Allogeneic hemopoietic stem cell transplantation in solid tumors.

The existence of a graft versus tumor (GVT) effect of donor-derived T cells after allogeneic hematopoietic stem cell transplantation is well established as a critical component for the success of the procedure in several hematologic malignancies. Although it has been suggested that a GVT effect might also be generated in patients affected by refractory solid tumors, the morbidity of conventional allogeneic hematopoietic stem cell transplantation has limited its investigation in these diseases. Recently introduced allogeneic nonmyeloablative regimens have greatly decreased morbidity and mortality related to transplants which retain a powerful GVT. On this basis, it has become possible to explore the existence of alloreactivity toward solid tumors. The present article reviews the early clinical results of this novel immunotherapeutic approach for solid tumors.

Prenatal diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies.

In the present study, we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women, 13 non-pregnant women who had given birth to male offsprings, 12 women who had never been pregnant, and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown "in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies, it does not seem to affect this new system of enrichment, culture, and FISH analysis of CD34+ fetal stem cells.

Interphase FISH for Y chromosome, VNTR polymorphisms, and RT-PCR for BCR-ABL in the monitoring of HLA-matched and mismatched transplants.

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.

A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells.

A new monoclonal antibody (MUM1p) was used to study the cell/tissue expression of human MUM1/IRF4 protein, the product of the homologous gene involved in the myeloma-associated t(6;14) (p25;q32). MUM1 was expressed in the nuclei and cytoplasm of plasma cells and a small percentage of germinal center (GC) B cells mainly located in the "light zone." Its morphologic spectrum ranged from that of centrocyte to that of a plasmablast/plasma cell, and it displayed a phenotype (MUM1(+)/Bcl-6(-)/Ki67(-)) different from that of most GC B cells (MUM1(-)/Bcl-6(+)/Ki67(+)) and mantle B cells (MUM1(-)/Bcl-6(-)/Ki67(-)). Polymerase chain reaction (PCR) analysis of single MUM1(+ )cells isolated from GCs showed that they contained rearranged Ig heavy chain genes with a varying number of V(H) somatic mutations. These findings suggest that these cells may represent surviving centrocytes and their progeny committed to exit GC and to differentiate into plasma cells. MUM1 was strongly expressed in lymphoplasmacytoid lymphoma, multiple myeloma, and approximately 75% of diffuse large B-cell lymphomas (DLCL-B). Unlike normal GC B cells, in which the expression of MUM1 and Bcl-6 were mutually exclusive, tumor cells in approximately 50% of MUM1(+) DLCL-B coexpressed MUM1 and Bcl-6, suggesting that expression of these proteins may be deregulated. In keeping with their proposed origin from GC B cells, Hodgkin and Reed-Sternberg cells of Hodgkin's disease consistently expressed MUM1. MUM1 was detected in normal and neoplastic activated T cells, and its expression usually paralleled that of CD30. These results suggest that MUM1 is involved in the late stages of B-cell differentiation and in T-cell activation and is deregulated in DLCL-B. (Blood. 2000;95:2084-2092)

Lymphomas expressing ALK fusion protein(s) other than NPM-ALK.

The tumor cells in ALK-positive lymphoma ("ALKoma") usually express the product of the NPM-ALK chimeric gene, generated by the t(2;5) chromosomal translocation. However, 10% to 20% of ALK-positive lymphomas express ALK fusion protein(s) other than NPM-ALK, and in this report, we describe the immunohistologic and clinicopathologic features of 15 such cases. The absence of the NPM-ALK fusion gene was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in 8 cases and by fluorescence in situ hybridization (FISH) analysis in a further 2 cases. In each case, ALK staining was restricted to the cytoplasm and the N-terminus of NPM to the nucleus (contrasting with lymphomas expressing NPM-ALK in which cytoplasmic as well as nuclear labeling is seen). However, in the course of screening 53 ALK-positive lymphomas, 2 biopsies were found that had a "cytoplasm-only" ALK staining pattern but that nevertheless were shown to carry the (2;5) (by NPM staining and RT-PCR). The 15 cases resembled typical NPM-ALK-positive lymphomas in that all were of T or null phenotype, usually occurred in young male patients, and frequently presented with advanced disease associated with systemic symptoms and extranodal involvement. Moreover, their prognosis was excellent and indistinguishable from that of classical t(2;5)-positive tumors, but was clearly different from that of ALK-negative anaplastic large-cell lymphomas. These results suggest that lymphomas carrying variants of the NPM-ALK fusion protein can be detected by immunostaining for ALK and NPM and also that they can be grouped with classical t(2;5)-positive tumors as a single entity (ALK-positive lymphoma or "ALKoma") that shows a better prognosis than ALK-negative anaplastic large-cell lymphoma.

ALK+ lymphoma: clinico-pathological findings and outcome.

A distinct pathologic entity (ALK+ lymphoma) that is characterized by expression of the anaplastic lymphoma kinase (ALK) protein has recently emerged within the heterogeneous group of CD30(+) anaplastic large-cell lymphomas. Information on clinical findings and treatment outcome of ALK+ lymphoma is still limited, and no data are available concerning the value of the International Prognostic Index when applied to this homogeneous disease entity. To clarify these issues, a recently developed monoclonal antibody ALKc (directed against the cytoplasmic portion of ALK) was used to detect expression of the ALK protein in paraffin-embedded biopsies from 96 primary, systemic T/null anaplastic large-cell lymphomas, and the ALK staining pattern was correlated with morphological features, clinical findings, risk factors (as defined by the International Prognostic Index), and outcome in 78 patients (53 ALK+ and 25 ALK-). Strong cytoplasmic and/or nuclear ALK positivity was detected in 58 of 96 ALCL cases (60.4%), and it was associated with a morphological spectrum (common type, 82.7%; giant cell, 3.5%; lymphohistiocytic, 8. 6%; and small cell, 5.2%) that reflected the ratio of large anaplastic elements (usually showing cytoplasmic and nuclear ALK positivity) to small neoplastic cells (usually characterized by nucleus-restricted ALK expression). Clinically, ALK+ lymphoma mostly occurred in children and young adults (mean age, 22.01 +/- 10.87 years) with a male predominance (male/female [M/F] ratio, 3.0) that was particularly striking in the second-third decades of life (M/F ratio, 6.5) and usually presented as an aggressive, stage III-IV disease, frequently associated with systemic symptoms (75%) and extranodal involvement (60%), especially skin (21%), bone (17%), and soft tissues (17%). As compared with ALK+ lymphoma, ALK- cases occurred in older individuals (mean age, 43.33 +/- 16.15 years) and showed a lower M/F ratio (0.9) as well as lower incidence of stage III-IV disease and extranodal involvement at presentation. Overall survival of ALK+ lymphoma was far better than that of ALK- anaplastic large-cell lymphoma (71% +/- 6% v 15% +/- 11%, respectively). However, within the good prognostic category of ALK+ lymphoma, survival was 94% +/- 5% for the low/low intermediate risk group (age-adjusted International Prognostic Index, 0 to 1) and 41% +/- 12% for the high/high intermediate risk group (age-adjusted International Prognostic Index, >/=2). Multivariate analysis identified ALK expression and the International Prognostic Index as independent variables that were able to predict survival among T/null primary, systemic anaplastic large-cell lymphoma. Thus, we suggest that such parameters should be taken into consideration for the design of future clinical trials.

Role of T-helper type 2 cytokines in down-modulation of fas mRNA and receptor on the surface of activated CD4(+) T cells: molecular basis for the persistence of the allergic immune response.

The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation-induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL-2), IL-4, IL-5, transforming growth factor beta1, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro exposure of purified CD4(+) lymphocytes to allergen yielded only transient up-regulation of surface Fas but did not influence susceptibility to Fas-mediated cell death. T-helper type 2 cytokines (IL-4, IL-5, and GM-CSF) had a dose-dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure.

ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum.

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffm sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.

Defective expression of Fas messenger RNA and Fas receptor on pulmonary T cells from patients with asthma.

BACKGROUND: Inflammation at sites of target organs seems to be the pathologic hallmark of respiratory allergic diseases, but why this response cannot be turned off in atopic persons is not known. Programmed cell death (apoptosis) mediated by Fas/APO-1 (CD95), a 45-kD surface protein belonging to the tumor necrosis factor receptor family, is important in the resolution of all inflammatory immune responses. OBJECTIVE: To test whether the expression of Fas receptor is defective in allergen-specific pulmonary T lymphocytes from persons with asthma. DESIGN: 12-month prospective study. SETTING: University allergy and immunology clinic. PATIENTS: 12 untreated persons with newly diagnosed allergic asthma who underwent bronchoalveolar lavage. Ten normal persons served as controls. MEASUREMENTS: Fas receptor expression was studied by using surface double-color cytofluorometry on pulmonary and circulating T lymphocytes. Fas messenger RNA (mRNA) was searched for in bronchoalveolar lavage cells from patients and controls by reverse transcription polymerase chain reaction (PCR). In vitro induction of DNA fragmentation, as an expression of cell death induced by an IgM anti-Fas monoclonal antibody, was assessed by propidium iodide staining and agarose gel electrophoresis. In vitro modulation of surface Fas receptor was studied on pulmonary T lymphocytes stimulated with anti-CD3 monoclonal antibody and interleukin-2 or interleukin-4. RESULTS: Pulmonary T lymphocytes from patients as opposed to controls did not undergo DNA fragmentation after in vitro exposure to IgM anti-Fas. Other activation markers (CD25, HLA-DR, and CD45R0) were displayed, but surface Fas expression was always negative. A remarkable proportion of T cells from controls showed a clear double-staining pattern. Reverse transcription PCR for Fas mRNA yielded the same results. Circulating T lymphocytes from patients and controls included similar percentages of CD3+ Fas+ cells. Pulmonary T cells from both patients and controls showed upregulation of Fas receptor expression after in vitro anti-CD3 stimulation; co-culturing with interleukin-4 downmodulated surface Fas receptor expression on T cells from patients; it was less effective in controls. CONCLUSIONS: Hypoexpression of Fas mRNA and surface Fas receptor on pulmonary CD3+ T lymphocytes may explain the persistence of inflammatory cellular infiltrates in allergic bronchial asthma.

Bcl-6 protein expression in normal and neoplastic lymphoid tissues.

The human bcl-6 gene, which is rearranged in about 30% of diffuse large B-cell lymphomas (DLCL-B), encodes for a Kruppel-type zinc finger protein of 706 amino acids. In order to investigate the expression of the bcl-6 gene at the protein level, two monoclonal antibodies (PG-B6a and PG-B6p) directed against the human bcl-6 protein were generated by immunizing Balb/c mice with a recombinant protein corresponding to the amino-terminal region (amino acids 3-484) of bcl-6. PG-B6a (a = avian) recognized the most conserved bcl-6 epitope (expressed in many animal species, including avian). PG-B6p (p = paraffin) reacted with an epitope of bcl-6 partially resistant to fixatives and detectable on microwave-heated paraffin sections. At immunocytochemistry, bcl-6 localized in the nucleus with a microgranular or diffuse pattern. Strong nuclear expression of bcl-6 was mainly detected in normal germinal-center B-cells, whereas mantle- and marginal-zone B cells, as well as plasma cells and marrow B-cell precursors, did not express bcl-6. These immunohistological findings strongly suggest that bcl-6 may play a role as a regulator of germinal-center related functions. All MoAbs stained neoplastic cells of follicular lymphomas, DLCL-B, and Burkitt's lymphomas. In DLCL-B, bcl-6 expression was independent of bcl-6 gene rearrangements and did not correlate with expression of other markers or the proliferation index. Among low-grade B-cell lymphomas, immunostaining for bcl-6 proved useful for differentiating proliferation centers in B-CLL (bcl-2+/bcl-6-) from trapped germinal centers in mantle-cell lymphomas (bcl-2-/bcl-6+). Strong nuclear positivity for bcl-6 was consistently detected in tumor (L&H) cells of nodular, lymphocyte-predominant Hodgkin's disease (NLPHD). These results further support the concept that NLPHD is a histogenetically distinct (germinal-center derived) subtype of HD. Notably, the nuclei of reactive CD3+/ CD4+ T cells near to and rosetting around L&H cells in NLPHD were also strongly bcl-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classic HD, whose cellular background was made up of CD3+/CD4+ T cells showing the bcl-6-/CD40L+ phenotype. The above immunohistological findings suggest that (a) bcl-6 may play a role in regulating B-cell differentiation step(s) occurring within germinal centers; (b) deregulated bcl-6 expression caused by rearrangements may contribute to B-lymphomagenesis; (c) bcl-6 is possibly involved in the pathogenesis of NLPHD.

Microsatellite instability is associated with the histological features of the tumor in nonfamilial colorectal cancer.

Replication errors (RERs) at microsatellite loci were examined in 46 specimens of nonfamilial colorectal cancer. Somatic microsatellite alterations in at least two genetic loci, D11S904, D13S175, D2S123, and D10S197, consistent with a RER(+) phenotype were found in four cases (8.7%). Six additional cases (13%) showed alterations at a single locus. Mucinous differentiation was observed in 3 of 4 (75%) adenocarcinomas with a RER(+) phenotype and only in 19% (8 of 42) of RER(-) adenocarcinomas (P < 0.05). A distinct cap of inflammatory cells at the advancing edge of the tumor and Crohn's-like reaction in peritumoral stroma were histologically identified in 50 and 25% of RER(+) and in 5 and 0% of RER(-) tumors, respectively (P < 0.05). Also, the plexiform pattern of growth of carcinoma turned out to be significantly associated with the RER(+) phenotype (P < 0.05). Mucinous differentiation and stromal inflammatory reactions are frequent features of hereditary nonpolyposis colorectal cancer in which germ-line mutations of mismatch repair genes cause genetic instability. Our results indicate that a link exists between such histological features and somatic genetic instability consistent with a RER(+) phenotype also in nonfamilial colorectal cancer.

Monoclonal antibodies PG-B6a and PG-B6p recognize, respectively, a highly conserved and a formol-resistant epitope on the human BCL-6 protein amino-terminal region.

The human BCL-6 gene, which is rearranged in approximately 30% of diffuse large B cell lymphomas, encodes a 706-amino-acid nuclear protein of the Kruppel-type zinc finger transcription factors mainly expressed in normal germinal center B cells and related lymphomas. Four monoclonal antibodies (PG-B6, PG-B6a, PG-B6p, and PG-B6m), specifically directed against the human BCL-6 protein, were generated by immunizing BALB/c mice with a recombinant protein corresponding to the BCL-6 amino-terminal region (amino acids 3 to 484). The PG-B6 monoclonal antibody reacted with a BCL-6 epitope sensitive to fixatives and preserved in all mammalian species. PG-B6a (a is for avian) recognized the most evolutionarily conserved BCL-6 epitope (expressed in all animal species including avian). PG-B6p (p is for paraffin) recognized a fixative-resistant epitope of BCL-6 that was detectable on paraffin sections after microwave heating in 1 mmol/L EDTA buffer. PG-B6m (m is for mantle) was the least specific monoclonal antibody as, in addition to BCL-6, it reacted with a yet undefined antigen selectively located in the cytoplasm of mantle and marginal zone B cells. All monoclonal antibodies detected strong nuclear expression of BCL-6 in follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and nodular, lymphocyte-predominance Hodgkin's disease. In diffuse large B cell lymphomas, BCL-6 expression was independent of BCL-6 gene rearrangements and did not correlate with expression of other markers or the proliferation index. BCL-6 was not expressed in B-CLL, hairy cell leukemia, mantle-cell- and marginal-zone-derived lymphomas. Labeling of paraffin sections with PG-B6p proved useful for differentiating proliferation centers in B-CLL (BCl-2+/BCL-6-) from trapped germinal centers in mantle cell lymphomas (BCL-2-/BCL-6+) and for identifying neoplastic cells in cases of nodular, lymphocyte-predominance Hodgkin's disease. Because of their high specificity, wide reactivity in humans and animal species including avians (PG-B6a), and suitability for labeling routine paraffin sections (PG-B6p), the reagents described in this paper should prove valuable in both research and diagnostics.

Distinctive expression pattern of the BCL-6 protein in nodular lymphocyte predominance Hodgkin's disease.

The BCL-6 gene encoding a nuclear-located Kruppel-type zinc finger protein is rearranged in about 30% diffuse large B-cell lymphomas and is expressed predominantly in normal germinal center B cells and related lymphomas. These findings suggest that BCL-6 may play a role in regulating differentiation of normal germinal center B cells and that its deregulated expression caused by rearrangements may contribute to lymphomagenesis. This prompted us to investigate the expression of the BCL-6 protein in Hodgkin's disease (HD), focusing on the nodular lymphocyte predominance subtype (NLPHD), which differs from classical HD by virtue of the B-cell nature of the malignant cell population (so-called L&H cells) and its relationship with germinal centers. Forty-one HD samples (19 NLPHD, 12 nodular sclerosis, and 10 mixed cellularity) were immunostained with the monoclonal antibodies PG-B6 and PG-B6p that react with a fixative-sensitive and a formalin-resistant epitope on the aminoterminal region of the BCL-6 gene product, respectively. Strong nuclear positivity for the BCL-6 protein was detected in tumor (L&H) cells in all cases of NLPHD. In contrast, BCL-6 was expressed only in a small percentage of Hodgkin and Reed-Sternberg cells in about 30% of classical HD cases. Notably, the nuclei of reactive CD3+/CD4+ T cells nearby to and rosetting around L&H cells in NLPHD were also strongly BCL-6+, but lacked CD40 ligand (CD40L) expression. This staining pattern clearly differed from that of classical HD, whose cellular background was made up of CD3+/CD4+ T cells showing the BCL-6-/CD40L+ phenotype. These results further support the concept that NLPHD is an histogenetically distinct, B-cell-derived subtype of HD and suggest a role for BCL-6 in its development.

A specific monoclonal antibody (PG-B6) detects expression of the BCL-6 protein in germinal center B cells.

The BCL-6 gene is frequently involved in translocations occurring at the 3q27 locus and is rearranged in approximately 30% of diffuse large cell lymphomas and in a small fraction of follicular lymphomas. The BCL-6 gene encodes for a Kruppel-type zinc-finger protein, the cell/tissue expression and function of which is unknown. In this study, we describe a new monoclonal antibody (PG-B6) that is specifically directed against a fixative-sensitive epitope on the amino-terminal region of the BCL-6 protein. By immunocytochemical analysis, BCL-6 localizes in the nucleus where PG-B6 staining gives a microgranular/diffuse pattern with exclusion of the nucleoli. The main reactivity of PG-B6 in tonsil and spleen is with the nuclei of germinal center B cells, whereas B cells within the mantle and marginal zones do not express BCL-6. No other lymphoid cells in the tonsil express BCL-6 except for a subset of CD3+/CD4+ intrafollicular and interfollicular T cells. A few lymphoid cells of unknown phenotype express BCL-6 in the thymus. Extra-lymphoid BCL-6 expression includes a weak nuclear positivity of epithelia. In non-Hodgkin's lymphomas, BCL-6 expression parallels that observed in normal lymphoid compartments, eg, expression in germinal center-derived tumors (follicular and diffuse large cell lymphomas), but not in mantle cell and marginal zone lymphomas. In most diffuse large cell lymphomas, the BCL-6 protein is expressed at high levels in cases with or without BCL-6 gene rearrangements. These findings indicate that BCL-6 expression is specifically regulated during B lymphocyte development and suggest that BCL-6 may play a role during B cell differentiation in the germinal center.

Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features.

A recently described putative tumor suppressor gene, the cyclin-dependent kinase 4 inhibitor (p16), has been shown to be altered by deletions and/or point mutations in various human cancers. To assess the incidence and clinico-biologic correlations of p16 homozygous deletion in hemopoietic tumors, we studied a panel of 244 DNA samples representative of distinct acute (99 cases) and chronic (57 cases) leukemia subtypes, myelodysplastic (22 cases) and myeloproliferative (15 cases) syndromes, and lymphomas (51 cases). A 361-bp probe complementary to the p16 exon 2 gene sequences was generated by polymerase chain reaction and used in Southern blot hybridization against these tumor DNAs. Homozygous deletions of p16 (p16-/-) were detected in 10 of 58 (17%) cases of acute lymphoblastic leukemia (ALL) of either B or T lineage and in no other tumors. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 was also performed in 40 of the 58 ALL cases and in 16 lymphomas. In no cases were point mutations detected. The comparison of clinical features at presentation in p16-/- and in p16 germline ALL cases showed a greater leukemic cell mass (P = .001) and higher white blood cell counts (P = .01) in the former group. Two ALL cases in which diagnostic and relapse DNA samples were available showed p16-/- in both specimens. We conclude that homozygous p16 gene deletions characterize a subset of ALL with features of aggressive disease.

ALL-1 gene rearrangements in acute myeloid leukemia: association with M4-M5 French-American-British classification subtypes and young age.

We have analyzed by Southern blotting the ALL-1 (MLL, HRX, Hrtx 1) gene configuration in a series of 126 patients with acute myeloid leukemia (AML) representative of all ages and French-American-British Classification groups and correlated this genetic feature with clinical and biological features at diagnosis. ALL-1 gene rearrangements were detected in 17 of the 74 cases with M4-M5 (myelomonocytic and monocytic) AML and in 2 of the 52 cases with other leukemic subtypes (P < 0.01). Within the series of 74 M4-M5 patients, ALL-1 rearrangements were significantly associated with French-American-British Classification M5 (P = 0.009), high WBC (P = 0.002), and young age. In particular, all 5 infant (< 1.5 years) AML cases, 6 of the 19 (31%) patients between 1.5 and 18 years of age, and 6 of the 50 (12%) patients > 18 years old showed an altered ALL-1 genomic configuration (P < 0.001). Immunophenotypic characterization revealed coexpression of lymphoid and myeloid markers in 6 of 17 ALL-1 rearranged M4-M5 cases. The IgH gene configuration was studied in 77 of 126 AMLs. Five patients (6%) showed IgH clonal rearrangements and all were in the ALL-1 rearranged group (P < 0.0001). Our findings indicate that ALL-1 rearrangement is the commonest genetic alteration presently detectable in M4-M5 AML, particularly in childhood where it is found in up to one-third of all cases. The association of IgH rearrangements with ALL-1 alterations in AML, coupled to the frequent detection in this subset of lymphoid associated markers, further supports the origin of these tumors from a common multipotent precursor with bipotential lymphoid and monocytic differentiation capability.

Bone marrow stroma-dependent modulation of CD45R isoform expression on Abelson virus transformed pre-B cells.

The CD45 glycoprotein family exhibits cell-lineage-associated structural heterogeneity which is due, in part, to alternative pre-mRNA splicing. The Abelson murine leukemia (A-MuLV) preferentially transforms immature B cells that express a B-cell-specific high molecular weight CD45 isoform, called B220. However, we observed that A-MuLV-transformed cell lines are often B220- while maintaining high levels of "pan" CD45 expression. In vitro transformation of murine bone marrow revealed that the stromal microenvironment over which A-MuLV-transformed lymphoblasts are grown affected the B220 phenotype of the pre-B cells. Over a period of a few weeks, B220+ populations grown over a clonal stromal cell line gradually became B220-. However, the transition from a B220+ to B220- phenotype was dependent on the lot of fetal calf serum used. In contrast, cells grown over a heterogeneous bone marrow stroma maintained B220+ expression for long periods of time. The appearance of B220- cells in clonal B220+ populations indicated that the change in phenotype resulted in part from modulation of B220 expression. B220- B-cell lines did not express the high molecular weight CD45 RNA species indicating that the B220- phenotype was due to alternative pre-mRNA splicing. Finally, the shift from B220+ to B220- was not accompanied by changes in the stage of development of the cultures. These observations demonstrate that expression of B220 is not required for the continued proliferation of Abelson-transformed pre-B cells and is regulated by unknown environmental factors.

Simultaneous expression of RBTN-2 and BCR-ABL oncogenes in a T-ALL with a t(11;14)(p13;q11) and a late-appearing Philadelphia chromosome.

Cytogenetic analysis of a pediatric patient with T-cell acute lymphoblastic leukemia (T-ALL) revealed a mosaic karyotype, 47,XX,+17,t(11;14)(p13;q11)/47,XX,+17,t(9;22)(q34;q11),t(11;14) (p13;q11). DNA blot analysis was used to examine the break-point within the BCR gene on chromosome 22 and showed that the breakpoint occurred within the 20-kb minor breakpoint cluster region (m-bcr) located within the first intron of the BCR gene. Immunoprecipitation analysis demonstrated that the leukemic cells expressed the P185 BCR-ABL protein tyrosine kinase. P185 BCR-ABL has previously been shown to be expressed in most cases of Ph+ acute leukemia of myeloid and B-progenitor origin. Here, we demonstrate for the first time that P185 can also be expressed in the T-cell lineage. DNA blot hybridization was also used to characterize the t(11;14) translocation. This showed rearrangement on chromosome 11 within the T-ALLbcr region, upstream of the RBTN-2 gene. Polymerase chain reaction revealed the presence of RBTN-2 transcripts in the leukemic cells. Finally, comparison of the T-ALLbcr, BCR-ABL, IGH, TCR beta and gamma gene rearrangements in leukemic cells obtained at the time of diagnosis and at first relapse showed that relapse occurred in a leukemic clone indistinguishable from the major Ph+ clone involved at diagnosis. Together, these data support a multistep pathogenesis in which the Philadelphia (Ph) chromosome translocation appeared subsequent to the +17 and t(11;14) and imparted a growth advantage over the Ph-negative cells that carried these abnormalities.

Natural-killer-stimulatory effect of combined low-dose interleukin-2 and interferon beta in hairy-cell leukemia patients.

The association of low doses of interleukin-2 (IL-2; 5 IU/ml) and interferon beta (IFN beta; 10 IU/ml) induced an additive or synergic stimulatory effect on natural killer (NK) activity (32%) in peripheral blood samples from hairy-cell leukemia patients, both those with active disease and those in remission. The synergic NK stimulatory effect was more commonly found in samples from patients with active disease, while the additive effect was more frequent in the patients in remission. The IL-2/IFN beta combination provoked a nonadditive nonsynergic NK-stimulatory effect in a further 19.8% samples. The targets of the IL-2/IFN beta combination were typical NK cells, as shown by the fact that there was increased cytotoxicity (synergic, additive or nonadditive nonsynergic) against the K562, but not the Daudi cell line in peripheral blood mononuclear cell samples treated with the combination of the two cytokines. When CD16+/CD56+ or CD57+/CD16+/CD56+ cells were removed, the NK-stimulatory effect was lost. The fact that the NK-cell-enhancing activity of the IL-2/IFN beta combination was reduced when Percoll fractions 2 and 3 were used, but still persisted in 66% of tests, may have been due to cytotoxicity being higher in the untreated fractions 2 and 3 than in the untreated unfractionated samples. One of the factors responsible for the NK-stimulatory effect appears to be the capacity of the IL-2/IFN beta combination to trigger an increase in IFN gamma synthesis. If similar experiments give like results in samples from patients suffering from other B-cell lymphoproliferative, or HIV-associated disorders, all of which are characterized by a deficiency in NK activity, it should be possible to use low-dose IL-2/IFN beta to treat these disorders and, perhaps, residual neoplastic disease without exposing the patient to undue toxicity. Further, by testing other combinations one should be able to identify the lowest IL-2 and IFN beta doses that would effectively boost the additive or synergic effect in a greater number of cases.