The use of indocyanine green to detect sentinel nodes in breast cancer: a prospective study.

BACKGROUND: Although Indocyanine green (ICG) is used to find sentinel nodes (SN) in patients with breast cancer (BC), its role in clinical practice is still debated, and needs a definitive validation to be included in the standard approach to finding sentinel nodes in breast cancer. MATERIALS AND METHODS: To validate the ICG methods of detecting the SN in BC we have recently concluded a prospective validation trial. Patients with clinically node-negative, invasive early BC scheduled for breast surgery and SN biopsy were included in the trial. All the patients underwent SN detection using both the standard-of-care procedure using radioisotope technetium (99mTc) and the ICG, using the Photodynamic Eye camera. A comparison of the detection rate and the diagnostic accuracy of the two methods was performed to detect the equivalency of the two approaches. RESULTS: At the end of the enrollment, 301 patients were considered eligible and included in the trial, and 589 nodes were removed. Five hundred and eighty-three nodes (99%) were identified with ICG (median 2 nodes per patient) and 452 (76.7%) were identified with 99mTc (median 2 nodes per patient). A concordance index of 98.75% (CI, 95% = 97.1%-99.5%) was detected. The dosage given ranged from 0.3 to 1.4 ml. ICG was used in all patients eligible for SN biopsy without any significant acute side effects. CONCLUSIONS: The index of concordance between 99mTc and ICG seems to be extremely high, suggesting that ICG could be validated as an alternative method to 99mTc in the detection of SN in BC.

Management of hepatitis B virus reactivation in patients with hematological malignancies treated with chemotherapy.

INTRODUCTION: Hepatitis B virus (HBV) reactivation is a major cause of morbidity and mortality in patients with hematological malignancies who receive cytotoxic chemotherapy. We have therefore carried out a prospective observational study out to assess the incidence, prevalence, and clinical course ina cohort of these patients. METHODS: HBV and HCV markers and liver function indices were monitored prospectively in 318 consecutive patients(171 males, 147 females; mean age 57 years) with hematological malignancies, who had been referred to the Hematology Division, Perugia University, between October 2005 and March 2007 and followed up for at least 6 months. RESULTS: At diagnosis, 32 patients (10%) had received HBV vaccination; 30 were responders. At least one HBV marker was positive in 70/318 patients (22%): 14 (20%) were HBsAg-positive(HBV surface antigen-positive), 13 (19%) were only anti-HBc positive (antibodies to HB core antigen), and 43(61%)were anti-HBc and anti-HBs positive. Twelve HBsAg+ patients received nucleoside/nucleotide analogs (adefovir [six patients],lamivudine [four], and combined adefovir/lamivudine[two non-responders to lamivudine]). After 6 months of therapy, HBV-DNA was negative and transaminases were normal in nine of these 12 patients (adefovir [six], lamivudina[two], adefovir + lamivudina [one]). Seroreversion was achieved in 3/13 patients (23%) who were only anti-HBc positive;all were on rituximab therapy and received adefovir. Seroreversion was not observed in any of the 43 patients who were anti-HBc- and anti-HBs positive. CONCLUSIONS: Essential to the management of patients with hematological malignancies undergoing chemotherapy are surveillance and prophylaxis of HBV infection together with prompt administration of nucleoside/nucleotide analogs in cases of reactivation and/or seroreversion.

Vaccine therapy of B cell malignancies: different strategies for a novel approach.

This review deals with the theoretical principles and experimental results of immunotherapy for B cell malignancies, namely for non-Hodgkin lymphomas (NHLs) and multiple myeloma. Its focus is the use of vaccines in clinical practice with particular emphasis on the most recent developments and therapeutic opportunities arising from combination therapies. Previous studies will be reviewed and the present status of vaccine technology summarized.

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.

Expression of B7 co-stimulatory molecules and CD1a antigen by alveolar macrophages in allergic bronchial asthma.

BACKGROUND: Lung allergen recognition that takes place in the airways of asthmatic subjects is still a controversial matter. OBJECTIVE: We hypothesized that a rapid allergen recognition process requires the presence, at the mucosal surface, of professional APC, such as B7+ alveolar macrophages (AM) and/or CD1+ dendritic cells, which usually have a lower expression in the normal human lung. METHODS: Studies were performed on bronchoalveolar lavage (BAL) fluid collected from 10 untreated allergic subjects and 10 adult normal volunteers. Further controls consisted of five untreated pulmonary sarcoidosis (PS) and four extrinsic allergic alveolitis (EAA) individuals. To ascertain whether T helper 2-type cytokines or allergen influence B7 and CD1 antigen expression, in vitro studies were carried out using unprimed (naive) cord blood plastic-adherent monocytes. RESULTS: Cytofluorymetric analysis revealed that AM from asthmatics, unlike those from normal subjects or patients with PS or EAA, overexpressed B7-2, CD1a and, to a lesser extent, B7-1 surface molecules. Immunohistochemical studies confirmed the presence of CD1+ dendritic cells in the BAL fluid from asthmatic subjects. On in vitro cultured naive cord blood monocytes both purified Dermatophagoides pteronyssinus allergen and T-cell cytokines, i.e. IL-4 and granulocyte macrophage colony-stimulating factor, induced surface expression of B7-2 and CD1a receptors, whereas they had no appreciable effect on that of B7-1 membrane molecule. CONCLUSIONS: Taken together, these findings support the proposal that airways of atopic individuals are equipped with professional APC that synergize with allergen-specific T cells for the recognition of intact allergens. When the recognition process takes place, asthmatic symptoms could develop in genetically susceptible individuals.

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.

Immunocytochemical diagnosis of acute promyelocytic leukemia (M3) with the monoclonal antibody PG-M3 (anti-PML).

Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes, leading to the expression of the PML/RARalpha fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARalpha fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARalpha fusion protein (usually >/=50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARalpha. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.

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.

Targeted antibodies in the treatment of lymphomas.

Monoclonal antibodies coupled to drugs and toxic agents (immunotoxins) or radionuclides (radioimmunoconjugates) represent new tools for immunotherapy of haematological malignancies. Immunotoxins constructed with toxins of either plant or bacterial origin have shown a powerful antitumor activity both in vitro and in mice with severe combined immunodeficiency bearing various kinds of leukaemias and lymphomas. Preliminary clinical trials have shown an activity of these compounds at least in a proportion of patients. However, tumour responses have generally been partial and transient. The main problems with immunotoxin therapy remain the inability of immunotoxins to target tumour cells in the presence of a high burden of disease, the host immune response against both the antibody and the toxin moieties, which precludes repeated administration of immunotoxins, and the vascular leak syndrome. Targeting of tumour cells with specific antibodies armed with radionuclides (usually iodine-131 or yttrium-90) appears to be an even more attractive approach. Preliminary clinical studies have clearly demonstrated the ability of radioimmunoconjugates, especially when administered at high dose followed by bone marrow rescue, to induce durable complete remission in patients with non-Hodgkin's lymphomas refractory to conventional therapies. Radioimmunotherapy also overcomes the antigenic heterogeneity of the tumour cell population, since antigen negative tumour cells will be irradiated by the nearby targeted antigen-positive cells. Efforts should now be focused on defining more precisely the optimal clinical setting for administration of immunotoxin and radioimmunoconjugates (e.g. minimal residual disease), to reduce the immunogenicity of these compounds and solve the problem of vascular leak syndrome.

Heterogeneous nuclear expression of the promyelocytic leukemia (PML) protein in normal and neoplastic human tissues.

The RING-finger promyelocytic leukemia (PML) protein is the product of the PML gene that fuses with the retinoic acid receptor-alpha gene in the t(15; 17) translocation of acute promyelocytic leukemia. Wild-type PML localizes in the nucleus with a typical speckled pattern that is a consequence of the concentration of the protein within discrete subnuclear domains known as nuclear bodies. Delocalization of PML from nuclear bodies has been documented in acute promyelocytic leukemia cells and suggested to contribute to leukemogenesis. In an attempt to get new insights into the function of the wild-type PML protein and to investigate whether it displays an altered expression pattern in neoplasms other than acute promyelocytic leukemia, we stained a large number of normal and neoplastic human tissues with a new murine monoclonal antibody (PG-M3) directed against the amino-terminal region of PML. As the PG-M3 epitope is partially resistant to fixatives, only cells that overexpress PML are detected by the antibody in microwave-heated paraffin sections. Among normal tissues, PML was characteristically up-regulated in activated epithelioid histiocytes and fibroblasts in a variety of pathological conditions, columnar epithelium in small active thyroid follicles, well differentiated foamy cells in the center of sebaceous glands, and hypersecretory endometria (Arias-Stella). Interferons, the PML of which is a primary target gene, and estrogens are likely to represent some of the cytokines and/or hormones that may be involved in the up-regulation of PML under these circumstances. In keeping with this concept, we found that PML is frequently overexpressed in Hodgkin and Reed-Sternberg cells of Hodgkin's disease, a tumor of cytokine-producing cells. Among solid tumors, overexpression of PML was frequently found in carcinomas of larynx and thyroid (papillary), epithelial thymomas, and Kaposi's sarcoma, whereas carcinomas of the lung, thyroid (follicular), breast, and colon were frequently negative or weakly PML+. We did not observe any changes in the levels of PML expression as the lesion progressed from benign dysplasia to carcinoma. Our immunohistological data are consistent with the hypothesized growth suppressor function of PML and strongly suggest that PML expression levels are likely to be modulated by a variety of stimuli, including cytokines and hormones.

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.

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.

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.

Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia.

PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).

Antitumor activity of anti-CD30 immunotoxin (Ber-H2/saporin) in vitro and in severe combined immunodeficiency disease mice xenografted with human CD30+ anaplastic large-cell lymphoma.

To develop a novel adjunctive therapy for CD30 (Ki-1)+ anaplastic large-cell lymphoma (ALCL), we investigated in preclinical studies the antitumor activity of an immunotoxin (IT) constructed by coupling the plant ribosome-inactivating protein saporin (SO6) to the monoclonal antibody (MoAb) Ber-H2 that is directed against the CD30 molecule, a new member of the tumor necrosis factor receptor (TNFR) super-family. The activity of Ber-H2/SO6 IT was tested both in vitro against the CD30+ ALCL-derived cell line JB6 and in vivo using our severe combined immunodeficiency disease (SCID) mouse model of human xenografted CD30+ ALCL. In vitro, the Ber-H2/SO6 IT was selectively and highly toxic to the JB6 cell line [50% inhibiting concentration (IC50), 3.23 x 10(-12) mol/L as SO6]. In vivo, a 3-day treatment with nontoxic doses of Ber-H2/SO6 (50% of LD50) induced lasting complete remissions (CR) in 80% of mice when started 24 hours after tumor transplantation. In contrast, injection of the IT at later stages of tumor growth (mice bearing subcutaneous tumors of 40- to 60-mm3 volume), induced CR in only 6 of 21 (approximately 30%) mice and significantly delayed tumor growth rate (P < .01). This finding suggests that maximum effect of the anti-CD30 IT is observed when tumor cell burden is small. Persistent tumors from IT-treated mice consisted of CD30+ cells, thus excluding the possibility that selection of CD30-negative mutant clones during IT therapy was responsible for resistance to treatment. We conclude that Ber-H2/SO6 IT is an effective agent against CD30+ ALCL growing in SCID mice, suggesting its possible role as adjuvant therapy in patients with CD30+ ALCL refractory to standard treatments.

PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD68 molecule.

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.