ALBERT

Description: Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, and eradication of Helicobacter pylori (HP) has been demonstrated to be an effective and tolerable firstline treatment for the patients infected with HP in Japan. However the mechanism has still remained to be uncovered. Recently CD4+CD25high+Foxp3+ regulatory T cells (Tregs), which regulate autoreactive T cells, have been identified, and suggested to play an important role in pathogenesis of ITP, as well as other autoimmune disorders. It has been shown that Tregs are reduced and suppressed in the ITP patients. And a recent report has demonstrated that the defective Tregs are restored upon the rituximab treatment. However, the effects of HP eradication therapy on Tregs have not been determined. The aim of this study is to investigate the circulating Tregs in ITP patients treated with HP eradication. And we also attempted to elucidate the mechanisms of the treatment. The peripheral blood CD4+CD25high+ Tregs were measured by flow cytometry before and after the treatment in 21 Japanese adults with HP-positive chronic ITP. We also confirmed the expression of Foxp3 in this cellular population with the permeabilized mononuclear cells. Among 21 patients, the platelet counts increased in 13 cases (responders), but not in 9 cases (non-responders). In responders the numbers of Tregs have been restored, but not in non-responders after the treatment. It is interesting here that the amounts of Tregs were still transiently elevated in an early phase (2–3weeks) after the treatment in some non-responders without recovery of the platelet counts. Furthermore, in three cases, who failed in pylorus elimination, Tregs were also transiently increased in number associated with brief recovery of the platelet counts, and reduced to the initial level in about two months. After the successful re-eradication, the numbers of Tregs and platelets have been restored. From these results it is shown that HP eradication can modulate Tregs to increase the platelet counts for ITP patients. We also demonstrate that the increase of Tregs by HP eradication was more rapid than that by rituximab, which required about three months. Further, it might be suggested that there are two phases of the therapy with HP eradication for ITP. In an initial stage, the therapy itself could have an effect modulating the immune systems to potentiate Tregs. Some drugs, such as macrolide antibiotics including clarithromycin have been demonstrated to be a potent immunomodulator. However, this phase is not sufficient for the successful treatment, as shown in the cases of failure in HP elimination. In a retentive stage, extermination of HP is also necessary for sustainment of restored Tregs.

Description: Abstract 5000 AIM The anti-CD20 antibody Rituximab has improved the disease-free survival and overall survival of patients with several types of B-cell lymphoma, in which CD20 is expressed on the cell surface and in the cytoplasm. Flow cytometry indicated that CD20 expression varies from null, weak to strong. Despite its clinical importance, the influence of CD20 expression levels on lymphoma therapy has not been well elucidated. Patients, Material and Methods 214 cases were analyzed, all newly diagnosed with B-cell lymphoma at our institute from January in 2002 to April in 2009. All were biopsied and analyzed by flow cytometry. The biopsy specimens were fixed in formalin and stained with Hematoxylin-Eosin; they were also immunostained. Histological subtypes were defined according to the World Health Organization Classification Ver. 3. The mean florescence intensities of CD20 and CD19 were determined by flow cytometry, and cytoplasmic expression of CD20 was determined by immunohistochemistry. 1) The cases were categorized as follows: A: CD20-negative, B: CD20/19 less than 20%, C: CD20/19 20-50% and D: CD20/19 more than 50%. Patients' backgrounds, pathological diagnoses, primary lesions, etc. were also analyzed. 2) Among DLBCL cases, 76 treated with R-chemo were selected and analyzed with respect to treatment response and overall survival. Results 1) Diagnoses: DLBCL 128, Follicular lymphoma 58, MALT 7, CLL 4 and so on. Among the DLBCL cases, immunohistochemistry indicated six (4%) were CD20-negative and three were CD20-positive; the ages in the CD20-negative DLBCL group tended to be high (74.28 vs 64.36, p=0.06, t-test). Weak CD20 expression was observed in 15 cases (B: 4, C:11). No statistically significant differences were seen with respect to gender, IPI, clinical stage, biopsy lesion, CD5 expression or Karyotype. No FL cases were CD20-negative, but two ( 3.4%) showed low CD20 expression. 2) Kaplan-Meier analysis of 76 cases of DLBCL treated with R-Chemo showed a significantly lower overall survival for Group A, (CD20-negative) while there was no significant difference in overall survival of Groups B+C(CD20-weak) and D(CD20-normal). Our results indicate that even patients with lower levels of CD20 expression in B-cell lymphoma may respond favorably to anti-CD20 therapy. Further studies will be necessary to explore this possibility. Disclosures No relevant conflicts of interest to declare.

Description: It is sometimes confusing to distinguish idiopathic thrombocytopenic purpura (ITP) from thrombocytopenia due to dysmegakaryopoiesis, as seen in myelodysplastic syndrome (MDS) patients, especially MDS with isolated thrombocytopenia. In this study, we investigated the useful parameters for the different diagnosis of thrombocytopenia. The number of reticulated platelets reflects the rate of thrombopoiesis, and this clinical utility has been established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, such as autoimmune thrombocytopenic purpura (AITP). However, the number of reticulated platelets has not been well investigated in the patients with myelodysplatsic syndrome (MDS), while some of them are misdiagnosed as ITP. The aim of this study is to evaluate the diagnostic utility of the measurements of reticulated platelets as well as other parameters of platelets, such as MPV (mean platelet volume), P-LCR (platelet larger cell ratio) and PDW (platelet distribution width). The reticulated platelets, expressed as the immature platelet fraction (IPF) were determined in 108 ITP and 57 MDS patients using the Sysmex XE-2100 blood cell counter with upgraded software (Sysmex, Kobe, Japan). This system enabled rapid, inexpensive, automated, stable measurements of reticulated platelets compared with the flow cytometry system, of which consensus method has not yet been identified to provide acceptable intra- and inter-laboratory results. The platelet counts in ITP and MDS patients were equivalent (ITP, 7.99 ± 0.40 × 104/μL; MDS, 8.05 ± 0.57× 104/μL). The IPF values in ITP patients (10.4 ± 0.61%) were significantly higher than those in MDS (5.82 ± 0.63%), and the inverse correlation between the IPF and the platelet counts was observed among the ITP patients, but not among the MDS. Both MPV and PDW in MDS (10.6 +/− 0.15 fL and 12.2+/−0.41 fL, respectively) were significantly higher than in ITP (7.7 +/− 0.38 fL and 9.4 +/− 0.48 fL, respectively), while P-LCR in MDS (28.7 +/− 1.2%) and ITP (23.6 +/− 1.3%) were not significantly different. Although MPV was correlated with IPF among either group, the correlation between IPF and either PDW or P-LCR was weak among MDS (IPF × PDW, r=0.673; IPF × P-LCR, r=0.660) compared with ITP (IPF × PDW, r=0.779; IPF × P-LCR, r=0.803). Next we precisely investigated the clinical features of the minor population of MDS with higher IPF. Most of these patients revealed the significantly higher values of PAIgG (Platelet-associated IgG) and/or poor response to the blood transfusion, suggesting the possibility of associated autoimmune mechanisms. The patients of MDS in overt leukemic stage also recorded higher IPF even if they had no or few blood transfusion. The IPF would be a useful parameter to distinguish ITP from MDS with isolated thrombocytopenia, which has been shown to have a favorable prognosis.

Description: Rituximab is commonly used for treatment of B-cell leukemia/lymphoma and its efficiency on CD20+ B-cell malignancies has been established. On the other hand, the effect of rituximab on CD20+ T cell leukemia/lymphoma is unclear. Two patients CD20+ T-cell malignancy (1 with T-cell prolymphocytic leukemia (T-PLL) and 1 with peripheral T-cell lymphoma, unspecified (PTCL-u)) were recently treated with rituximab without appreciable effects. The treatment failure prompted a study of the mechanisms underlying the resistance to rituximab therapy using leukemia/lymphoma cells of these patients and another CD20+ T-PLL patient. Patient 1: A 68-year old Japanese male was diagnosed as having T-PLL (small cell variant) characterized by CD2+CD3+CD4− CD5+CD8+CD20+TCR-Vβ8+. Southern blotting of these T-PLL cells showed a TCR-β gene rearrangement. The systemic lymphadenopathy and high-fever of the patient were ameliorated by the administration of prednisolone (20 mg/d). Rituximab was administered weekly, but no clinical improvement was observed. In vitro treatment of the T-PLL cells with rituximab did not show any complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Patient 2: A 75-year old Japanese male was diagnosed as having T-PLL (small cell variant) with a phenotype of CD2+CD3+CD4+CD5+CD8+CD20+. Southern blotting of these T-PLL cells showed a TCR-β gene rearrangement. The patient is being followed without treatment because his pancytopenia is non-progressive. Patient 3: A 67-year old Japanese male developed a left thigh tumor which proved to be PTCL-u. The phenotype of lymphoma cells was CD2+CD3−CD4−CD5−CD8+CD20+ and Southern blotting of these cells showed a TCR-β gene rearrangement. The patient’s PTCL-u was refractory to CHOP and rituximab plus EPOCH therapy. The reactivity of various monoclonal Abs specific to CD20 molecules against these patients’ leukemia/lymphoma cells is summarized in Table 1. CD20 molecules of these 3 patients’ leukemia/lymphoma cells were detectable by flow cytometry using anti-CD20 mAbs (L27 and rituximab) and also by Western blotting using anti-CD20 mAbs which recognize N-terminal region of CD20. However, the CD20 molecules could not be revealed either by immunohistochemistry using anti-CD20 mAbs (L26) or by Western blotting using anti-CD20 mAbs which recognize C-terminal region of CD20. The sequence of the full-length CD20 cDNA derived from Patient 1’s T-PLL cells proved to be intact. These results indicate that the CD20 molecules of the 3 patients’ leukemia/lymphoma cells have defects in the cytoplasmic C-terminal region which may impair the cytotoxic effect of rituximab such as CDC and ADCC and the defect may be due to post translational changes such as phosphorylation of serine and threonine residues. Table 1. Reactivity to different Abs specific to CD20 Pt Age Gender L27 (FACS) Rituximab (FACS) L26 (IHC) C-terminal Ab (WB) N-terminal Ab (WB) FACS, flow cytometry; IHC, immunohistochemistry; WB, Western blotting; +, positive; −, negative; ND, not determined 1 68 M + + − − + 2 75 M + + − − + 3 67 M + ND − − +

Description: The case is a 27-year-old man, who referred to us presenting the mild bleeding tendency after extraction of a tooth. Bleeding time was 3 minutes by the Duke method, and his platelet count was slightly decreased (7x10 10 /l). His mother also has the similar platelet abnormality. They have no albinism. The peripheral smear demonstrated the abnormal platelet morphology with hypo or agranular and large forms. Almost normal platelets were also present on the smear. A platelet adhesion test showed the impaired collagen adhesion. A platelet aggregation study revealed the abnormally poor responses to adenosine diphosphate, collagen, arachidonate, U-46619, and a relatively retained response to ristocetin. Electron microscopy of the platelets demonstrated 2 populations: one with almost normal distribution of the granules, the other with marked decrease in both α and dense granules, even though the former population had the abnormal granules and the obvious vacuoles in the platelets. Flow cytometry revealed the reduced Mepacrine positive platelets and the decreased surface CD62P induced with phorbor ester in the patient. The patient platelets contained the reduced concentration of both adenosine triphosphate and serotonin, compared with the normal control. The cytoskeleton structures were examined with immunocytochemistry using the specific antibodies. Most platelets of the patient lost the typical coiled marginal bands and showed the yarnball-like structures of the microtubules, although the actin microfilaments were relatively retained. In other words, the platelets even with the almost normal distribution of the granules have a disturbed tubulin organization, which were not apparent in the leukocytes. These results suggested that some platelet-specific proteins, such as β1 tubulin, involved in the formation of the microtubules could be causative for this hereditary storage pool deficiency.