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Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Th17 cells have never been explored in human graft-versus-host disease (GVHD). We studied the correlation between the presence of Th17 cells with histologic and clinical parameters. We first analyzed a cohort of 40 patients with GVHD of the gastrointestinal tract. Tumor necrosis factor (TNF), TNF receptors, and Fas expression, and apoptotic cells, CD4(+)IL-17(+) cells (Th17), and CD4(+)Foxp3(+) cells (Treg) were quantified. A Th17/Treg ratio less than 1 correlated both with the clinical diagnosis (P < .001) and more than 2 pathologic grades (P < .001). A Th17/Treg ratio less than 1 also correlated with the intensity of apoptosis of epithelial cells (P = .03), Fas expression in the cellular infiltrate (P = .003), TNF, and TNF receptor expression (P < .001). We then assessed Th17/Treg ratio in 2 other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio less than 1 correlated with the pathologic grade of GI GVHD. Finally, 15 patients with skin GVHD and 11 patients with skin rash but without pathologic GVHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GVHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathologic in situ biomarker of GVHD.

The expression of bcl-x(L), an antiapoptotic member of the bcl-2 family, has been correlated with poor prognosis in nodal follicular lymphomas (NFLs). So far, it has not been studied in primary cutaneous follicle center lymphomas (PCFCLs), which, compared with NFLs, express less frequently t(14;18)(q32;q21) and bcl-2. Using real-time PCR we measured bcl-xL and bcl-2 gene expression levels in laser-microdissected lymphoma cells of 20 PCFCL frozen sections. Numbers of apoptotic cells labeled by TUNEL assay were negatively correlated with bcl-xL expression levels (r=-0.840, P<0.005). Bcl-xL expression was significantly higher in biopsies of patients who developed relapse or disease progression later compared with patients who did not (P=0.022), and higher levels of bcl-xL gene expression were significantly correlated with shorter progression-free survival (PFS) (P=0.017). None of these features was correlated with bcl-2 gene expression levels. Our findings indicate that bcl-xL overexpression is inversely correlated with PFS in PCFCL. Moreover, the inverse correlation between bcl-xL expression levels and apoptotic cell numbers suggests that bcl-xL, through its antiapoptotic effect, might contribute to tumor cell survival in PCFCL.

In animal models, tissue stem cells were proposed to exhibit an unexpected level of plasticity, although issues on cell fusions have lead to some controversies. Only transplantation experiments using genetically distinct recipients and donors can unequivocally show these changes in cell fate. We have analyzed oral squamous cell carcinomas arising in 8 long-term survivors of allogeneic bone marrow transplantation, in whom chronic graft-versus-host disease greatly favors development of squamous cell carcinomas, possibly as a consequence of lichenoid mucosal inflammation. With the use of 2 independent methods, (1) combined immunostaining and fluorescent in situ hybridization (FISH) analysis for X and Y chromosomes sequences in sex-mismatched grafts and (2) comparison of microsatellite typing of laser-microdissected tumor, donor, and recipient cells, in all tumors, we showed that 4 of these 8 epithelial tumors actually arose from the engrafted allogeneic bone marrow. Thus, donor-derived bone marrow cells, whether hematopoietic or mesenchymal, recruited to sites of chronic mucosal inflammation yielded epithelial tumors. Our observations therefore show that marrow cells in humans have a major role in epithelial cancer formation after allogeneic transplantation.

The positive regulatory domain I (PRDM1) is a master regulator of terminal B-cell differentiation. However, PRDM1 is not B-cell specific. To determine its role in T-cell lymphoma, PRDM1 expression was investigated in 60 patients. PRDM1alpha and PRDM1beta transcripts were detected in laser-microdissected T-lymphoma cells in 27 and 14 patients, respectively, mostly in cases with IRF4 expression. PRDM1beta was associated with increased c-MYC expression. PRDM1beta-positive patients displayed advanced Ann Arbor stage and high-risk International Prognostic Index and were linked to short survival times. In vitro, PRDM1beta was related to resistance to chemotherapeutic agents and could be down-regulated by the proteasome inhibitor bortezomib. Kinetic studies showed that bortezomib down-regulation of PRDM1beta preceded decreased IRF4 and c-MYC expression. An earlier retaining of cytoplasmic IkappaBalpha in bortezomib-treated cells was revealed, concomitant with blockade of NF-kappaB nuclear translocation. These results demonstrate the involvement of PRDM1beta in T-cell lymphoma, with possible therapeutic interference by the proteasome inhibitor.

The positive regulatory domain I (PRDM1) is a master regulator in the differentiation of mature B lymphocytes to plasma cells. It has 2 isoforms, PRDM1alpha and PRDM1beta, and is regulated by the transcriptional regulator nuclear factor kappa (NF)-kappaB. PRDM1 protein expression was recently demonstrated in a subset of diffuse large B-cell lymphoma (DLBCL) with aggressive behavior, a type of lymphoma for which rituximab associated with chemotherapy (R-CHOP) is now widely indicated. Using laser microdissection combined with reverse transcription-polymerase chain reaction (RT-PCR) amplification, PRDM1 gene expression was assessed in 82 DLBCL patients. The results showed that both PRDM1alpha and PRDM1beta transcripts were expressed in microdissected lymphoma cells only in the non-germinal center B-cell-like (non-GCB) subtype of DLBCL. PRDM1beta gene expression was correlated with short survival time in the non-GCB patients treated with CHOP but not with R-CHOP. In vitro, B-lymphoma cells resistant to chemotherapy expressed PRDM1beta. Rituximab suppressed PRDM1beta expression, which was concomitant with NF-kappaB inactivation. The value of PRDM1beta expression as a prognostic marker in non-GCB DLBCL might thus be considered. This study confirms the efficiency of rituximab on DLBCL and allows a better understanding of one of its biologic actions.

Graft-versus-host disease (GvHD)-induced apoptosis of the skin targets both epidermal keratinocytes and dermal endothelial cells. We studied the donor-versus-recipient origin of GvHD of these target cells in skin of 18 sex-mismatched hematopoietic stem-cell transplant (HSCT) recipients. Combining XY fluorescence in situ hybridization (FISH) and double immunostaining, and further 3D tissue Z-stack analysis, we found keratinocytes and endothelial cells of donor origin, but only in patients with GvHD. Using terminal dUTP nick-end labeling (TUNEL) assay on sister sections, we found a correlation between the numbers of chimeric and apoptotic epidermal and endothelial cells. Moreover, donor-derived cells were more numerous and preferentially distributed in the areas of severe GvHD damage in biopsies performed early in the course of GvHD, whereas they were less numerous and found in the whole epidermis in late biopsies. Because donor-derived cells were found at the site and at the time of maximum tissue damage, they could contribute to epidermal and microvessel repair.

In an experimental model we demonstrated that endothelial cells of all organs are targets of the alloimmune reaction. Here, in 68 digestive biopsies, we found endothelial lesions by immunohistochemistry and ultrastructure in patients with severe acute graft-versus-host disease (GVHD). In contrast, no such endothelial cell alterations were found either in patients without GVHD or in nongrafted controls. In the biopsies with severe GVHD lesions, ultrastructure showed rupture of the capillary basal membrane and extravased red blood cells. These pericapillary hemorrhages were highly correlated with GVHD severity. In a separate cohort of 39 patients who underwent an allogeneic transplantation after a nonmyeloablative conditioning, 8 patients had intestinal biopsies. Three of these latter patients had both severe pathologic lesions of GVHD and similar endothelial lesions, thus, strengthening the concept that endothelial lesions are linked to GVHD severity and not to the intensity of the conditioning regimen. (Blood. 2004;103:4681-4684)

bcl-xL, a member of the Bcl-2 family, exerts an antiapoptotic effect on lymphocytes. To assess its clinical significance in patients with follicular lymphoma, realtime quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of bcl-xL gene expression was investigated in whole lymph node sections and laser-microdissected lymphoma cells of 27 patients. Compared with 10 patients with reactive follicular hyperplasia, the bcl-xL gene was overexpressed in patients with follicular lymphoma at a higher level in microdissected lymphoma cells. The bcl-xL gene level correlated with the number of apoptotic lymphoma cells labeled by terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) assays (r = -0.7736). Clinically, a high bcl-xL level was significantly associated with multiple sites of extranodal involvement (P =.0020), elevated lactate dehydrogenase level (P =.0478), and an International Prognostic Index indicating high risk (P =.0235). Moreover, bcl-xL gene overexpression was linked to short overall survival times (P =.0129). The value of bcl-xL gene expression as a prognostic marker in follicular lymphoma should thus be considered.