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Tγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαβ LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαβ cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2- cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαβ cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαβ LGLL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Epstein-Barr virus (EBV)-DNA was prospectively analyzed in plasma and mononuclear cells (MNCs) from peripheral blood in patients with extranodal natural killer (NK)/T-cell lymphoma, nasal type, to evaluate the clinical significance for diagnosis, monitoring the tumor burden, and prognostication. Thirty-three patients were enrolled, and 32 were evaluable. Pretreatment plasma and MNC EBV-DNA was detectable in 14 (range, 50-71 000 copies/mL) and 6 patients (range, 20-780 copies/μg DNA), respectively, and both were well correlated (r = 0.8741, P < .0001). Detectable plasma EBV-DNA was associated with higher clinical stage (P = .02), presence of B symptoms (P = .02), worse performance status (P = .02), and higher serum soluble IL-2 receptor level (P < .0001). Twenty-two patients attained complete response. Plasma EBV-DNA level was significantly higher in nonresponders than in responders (mean, 16,472 vs 2,645 copies/mL; P = .02). Multivariate analysis showed clinical stage (hazard ratio, 9.0; 95% confidence interval, 1.8%-45.0%) and pretreatment plasma EBV-DNA (hazard ratio, 10.6; 95% confidence interval, 1.3%-87.0%) were significant prognostic factors. Three-year overall survival of plasma EBV-DNA positive and negative patients was 42.9% and 94.4%, respectively (P = .0009). Plasma was a preferable sample for this purpose in NK/T-cell lymphoma, nasal type, and EBV-DNA level was a good indicator for response and overall survival.

Diagnostic errors in distinguishing between malignant and reactive processes can cause serious clinical consequences. We report 10 cases of unrecognized self-limited natural killer-cell proliferation in the stomach, designated as lymphomatoid gastropathy (LyGa). This study included 5 men and 5 women (age, 46-75 years) without any gastric symptoms. Gastroscopy showed elevated lesion(s) (diameter, ∼ 1 cm). Histologically, medium-sized to large atypical cells diffusely infiltrated the lamina propria and, occasionally, the glandular epithelium. The cells were CD2(+/-), sCD3(-), cCD3(+), CD4(-), CD5(-), CD7(+), CD8(-), CD16(-), CD20(-), CD45(+), CD56(+), CD117(-), CD158a(-), CD161(-), T cell-restricted intracellular antigen-1(+), granzyme B(+), perforin(+), Epstein-Barr early RNA(-), T-cell receptor αβ(-), and T-cell receptor γδ(-). Analysis of the 16 specimens biopsied from 10 patients led to a diagnosis of lymphoma or suspected lymphoma in 11 specimens, gastritis for 1 specimen, adenocarcinoma for 1 specimen, and LyGa or suspected LyGa for 3 specimens. Most lesions underwent self-regression. Three cases relapsed, but none of the patients died. According to conventional histopathologic criteria, LyGa is probably diagnosed as lymphoma, especially as extranodal natural killer/T-cell lymphoma, nasal type. However, LyGa is recognized as a pseudomalignant process because of its clinical characteristics. The concept of LyGa should be well recognized.

Bortezomib is a novel proteasome inhibitor with significant antimyeloma activity. Its frequent adverse effects are manageable, including gastrointestinal symptoms, peripheral neuropathy, and thrombocytopenia. Severe lung toxicity has not previously been reported. Between June 2004 and September 2005, 13 Japanese patients with multiple myeloma were treated with bortezomib in Toranomon Hospital, Juntendo University School of Medicine, and Jichi Medical School. Four of them developed severe pulmonary complications, and 2 died of respiratory failure without progression of underlying disease. To our knowledge, this is the first report on life-threatening pulmonary adverse effects after bortezomib therapy. Previous clinical studies on bortezomib, mostly in the United States and Europe, have shown low incidences of pulmonary adverse effects. Our study suggests that bortezomib can cause serious lung injury, and that its incidence might vary among different ethnicities. Clinicians need to be alert to the possibility.

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the MDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3(-)CD16(+) or CD3(-)CD56(+) lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% +/- 0. 9%, 93.4% +/- 3.1% and 36.9% +/- 11.7%, respectively, by 1 micromol/L CsA, and 80.2% +/- 3.6%, 91.7% +/- 2.6% and 32.7% +/- 10. 1%, respectively, by 1 micromol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.

Both tumor necrosis factor alpha (TNFalpha) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, we examined the ameliorating effects of neutralizing anti-FasL and/or anti-TNFalpha monoclonal antibody (MoAb) in a lethal acute GVHD model in mice. Whereas the treatment with either anti-FasL or anti-TNFalpha MoAb alone significantly delayed the mortality and improved the body weight, a complete protection was achieved by the administration of both MoAbs. Pathological examination indicated differential effects of anti-FasL or anti-TNFalpha MoAb on GVHD-associated pathologies. Hepatic lesion was improved by anti-FasL but not anti-TNFalpha MoAb. In contrast, intestinal lesion was improved by anti-TNFalpha but not anti-FasL MoAb. Cutaneous and splenic lesions were improved by either MoAb. The combination of both MoAbs improved all these lesions. These results indicate that FasL and TNFalpha differentially contribute to the GVHD pathologies and a complete protection from mortality can be achieved by neutralization of both FasL and TNFalpha.

We related cellular content of DNA topoisomerase (topo) IIalpha and IIbeta with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIalpha increased especially in late S to G2/M phases, although the topo IIbeta level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIalpha but not the topo IIbeta level. A new G2/M population containing virtually no topo IIalpha appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIalpha or IIbeta staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIbeta epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIalpha epitope and another topo IIbeta epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIbeta protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIalpha remained of its original size, although both topo IIalpha and IIbeta left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIalpha and IIbeta in growing HL-60 cells. Although topo IIalpha and IIbeta were distributed throughout the cell during mitosis, only topo IIalpha was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.

Tumor necrosis factor (TNF) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD), which is a major complication after allogeneic bone marrow transplantation. We examined here the ameliorating effect of a metalloproteinase inhibitor (KB-R7785) that inhibits TNF-alpha and FasL release in a lethal acute GVHD model in mice. Administration of KB-R7785 into (BALB/c x C57BL/6) F1 that received C57BL/6 spleen cells markedly reduced the mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. The ameliorating effect of KB-R7785 was superior to that of anti-TNF-alpha antibody. Our results suggest that KB-R7785 could be a potent therapeutic agent for GVHD.

The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/microL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) beta gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR V beta gene regions, we identified a unique TCR V beta on GL from each of three patients studied. In two patients with CD3- LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/microL. These data demonstrate that newer techniques such as MoAbs against V beta gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.