B ALL where blast cells were CD10 negative and CD19 positive. Class II antigens were present in 80% and C19 in 70%. Eight patients had successful karyotype analysis at diagnosis, and an additional patient at first relapse. Seven of these karyotypes showed a (4;11) (q21;q23) translocation. Most of the patients (70%) were young females, and they often presented with organomegaly. Six patients were less than 1 year old. The white cell count was over 100 x 10(9)/l in 48% of the cases. The FAB type was L2 in 56% of the patients. The most striking features were the poor response to therapy and survival. Six patients never attained complete remission and nine patients relapsed, most of them during the first year after diagnosis. Allogeneic bone marrow transplantation was performed in three children, of whom two are still alive 2 years after diagnosis. This study emphasizes the prognostic value of immuno-phenotypic and karyotypic investigations of ALL.     

Prognostic significance of terminal transferase activity in childhood acute lymphoblastic leukemia: a prospective analysis of 164 patients

Whether the level of terminal deoxynucleotidyl transferase (TdT) activity in mononuclear cells from bone marrow and peripheral blood has prognostic significance has been analyzed prospectively in 164 children with T and non-T, non-B marked acute lymphoblastic leukemia (ALL). TdT was measured at diagnosis to assess its value as a predictor of duration of remission and length of survival. It was measured repeatedly during remission to assess whether it could predict relapse. Ninety-seven percent of the children achieved a complete remission of their disease, and 40% relapsed during the study. The level of TdT activity in blasts at diagnosis varied 1000-fold from patient to patient. There was no statistically significant relationship between TdT activity in cells at diagnosis and the achievement of complete remission, the duration of remission, or length of survival. TdT activity was significantly increased in the bone marrow of 65% of patients at the time of marrow morphological relapse, but was rarely increased in marrow from patients with isolated testicular or central nervous system relapse. Wide fluctuations in TdT activity were characteristically seen in mononuclear cells from the marrow and peripheral blood of patients with ALL at all stages of their disease. An isolated high value of TdT activity in the bone marrow or peripheral blood cannot be taken as evidence of impending relapse. Quantitative measurements of TdT activity alone on mononuclear cells from bone marrow and peripheral blood are helpful in differential diagnosis, but cannot guide therapy of children with ALL.     

Acute lymphoblastic leukemia with pre-B-cell characteristics.

Blast cells from 6 of 50 patients with acute lymphoblastic leukemia (ALL) displayed intracytoplasmic mu chains in the absence of detectable light chains and surface immunoglobulins. These cells also expressed lalike and common ALL antigens. Terminal deoxynucleotidyltransferase was detectable in 2 of 5 cases tested. These blast cells are probably related to early B-cell precursors (pre-B cells). In 4 of 6 cases the disease had a tumoral presentation; the prognostic significance of this new subgroup, which accounts for 20% of patients with non-T non-B ALL, remains to be established.     

Phenotypic analysis of acute lymphoblastic leukemia (ALL) cells which are classified as non-T non-B and negative for common ALL antigen

When phenotypic marker analysis of acute lymphoblastic leukemia (ALL) cells (102 cases) was performed, a group of ALL cells (15 cases) classified as non-T non-B, and negative for common-ALL antigen (CALLA) was characterized in a focused manner. "Non-T non-B" was defined as negative for T cell properties such as E-rosetting or reactivity with anti-human T-cell monoclonal antibodies (T101, WT1), and absence of any B-cell characteristics (cell surface and/or cytoplasmic immunoglobulin and reactivity with B1 monoclonal antibody). Despite their marked heterogeneity, CALLA(-) non-T non-B ALL cells revealed three different phenotypic patterns in terms of presence of terminal deoxynucleotidyl transferase (TdT) and of reactivity with antimyeloid (MCS1) or myelomonocyte (MCS2 and OKM1) monoclonal antibodies. Four of 15 cases reacted with some myeloid-specific antibodies, but were negative for TdT. Six cases had both MCS2 antigen and TdT. The remaining five cases expressed no myeloid antigens, but were positive for TdT with some exceptions. These findings showed that acute leukemias with myeloid antigens might be involved in CALLA(-) non-T non-B ALL having no relationship to the presence of TdT, and, furthermore, that the blasts with simultaneous expression of TdT and myeloid-specific antigen (MCS2) might represent an immature stage in hematopoietic differentiation closely corresponding with the bifurcation of the lymphocyte/myeloid pathway. Alternatively, only five cases remained "unclassified leukemia." We therefore think that the detailed examination of CALLA(-) non-T non-B ALL cells using myeloid specific antibodies is helpful in clarifying the characteristics of myeloid precursors and the common bipotential stem cell of lymphoid and myeloid progenitors.     

Philadelphia chromosome positive childhood acute lymphoblastic leukemia: clinical and cytogenetic characteristics and treatment outcome. A Pediatric Oncology Group study

Among 3,638 children with acute lymphoblastic leukemia (ALL) entered on Pediatric Oncology Group (POG) protocols between June 1981 and April 1989, successful cytogenetic studies were available for 2,519, 58 (2.3%) of which had the Philadelphia (Ph) chromosome detected. Features associated with the presence of the Ph chromosome were high leukocyte count (median, 33 x 10(9)/L), older age median, 9.6 years), a higher proportion of French-American-British L2 morphology, and a lower frequency of mediastinal mass. Immunologic marker studies at diagnosis in 56 Ph+ cases identified early pre-B ALL in 42 cases (75%), pre-B-cell in 9 (16%), and T-cell in 5 (9%). This distribution is similar to that found in Ph+ ALL. Intensive multiagent chemotherapy induced complete remissions in only 78% of eligible Ph+ patients compared with 96% of those without an identified Ph chromosome (P less than .001). Of 44 eligible Ph+ patients treated on POG frontline protocols for children with non-T, non-B-cell ALL, 27 have failed therapy, compared with 520 of 1,892 without an identified Ph chromosome (logrank P less than .001). Ph+ ALL is an aggressive form of acute leukemia that frequently presents in older children with a high leukocyte count, FAB L2 morphology, and a pseudodiploid karyotype, and becomes multidrug-resistant early. Thus, Ph+ cases require early identification to permit treatment with intensive induction regimens and experimental approaches such as bone marrow transplantation.     

Terminal Transferase Enzyme Assay and Immunological Membrane Markers in the Diagnosis of Leukaemia: a Multiparameter Analysis of 300 Cases

Multiparameter analyses have been carried out with recently developed enzyme and membrane markers in 300 patients with various leukaemias including ALL, AML, but excluding Ph¹ positive leukaemias.
TdT enzyme levels were particularly valuable in the differential diagnosis of adult acute lymphoid and myeloid leukaemias. The levels were raised in 108 (94%) of the 115 patients who were considered to be non-T, non-B ALL on membrane marker and morphological analysis; all seven cases giving negative TdT results in this group were young children. Unexpectedly high levels were seen only in three (4.1%) of 73 cases of acute myeloid leukaemia verified by histochemistry and membrane markers. Anti-ALL serum was a most useful reagent in childhood leukaemias but blasts from 19 patients (10% of childhood ALL cases and 29% of adult ALL cases) failed to react with the serum in spite of TdT positivity. Strongly ALL⁺ blasts were seen only in non-T, non-B ALL and some undifferentiated leukaemias. Weakly ALL⁺ blasts were seen in seven of 32 cases of thymic ALL (Thy-ALL) but in other respects these blasts expressed Thy-ALL features, such as strong reactivity with anti-T cell (HuTLA) serum, negativity with anti-Ia-like serum and raised TdT. The combination of tests was particularly useful in 32 cases of undifferentiated leukaemia: in 10 of these cases TdT positivity indicated the probable 'lymphoblast', nature of blast cells: the remaining 22 cases remained unclassifiable with the markers used. The analysis revealed other interesting variant forms of leukaemias.     

Ultrastructural study of acute lymphocytic leukemia: comparison with immunologic studies.

Leukemic cells from 29 cases of acute lymphocytic leukemia (ALL), studied for T and B cell markers by the use of sheep erythrocyte rosetting and surface immunoglobulin determinations, were examined by electron microscopy. The majority of patients (76%) were found to have non-T, non-B neoplasms composed predominantly of relatively small, inactive-appearing cells with frequent nuclear folds. T cell cases (21%) were associated with mediastinal masses and were predominantly composed of large, active-appearing cells with nuclear irregularity and little rough endoplasmic reticulum. One case of B cell origin was not morphologically distinct from the non-T, non-B cell cases.     

Immunological subclassification of non-T, non-B acute lymphoblastic leukaemia in childhood

41 cases of non-T, non-B acute lymphoblastic leukaemia (ALL) were classified by immunological criteria using a panel of monoclonal antibodies against common ALL antigen (cALLa), p24, p20 and HLA-DR antigens. Out of 41 cases, cALLa, p24, p20 and HLA-DR antigens were positive in 31 cases, 34 cases, 35 cases and 41 cases, respectively. 4 cases expressing cytoplasmic mu heavy chains were included in the group of common ALL. We demonstrated that a majority of non-T, non-B ALL would be derived from B-lineage cells. The expression of p24 and p20 was followed by the expression of cALLa and the synthesis of cytoplasmic immunoglobulin. These items of evidence support the idea that the expression of cALLa, p24, p20 and HLA-DR antigens on ALL cells would be universal in the USA, Europe and Japan. Although morphologically identical, non-T, non-B ALL cases could be subdivided into phenotypically-defined subgroups on the basis of cALLa, p24, p20 and HLA-DR antigens.     

Rearrangement of the T cell receptor gamma-chain gene in childhood acute lymphoblastic leukemia

We analyzed rearrangements of the T cell receptor gamma-chain (T gamma) gene as well as rearrangements of the T cell receptor beta-chain (T beta) gene and immunoglobulin heavy-chain (IgH) gene in 68 children with acute lymphoblastic leukemia (ALL). All 15 patients with T cell ALL showed rearrangements of both T beta and T gamma genes. Twenty-four of 53 non-T, non-B ALL patients (45%) showed T gamma gene rearrangements and only 14 of these also showed T beta gene rearrangements. Only a single patient rearranged the T beta gene in the absence of T gamma gene rearrangement. The rearrangement patterns of the T gamma gene in non-T, non-B ALL were quite different from those observed in T cell ALL, as 20 of 23 patients retained at least one germline band of the T gamma gene. In contrast, all T cell ALL patients showed no retention of germline bands. These data indicate that rearrangement of the T gamma gene is not specific for T cell ALL. Further, the results also suggest that T gamma gene rearrangement precedes T beta gene rearrangement. The combined analysis of rearrangement patterns of IgH, T beta, and T gamma genes provides new criteria for defining the cellular origin of leukemic cells and for further delineation of leukemia cell heterogeneity.     

Immunological reclassification of 22 children with a former diagnosis of non-T, non-B ALL

Stored peripheral blood or bone marrow mononuclear cells from 22 pediatric patients with verified acute lymphoblastic leukemia (ALL) previously classified as non-T, non-B ALL were re-investigated by flow cytometric analysis by means of a panel of B cell-specific and -associated monoclonal antibodies (moabs) using a new analytical method described by Platz et al, the so-called Delta Channel Value method. All 22 patients were immunologically re-characterized as pre-B ALL. The reproducibility between the first (acute) and subsequent re-analysis was almost complete. 20 of the tumor cell populations could be assigned to the B cell differentiation scheme recently proposed by Nadler et al. This scheme operates with four stages of pre-B cell differentiation and each stage is defined by the expression of one to four of the following markers: HLA-DR, CD19, CD10 and CD20. Two additional markers, CD24 and CD22, were investigated in our study and allowed further subdivision of the four subgroups proposed by Nadler et al. The composition of a panel of moabs for routine classification of pre-B ALL is proposed.     

Leukaemia-associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was <1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of >1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells >1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.     

Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6.

In this study, a monoclonal antibody (mAb) termed SN6 was generated by immunizing a mouse with a non-T-cell leukemia antigen preparation isolated from cell membranes of leukemia cells derived from a patient (FJ) with non-T/non-B-cell-type acute lymphoblastic leukemia (ALL). SN6 was tested against a variety of cultured and uncultured human cell specimens by using a sensitive cellular radioimmunoassay. Among the 26 cultured malignant and nonmalignant cell lines tested, SN6 reacted with all of the 6 leukemic non-T/non-B (including pre-B)-cell lines tested--i.e., KM-3, NALM-16, REH, NALL-1, NALM-1, and NALM-6. Of these cell lines, 5 were derived from individual patients with ALL; the remaining 1 was from a patient with chronic myelocytic leukemia in blast crisis. In addition, SN6 reacted with 3 of 3 leukemic myelo-monocytic cell lines tested--i.e., ML-2, HL-60, and U937. SN6 did not react with any other cell lines. A consistent result was obtained with 42 fresh (uncultured) cell specimens derived from individual patients with several different types of leukemias. SN6 reacted with 11 of 16 non-T/non-B (including pre-B)-cell ALL specimens. In addition, it reacted with various myelo-monocytic leukemia cell specimens to various degrees. SN6 did not show a significant reaction with normal peripheral blood cells tested, which included B cells, T cells, granulocytes, monocytes, and erythrocytes. However, it reacted with a small population (approximately 1% as determined by immunofluorescence staining) of normal bone marrow cells. The approximate molecular mass of the glycoprotein antigen defined by SN6 was determined to be 160,000 by radioimmunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Only one component of 80,000 daltons was formed upon reduction of the 160,000 molecular mass antigen. Therefore, this antigen is apparently a homodimer of a 80,000-dalton subunit. This conclusion was further corroborated by two-dimensional gel analysis, which showed a single well-defined spot for the reduced antigen. We designate this distinct human leukemia-associated cell surface antigen "GP160."     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

Summary bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph¹)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged J_H genes. On analysis of molecular events associated with the Ph¹ chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph¹-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph¹-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph¹-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.     

The genotype and phenotype of T cell and non-T, non-B acute lymphoblastic leukemia

We used Southern blot analysis to investigate the T cell receptor and immunoglobulin (Ig) genes in tumor DNA derived from 13 patients with acute lymphoblastic leukemia (ALL). Rearrangement of both alleles of the T cell receptor beta chain gene was demonstrated in the three individuals with T cell ALL, while the Ig genes remained in germline configuration. In contrast, in nine of ten patients with non-T, non-B ALL (seven common ALL antigen [CALLA]-positive), Ig genes were rearranged while the T cell receptor genes were intact: the noteworthy exception was a CALLA-positive individual in whom one allele of the T cell receptor was rearranged and in whom a minor rearranged Ig band was detected as well. This exception indicates that CALLA-positive non-T, non-B ALL includes proliferations that have undergone an initial genetic step toward T cell differentiation. With probes now available for both the Ig and T cell receptor genes, analysis of genomic tumor DNA is useful in all variants of ALL.