Sequential chemotherapy/radiotherapy was comparable with concurrent chemoradiotherapy for stage I/II NK/T-cell lymphoma.

Background: In stage I/II natural killer (NK)/T-cell lymphoma, concurrent chemoradiotherapy (CCRT) had previously been shown to result in superior outcome compared with anthracycline-containing regimens, which have since been considered ineffective. The role of CCRT in comparison with approaches employing nonanthracycline-containing chemotherapy (CT) and sequential radiotherapy (RT) in such patients remains to be defined. Patients and methods: Three hundred and three untreated patients (207 men, 96 women; median age: 51, 18-86 years) with stage I/II NK/T-cell lymphoma who had received nonanthracycline-containing regimens were collected from an international consortium and retrospectively analyzed. Treatment included single modality (CT and RT), sequential modalities (CT + RT; RT + CT) and concurrent modalities (CCRT; CCRT + CT). The impact of clinicopathologic parameters and types of treatment on complete response (CR) rate, progression-free-survival (PFS) and overall-survival (OS) was evaluated. Results: For CR, stage (P = 0.027), prognostic index for NK/T-cell lymphoma (PINK) (P = 0.026) and types of initial treatment (P = 0.011) were significant prognostic factors on multivariate analysis. On Cox regression analysis, ECOG performance score (P = 0.021) and PINK-EBV DNA (PINK-E) (P = 0.002) significantly impacted on PFS; whereas ECOG performance score (P = 0.008) and stage (P < 0.001) significantly impacted on OS. For comparing CCRT ± CT and sequential CT + RT, CCRT ± CT patients (n = 190) were similar to sequential CT + RT patients (n = 54) in all evaluated clinicopathologic parameters except two significantly superior features (higher proportion of undetectable circulating EBV DNA on diagnosis and lower PINK-E scores). Despite more favorable pre-treatment characteristics, CCRT ± CT patients had CR rate, PFS and OS comparable with sequential CT + RT patients on multivariate and Cox regression analyses. Conclusions: In stage I/II NK/T-cell lymphomas, when effective chemotherapeutic regimens were used, CCRT and sequential CT + RT gave similar outcome.

Rapidly progressive fatal hemorrhagic pneumonia caused by Stenotrophomonas maltophilia in hematologic malignancy.

BACKGROUND: Pneumonia caused by Stenotrophomonas maltophilia is rare, but can be lethal in severely immunocompromised patients. However, its clinical course remains unclear. PATIENTS AND METHODS: Patients with pneumonia caused by S. maltophilia in Toranomon Hospital (890 beds, Tokyo, Japan) were reviewed retrospectively between April 2006 and March 2010. RESULTS: During the study period, 10 cases of S. maltophilia pneumonia were identified. Seven patients had acute myeloid leukemia, 2 had myelodysplastic syndrome, and 1 had malignant lymphoma. All patients developed symptoms after allogeneic hematopoietic stem cell transplantation (HSCT). Five patients received first cord blood transplantation (CBT), 4 patients received second CBT, and 1 patient received first peripheral blood stem cell transplantation (PBSCT). The overall incidence of S. maltophilia pneumonia among 508 patients who received HSCT during the period was 2.0%. The incidence was 0% (0/95) in patients after bone marrow transplantation, 0.8% (1/133) after PBSCT, and 3.2% (9/279) after CBT. Pneumonia developed a median of 13.5 days (range, 6-40) after transplantation. At onset, the median white blood cell count was 10/µL (range, 10-1900), and the median neutrophil count was 0/µL (range, 0-1720). In all patients, S. maltophilia bacteremia developed with bloody sputum or hemoptysis. The 28-day mortality rate was 100%; the median survival after onset of pneumonia was 2 days (range, 1-10). CONCLUSIONS: Hemorrhagic S. maltophilia pneumonia rapidly progresses and is fatal in patients with hematologic malignancy. Attention should be particularly paid to the neutropenic phase early after HSCT or prolonged neutropenia due to engraftment failure. A prompt trimethoprim-sulfamethoxazole-based multidrug combination regimen should be considered to rescue suspected cases of S. maltophilia pneumonia in these severely immunosuppressed patients.

Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis.

X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands, no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.

FDG-PET in T-cell and NK-cell neoplasms.

BACKGROUND: A growing number of studies demonstrate the utility of (18)fluoro-2-deoxyglucose positron emission tomography (FDG-PET) in the management of malignant lymphoma. The results of FDG-PET, however, have not been studied extensively for T-cell and natural killer (NK)-cell neoplasms. PATIENTS AND METHODS: We retrospectively evaluated pretreatment FDG-PET scans in 41 patients with T/NK-cell neoplasms diagnosed according to the World Health Organization (WHO) classification. Histological subtypes frequently included were peripheral T-cell lymphoma, unspecified (PTCLu, n = 11), extranodal NK/T-cell lymphoma, nasal type (ENKL, n = 8), primary cutaneous anaplastic large cell lymphoma (C-ALCL, n = 5), and angioimmunoblastic T-cell lymphoma (AILT, n = 4). RESULTS: FDG-PET detected a lymphoma lesion in at least one site in 36 out of 41 patients. The positive rate was equally high in most histological subtypes except for cutaneous lymphomas: PTCLu 91%, ENKL 100%, C-ALCL 60%, AILT 100%. All the patients without an FDG-avid lesion had lesions restricted to skin. Among patients who had cutaneous lesions, only 50% had FDG-avid cutaneous lesions, all of which were tumorous. The positive rate of FDG-PET for bone marrow involvement was only 20%. CONCLUSION: T/NK-cell neoplasms incorporated in this study were generally FDG-avid except for cutaneous lesions and bone marrow involvement.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

Mutations of the Smad4 gene in acute myelogeneous leukemia and their functional implications in leukemogenesis.

The Smad family proteins are critical components of the transforming growth factor (TGF)-beta signaling pathway. TGF-beta is a multipotent cytokine that elicits many biological functions. In particular, TGF-beta exhibits effects on the cell cycle manifested by G1-phase arrest, differentiation, or apoptosis of several target cells, suggesting that disruption of TGF-beta signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (Delta(483 - 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The Delta(483 - 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-beta signaling. This is the first report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-beta signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 - 96.

Bicarbonate ion-ATPase in rat liver cell fractions.

The distribution of HCO3MINUS-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3MINUS-ATPase activity. Approximately 85% of the total HCO3minus-ATPase activity of the post 6000 times g-min supernatant was recovered in the mitochondrial fraction. The properties of this mitochondrial HCO3minus-ATPase were not distinguishable from those of the various microsomal HCO3minus-ATPases previously described by other investigators.

Cardiac complications after haploidentical HLA-mismatched hematopoietic stem cell transplantation using in vivo alemtuzumab.

Alemtuzumab is a humanized monoclonal antibody directed against human CD52 with a strong lympholytic effect. We have performed unmanipulated hematopoietic stem cell transplantation (HSCT) from 2- or 3-locus-mismatched family donors in 14 patients using in vivo alemtuzumab. All achieved complete donor cell engraftment and grade III-IV acute graft-versus-host disease was observed in only one patient. However, eight of the 14 patients developed grade II-IV cardiac complications according to Bearman's criteria. Next, we retrospectively analyzed the records of 142 adult patients who underwent allogeneic HSCT from 1995 to 2004 to evaluate whether the use of alemtuzumab was an independent risk factor for cardiac complications. Among several factors that increased the incidence of grade II-IV cardiac complications with at least borderline significance, a multivariate analysis identified the cumulative dose of anthracyclines (P=0.0016) and the use of alemtuzumab (P=0.0001) as independent significant risk factors. All of the cardiac complications in the alemtuzumab group were successfully treated with diuretics and/or catecholamines. Patient selection and close monitoring of cardiac function may be important in HLA-mismatched HSCT using in vivo alemtuzumab.

High-grade cytomegalovirus antigenemia after hematopoietic stem cell transplantation.

Clinical impact of high-grade (HG) cytomegalovirus (CMV) antigenemia after hematopoietic stem cell transplantation has not been clarified. Therefore, in order to investigate the risk factors and outcome for HG-CMV antigenemia, we retrospectively analyzed the records of 154 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2002 at the University of Tokyo Hospital. Among 107 patients who developed positive CMV antigenemia at any level, 74 received risk-adapted preemptive therapy with ganciclovir (GCV), and 17 of these developed HG-antigenemia defined as > or = 50 positive cells per two slides. The use of systemic corticosteroids at > or = 0.5 mg/kg/day at the initiation of GCV was identified as an independent significant risk factor for HG-antigenemia. Seven of the 17 HG-antigenemia patients developed CMV disease, with a cumulative incidence of 49.5%, which was significantly higher than that in the low-grade antigenemia patients (4%, P<0.001). However, overall survival was almost equivalent in the two groups. In conclusion, the development of HG-antigenemia appeared to depend on the profound immune suppression of the recipient. Although CMV disease frequently developed in HG-antigenemia patients, antiviral therapy could prevent a fatal outcome.

Effect of graft-versus-host disease on the outcome of bone marrow transplantation from an HLA-identical sibling donor using GVHD prophylaxis with cyclosporin A and methotrexate.

The effect of graft-versus-host disease (GVHD) on relapse incidence and survival has been analyzed in several studies, but previous studies included heterogeneous patients. Therefore, we analyzed the data of 2114 patients who received unmanipulated bone marrow graft from an HLA-identical sibling donor with a GVHD prophylaxis using cyclosporin A and methotrexate. Among the 1843 patients who survived without relapse at 60 days after transplantation, 435 (24%) developed grade II-IV acute GVHD. Among the 1566 patients who survived without relapse at 150 days after transplantation, 705 (47%) developed chronic GVHD. The incidence of relapse was significantly lower in patients who developed acute or chronic GVHD, but disease-free survival (DFS) was significantly inferior in patients who developed acute GVHD. A benefit of 'mild' GVHD was only seen in high-risk patients who developed grade I acute GVHD. The strongest association between GVHD and a decreased incidence of relapse was observed in patients with standard-risk acute myelogenous leukemia/myelodysplastic syndrome. In conclusion, the therapeutic window between decreased relapse and increased transplant-related mortality due to the development of GVHD appeared to be very narrow.

Identification of a novel fusion gene, TTL, fused to ETV6 in acute lymphoblastic leukemia with t(12;13)(p13;q14), and its implication in leukemogenesis.

ETS variant gene 6 (ETV6)/translocation, ETS, leukemia (TEL)-involving chromosomal translocations are frequently observed in various hematologic neoplasms. We describe here a novel ETV6-involving translocation, t(12;13)(p13;q14), found in the case of acute lymphoblastic leukemia, in which ETV6 fused with a previously unknown gene, named Twelve-thirteen Translocation Leukemia gene (TTL), at 13q14. TTL was weakly but ubiquitously expressed in normal human tissues as detected by reverse transcribed-PCR. Three TTL splicing forms were identified, TTL-T from a human testis cDNA library, with an open-reading frame of 402 bp encoding 133 amino acids (aa), and TTL-B1 and -B2 from a human brain cDNA library. These proteins have no homology to known proteins. In leukemic cells from the patient, both reciprocal fusion transcripts, ETV6/TTL and TTL/ETV6, were expressed. The predominant fusion transcript, TTL/ETV6-1, encodes a predicted 530 aa fusion protein containing 89 aa of the N-terminal TTL fusing to the helix-loop-helix domain and ETS-binding domain of ETV6. Although the function of TTL is yet to be elucidated, our findings will provide another insight into the molecular pathogenesis of leukemia having ETV6-involving translocations.

Oral manifestations of chronic graft-v-host disease.

Sixty patients were studied 180 to 500 days after allogeneic marrow transplantation to determine if late oral abnormalities were associated with the presence of chronic graft-v-host disease (GVHD). Lip and intraoral mucosal surfaces were evaluated for color, keratinization, atrophy, and erythema. Subjective complaints of oral pain and xerostomia were also recorded. Abnormalities were scored on a scale of 0 to 3 and tested for association with GVHD by chi 2 test. Oral manifestations most strongly associated with chronic GVHD included atrophy and erythema or lichenoid lesions of the buccal and labial mucosa and oral pain. Oral manifestations resembled several naturally occurring autoimmune disorders. Recognition of these changes can aid in the clinical diagnosis and assessment of established chronic GVHD.

Distribution of a Ca2+ storing site in PtK2 cells during interphase and mitosis. An immunocytochemical study using an antibody against calreticulin.

To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.

Radioprotection by WR-2721 of gamma-irradiated rat parotid gland: effect on gland weight and secretion at 8-10 days post irradiation.

Changes in rat parotid salivary gland weight and functional parameters were evaluated at 8 to 10 days post irradiation in WR-2721 protected and non-protected animals following exposure to a single 15.3 Gy dose of Cs-137 radiation to the head. Glandular fluid secretory capacity was assessed by maximum flow rate, total volume of saliva and duration of secretion following pilocarpine stimulation. Protection against radiomucositis was also evaluated indirectly by daily monitoring of food and water intake, body weight and paraoral symptomatology. WR-2721 provided a significant degree of protection for all glandular functional parameters as well as gland weight. Relative protective factors (RPF) were computed for irradiated protected and non-protected animals compared to their sham-irradiated, pair-fed controls. The calculated RPFs were: Gland weight 1.9, maximum flow rate 2.9, volume of saliva 2.1 and duration of secretion 2.1 for a mean "relative protection" of 2.25. Substantial protection against radiomucositis in protected animals was evident by a progressive gain in body weight and lack of oral signs and symptoms as compared to non-protected animals. Protection against radiomucositis and preservation of residual parotid gland secretory capacity as determined by functional parameters suggests that WR-2721 may be of significant benefit in alleviating oral symptoms and maintaining salivary gland function for patients receiving radiotherapy for head and neck tumors.

Disordered salivary immunoglobulin secretion and sodium transport in human chronic graft-versus-host disease.

Whole saliva samples and lip biopsies were collected from 12 allogeneic bone marrow transplant recipients who developed extensive chronic graft-versus-host disease (GVHD) and from 10 healthy allogeneic and syngeneic recipients without GVHD. Six of ten biopsies from patients with chronic GVHD had lichenoid stomatitis or sialadenitis, or both, with sialodochitis. Seven of nine biopsies from patients free of chronic GVHD were entirely normal, and two had either mild glandular or mucosal changes. Salivary gland involvement in chronic GVHD was associated with decreased or absent levels of salivary IgA and inorganic phosphate, decreased salivary flow rates, and increased concentrations of salivary sodium, albumin, and IgG. The most striking abnormalities were found in patients with histologic evidence of sialadenitis. In contrast, marrow transplant recipients without chronic GVHD had normal salivary immunoglobulin and electrolyte levels. Secretory IgA deficiency may contribute to the frequent sinobronchial infections observed in patients with chronic GVHD.

The predictive value of elevated labial saliva sodium concentration: its relation to labial gland pathology in bone marrow transplant recipients.

Labial minor gland salivary flow rate and sodium concentration were analyzed in relation to 1) histologic findings in labial biopsy specimens and 2) the occurrence of chronic graft-versus-host disease (GVHD) in patients who received bone marrow transplants. Biopsy specimens and samples were obtained from 61 recipients of marrow transplants (including three twins) 51 to 1,260 days post transplantation. Labial saliva sodium concentrations were elevated in some patients, and these increases were associated with inflammation and destruction of minor salivary gland acini and ducts by chronic GVHD or other factors. The predictive value of the salivary sodium changes in evaluating labial salivary gland pathologic changes was 91 per cent, and the sensitivity was 74 per cent. Thus, if a transplant recipient is found to have an elevated labial saliva sodium level, then the probability that he has pathologic labial gland changes is 91 per cent. When analyses were restricted to include only patients who received no irradiation during transplantation, then elevated labial saliva sodium concentration was significantly associated with the occurrence of chronic GVHD. The sensitivity of this relationship was 42 per cent, but the predictive value was 100 per cent. Thus, if a nonirradiated transplant recipient is found to have an elevated labial saliva sodium concentration, then it is virtually certain that he has chronic GVHD. We found no significant changes in labial saliva flow rates in these bone marrow transplant recipients.

Predictors for severe cardiac complications after hematopoietic stem cell transplantation.

The value of pre-transplant factors for predicting the development of cardiac complications after transplantation has been inconsistent among studies. We analyzed the impact of pre-transplant factors on the incidence of severe cardiac complications in 164 hematopoietic stem cell transplant recipients. We identified eight patients (4.8%) who experienced grade III or IV cardiac complications according to the Bearman criteria. Seven died of cardiac causes a median of 3 days after the onset of cardiac complications. On univariate analysis, both the cumulative dose of anthracyclines and the use of anthracyclines within 60 days before transplantation affected the incidence of severe cardiac complications (P=0.0091 and 0.011). The dissociation of heart rate and body temperature, which reflects "relative tachycardia", was also associated with a higher incidence of cardiac complications (P=0.024). None of the variables obtained by electrocardiography or echocardiography were useful for predicting cardiac complications after transplantation, although the statistical power might not be sufficient to detect the usefulness of ejection fraction. On a multivariate analysis, the cumulative dose of anthracyclines was the only independent significant risk factor for severe cardiac complications. We conclude that the cumulative dose of anthracyclines is the most potent predictor of cardiac complications and the administration of anthracyclines should be avoided within two months before transplantation.

Increased soluble Fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a major complication following allogeneic bone marrow transplantation (BMT). Recently, accumulating evidence indicates that the Fas/Fas ligand (FasL) system is implicated in the pathogenesis of aGVHD in murine models. We determined the serum levels of soluble FasL (sFasL) in BMT recipients using an enzyme-linked immunosorbent assay. The serum sFasL was suppressed during the period of myelosuppression following the preparative regimen and subsequently increased with hematopoietic reconstitution after BMT. In patients with aGVHD, the serum sFasL level was significantly higher than in those without aGVHD. In the mixed lymphocyte reaction assay, sFasL in the supernatants was increased with a significant correlation to the level of 3H-thymidine uptake. Our findings suggest that the Fas/FasL system is activated by allogeneic stimulation and may have close correlation to the development of aGVHD in human BMT.