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Immune-related pneumonitis is an important toxicity associated with checkpoint inhibitor therapy with anti–PD-1 or anti–PD-L1 antibodies, often necessitating discontinuation of treatment. Development of methods to mitigate checkpoint inhibitor–related pneumonitis is required. The contributions of PD-L1, PD-L2, and VEGF to the pathogenesis of pneumonitis were examined in an IL2- plus IL18-induced mouse pneumonitis model (IL pneumonitis model). Furthermore, the incidences of pneumonitis were retrospectively examined in patients with non–small cell lung cancer treated with the anti–PD-L1 mAb atezolizumab plus chemotherapy, with or without the anti-VEGF mAb bevacizumab, in the phase III IMpower150 trial. PD-1 signal blockade by anti–PD-L1 and anti–PD-L2 antibodies aggravated pneumonitis in the IL pneumonitis model. An anti-VEGF antibody prevented PD-1 signal blockade from aggravating pneumonitis in this model. PD-1 signal blockade induced interstitial T-cell infiltration in the lungs, but VEGF blockade did not affect this T-cell infiltration. The anti-VEGF antibody protected against vascular-to-alveolar leakage of protein and fluid due to PD-1 signal blockade in a murine model. In the IMpower150 trial, incidence rates of pneumonitis of any grade were 4.3% in the group without bevacizumab and 2.8% in the group with bevacizumab. In patients with pneumonitis, outcomes of “Not recovered/Not resolved” were reported for 29.4% in the group without bevacizumab compared with 9.1% in the group with bevacizumab. Our findings suggest that anti-VEGF antibodies in combination with checkpoint inhibitors may be a treatment method that can control checkpoint inhibitor–related pneumonitis.

To investigate the use of high-dose therapy and autologous stem cell transplantation (ASCT) for relapsed or high-risk diffuse large B-cell lymphoma (DLBCL) between 1990 and 2007, we conducted a nationwide survey using the registry database of the Japan Society for Hematopoietic Cell Transplantation. Of the 1222 patients in the database, 576 (47%) received ASCT in first complete remission (CR1), 140 (12%) in first partial remission, 281 (23%) in sensitive relapse, 150 (12%) in resistant or sensitivity-unknown relapse, and 75 (6%) in primary refractory status. With a median follow-up of 22 months, the 2-year overall survival (OS) and progression-free survival rates were 71% and 68%, respectively. The cumulative incidences of 2-year non-relapse mortality and relapse/progression were 6% and 26%, respectively. Relapse/progression after ASCT in the rituximab era (2002-2007) was significantly lower than that in the pre-rituximab era (1990-2001; P < 0.001). Older age, male gender, poor performance status at ASCT, non-CR1 at ASCT, ASCT performed in 1990-2001, and LEED or MCEC regimen were adverse predictors of OS. Because ASCT for newly diagnosed high-risk DLBCL has not been performed recently, a registry database study to assess the impact of ASCT for relapsed or refractory DLBCL in the rituximab era is warranted.

Aim. Iron overload is a life-threatening disorder that can increase the risks of cancer, cardiovascular disease, and liver cirrhosis. There is also a risk of iron overload in patients with chronic kidney disease. In patients with renal failure, iron storage is increased due to inadequate iron utilization associated with decreased erythropoiesis and also to the inflammatory status. To evade the risk of iron overload, an accurate and versatile indicator of body iron storage in patients with iron overload is needed. In this study, we aimed to find useful iron-related parameters that could accurately reflect body iron storage in mice in order to construct a murine model of iron overload. Methods. To select an appropriate indicator of body iron status, a variety of parameters involved in iron metabolism were evaluated. Noninvasively measured parameters were R1, R2, and R2∗ derived from magnetic resonance imaging (MRI). Invasively measured parameters included serum hepcidin levels, serum ferritin levels, and liver iron contents. Histopathological analysis was also conducted. Results/Conclusion. Among the several parameters evaluated, the MRI T2∗ relaxation time was able to detect iron storage in the liver as sensitively as serum ferritin levels. Moreover, it is expected that using an MRI parameter will allow accurate evaluation of body iron storage in mice over time.

This multicenter phase II study (UMIN000008145) aims to investigate the efficacy and safety of six cycles of combination therapy (RBD) comprising rituximab, bendamustine, and dexamethasone (DEX) for relapsed or refractory (RR) indolent B-cell non-Hodgkin lymphoma (B-NHL) and mantle cell lymphoma (MCL). Although the initial study protocol comprised 20 mg/body DEX on days 1 and 2, and 10 mg/body on days 3-5 [high-dose (HD-) DEX group], the dose of DEX was later decreased to 8 mg/body on days 1 and 2 [low-dose (LD-) DEX group] due to frequent cytomegalovirus (CMV) antigenemia and recurrent retinitis. We enrolled 33 patients, and LD-DEX and HD-DEX were administered in 15 and 18 patients, respectively. The overall response and the 3-year progression-free survival rates were 88% and 75.5%, respectively. The leading adverse event was myelosuppression. Incidence of grade 3-4 leukocytopenia, neutropenia, and lymphocytopenia was 55%, 67%, and 91%, respectively. The most frequent nonhematological adverse events were CMV antigenemia and rash (33% and 30%, respectively). Incidence of CMV antigenemia over 10/100,000 white blood cells was significantly lower with LD-DEX than that with HD-DEX (P = 0.0127). In conclusion, RBD showed significant effectiveness for RR indolent B-NHL and MCL.

Anti-PD-L1 antibodies benefit many cancer patients, even those with “non-inflamed tumor”. Determining which patients will benefit remains an important clinical goal. In a non-inflamed tumor mouse model, we found that PD-L1 was highly expressed on antigen-presenting cells (APCs) especially on CD103+ CD11c+ dendritic cells in tumor-draining lymph nodes (dLNs), suppressing T-cell priming by APCs. In this model, anti-PD-L1 antibodies enhanced T-cell priming and increased CXCR3+ activated T-cells in dLNs, which was followed by the trafficking of T-cells to tumors in response to CXCR3 ligands. As predictive biomarker, each APCs-related gene expression (AP score) alone or T-cells trafficking-related chemokine gene expression (T score) alone were still less than perfect among the 17 mouse models examined. However a combining score of AP score and T score (AP/T score) precisely identified anti-PD-L1-sensitive tumors. In the phase 3 trial of atezolizumab vs docetaxel in advanced NSCLC patients (OAK), the AP/T score could identify atezolizumab-treated NSCLC patients who achieved significant improvement in overall survival. This biomarker concept would be a clinically valuable for prediction of anti-PD-L1 antibody efficacy.

We herein report the rare case of a 72-year-old female who presented with paraneoplastic pemphigus (PNP) and bronchiolitis obliterans (BO) associated with follicular lymphoma (FL), who was successfully treated with obinutuzumab (GA101; G) and bendamustine (B). The patient had severe erosive stomatitis and bilateral conjunctival hyperemia that persisted for more than 6 months. A huge mass was found in the abdominal cavity, and a biopsy revealed grade 1 FL (stage IV). Based on a lip biopsy result, the patient was diagnosed with PNP associated FL. The patient received bendamustine and obinutuzumab (BG) chemotherapy and FL and PNP responded very well, but BO was additionally associated during the course of BG. BO progressed without exacerbation as BG therapy progressed to a 2 year maintenance therapy with G, and combination of azithromycin, inhaled bronchodilator therapy, and corticosteroid. She was followed up at the outpatient department with no pulmonary function decline or FL and PNP recurrence. Our case suggests that BG could be a promising treatment option for PNP and BO.

The anti-VEGF humanized antibody bevacizumab suppresses various malignancies, but tumors can acquire drug resistance. Preclinical studies suggest myeloid-derived suppressor cells (MDSCs) may be associated with tumor refractoriness to anti-VEGF treatment. Here we report a novel mechanism of tumor escape from anti-VEGF therapy. Anti-VEGF treatment enhanced intratumoral recruitment of CD11bhigh/Gr-1high polymorphonuclear (PMN)-MDSCs in anti-VEGF-resistant Lewis lung carcinoma tumors. This effect was diminished by the anticancer agent capecitabine, a pro-drug converted to 5-fluorouracil, but not by 5-fluorouracil itself. This process was mediated by enhanced intratumoral granulocyte-colony stimulating factor expression, as previously demonstrated. However, neither interleukin-17 nor Bv8, which were previously identified as key contributors to anti-VEGF resistance, was involved in this model. Capecitabine eliminated PyNPase-expressing MDSCs from both tumors and peripheral blood. Capecitabine treatment also reversed inhibition of both antitumor angiogenesis and tumor growth under anti-VEGF antibody treatment, and this effect partially inhibited in tumors implanted in mice deficient in both PyNPases. These results indicate that intratumoral granulocyte-colony stimulating factor expression and CD11bhigh/Gr-1high PMN-MDSC recruitment underlie tumor resistance to anti-VEGF therapy, and suggest PyNPases are potentially useful targets during anti-angiogenic therapy.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Background Bevacizumab in combination with chemotherapeutics has shown significant survival benefit in clinical studies in patients with non-small cell lung cancer (NSCLC). Since bevacizumab was administered as standard treatment until disease progression, the importance of bevacizumab during the maintenance phase was not prospectively investigated in these studies. Materials and methods Three human NSCLC cell line xenograft models were used to investigate antitumor effect of bevacizumab and tumor microvessel density (MVD) was analyzed. Results In A549 and NCI-H292 models, bevacizumab maintenance following combination with paclitaxel exhibited stronger tumor suppression than its absence. In an NCI-H292 model, bevacizumab maintenance continuously inhibited increase of MVD. In an NCI-H2228 model following induction treatment with pemetrexed and bevacizumab, maintenance with pemetrexed plus bevacizumab, had stronger efficacy than pemetrexed alone and led to lower MVD and level of thymidylate synthase. Conclusion Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of maintenance treatment containing bevacizumab.

Silicon interposer (Si-IP) technology has been used in accelerated processing units such as graphic processing units in high-performance computing because it can package a system-on-chip and high bandwidth memories. However, the conventional Si-IP has difficulty developing larger packages because of the mismatch in the coefficient thermal expansions (CTE) of the Si-IP and the organic substrate. Therefore, the Si-IP has limited capacity for improving computing performance by the application which requires more chips. We developed a multilayer glass substrate (Glass-ST) that features a stacked glass core and propose to apply this Glass-ST to a computer board. The proposed structure has no CTE mismatch and can use high density wiring. Thus, the Glass-ST enables the assembly of more large chips than is possible using the conventional Si-IP. In this study, we prepared a 100X100 mm Glass-ST with a 5/5 µm line/space and 20 µmΦ vias. We mounted nine 21X21 mm chips with 40 µm pitch micro bumps. The results revealed that conformal plated through glass vias and a fine wiring pattern had been fabricated in the Glass-ST, and that the nine chips and Glass-ST were connected by micro bumps. The maximum warpage of the nine chips was 23 µm between temperatures of 30°C and 250°C. This means that the Glass-ST can mount chips with micro bumps due to the very slight resulting warpage. In addition, we performed thermomechanical simulation to investigate the stress experienced by the micro bumps. The results show that the maximum stresses of micro bumps with pitches ranging between 10 µm and 55 um are very similar to that of 40 µm pitch micro bumps with which the real sample was packaged. We believe the improvements in the computing performance are significant by the Glass-ST technology compared to that of the conventional Si-IP technology.

Vascular endothelial growth factor (VEGF)-neutralizing therapy with bevacizumab has become increasingly important for treating colorectal cancer. It was demonstrated that second-line chemotherapy together with bevacizumab after disease progression (PD) on first-line therapy including bevacizumab showed clinical benefits in metastatic colorectal and breast cancers (ML18147 trial, TANIA trial). One of the rationales for these trials was that the refractoriness to first-line therapy is caused by resistance to not so much bevacizumab as to the chemotherapeutic agents. Nevertheless, resistance to bevacizumab cannot be ruled out because VEGF-independent angiogenesis has been reported to be a mechanism of resistance to anti-VEGF therapy. In this study, we used a xenograft model with the human colon cancer HT-29 cells to investigate the mechanisms underlying the effect of continued administration of bevacizumab plus capecitabine even after resistance to bevacizumab was acquired. The combination of capecitabine plus bevacizumab exhibited significantly stronger antitumor and anti-angiogenic activities than did monotherapy with either agent. Capecitabine treatment significantly increased the intratumoral VEGF level compared with the control group; however, the combination with bevacizumab neutralized the VEGF. Among angiogenic factors other than VEGF, intratumoral galectin-3, which reportedly promotes angiogenesis both dependent on, and independently of VEGF, was significantly decreased in the capecitabine group and the combination group compared with the control group. In an in vitro experiment, 5-fluorouracil (5-FU), an active metabolite of capecitabine, inhibited galectin-3 production by HT-29 cells. These results suggested that capecitabine has a dual mode of action: namely, inhibition of tumor cell growth and inhibition of galectin-3 production by tumor cells. Thus, capecitabine and bevacizumab may work in a mutually complementary manner in tumor angiogenesis inhibition to overcome the resistance caused by angiogenic factors other than VEGF. These results suggest the clinical relevance and the mechanism of action of treatment with bevacizumab in combination therapy beyond PD.

To investigate the use of high-dose therapy and autologous stem cell transplantation (ASCT) for relapsed or high-risk diffuse large B-cell lymphoma (DLBCL) between 1990 and 2007, we conducted a nationwide survey using the registry database of the Japan Society for Hematopoietic Cell Transplantation. Of the 1222 patients in the database, 576 (47%) received ASCT in first complete remission (CR1), 140 (12%) in first partial remission, 281 (23%) in sensitive relapse, 150 (12%) in resistant or sensitivity-unknown relapse, and 75 (6%) in primary refractory status. With a median follow-up of 22 months, the 2-year overall survival (OS) and progression-free survival rates were 71% and 68%, respectively. The cumulative incidences of 2-year non-relapse mortality and relapse/progression were 6% and 26%, respectively. Relapse/progression after ASCT in the rituximab era (2002–2007) was significantly lower than that in the pre-rituximab era (1990–2001; P

Introduction Complications such as severe infection may occur during the chemotherapy of malignant lymphoma. Phlegmonous gastritis (PG) is a rare acute bacterial infection associated with high mortality, requiring early diagnosis, and prompt management. In addition, Guillain-Barre syndrome (GBS) occasionally requires early treatment and intensive care management due to the occurrence of severe neuropathy and respiratory failure. Patient concerns A 70-year-old male was diagnosed with primary gastric diffuse large B-cell lymphoma (DLBCL) after the detection of several polypoid tumors with ulcers. The patient underwent chemotherapy for DLBCL and exhibited adverse effects (i.e., fever, vomiting, epigastric pain, and neutropenia). Computed tomography indicated widespread thickening in the gastric wall. Furthermore, approximately 2 weeks later, the patient presented with gradual symmetric lower extremity weakness and respiratory failure due to paralysis of the respiratory muscle. Diagnoses DLBCL was diagnosed through a gastric tumor biopsy. On the basis of the computed tomography findings, a culture of gastric juice, nerve conduction studies, and clinical symptoms, this case of gastric lymphoma was complicated with PG and GBS. Interventions The patient was treated with antimicrobial therapy and administration of granulocyte colony-stimulating factor for PG, and with intravenous immunoglobulin and intensive care management for GBS. Outcomes Despite the aggressive progress of the condition, the patient improved without relapse of DLBCL. Conclusion PG was regarded as a precedent infection of GBS. In this article, we present the first reported case of gastric lymphoma complicated with PG and GBS.

Background Immune checkpoint inhibitors have marked antitumor effect. However, monotherapy benefits only a limited population of patients, and further improvement of their effects is required. Here the therapeutic effect and immune response during anti-PD-L1 plus cisplatin combination therapy were investigated in a mouse model. Materials and methods E.G7-OVA, expressing ovalbumin (OVA) gene as a model tumor antigen, was subcutaneously inoculated into syngeneic mice and treated with anti-PD-L1 with/without cisplatin. The tumor growth and activation status of immune cells were evaluated. Results The anti-PD-L1 plus cisplatin combination resulted in a potent antitumor effect leading to tumor shrinkage compared to anti-PD-L1 or cisplatin alone, even though each alone, significantly inhibited tumor growth compared to the control group. During treatment, all groups, including that treated with cisplatin alone, had increased CD8+ T-cell infiltration into tumor tissues compared with the control group, and the therapeutic effect was diminished by CD8+ cell depletion. Aside from its direct cytotoxic effect, cisplatin alone increased chemokine levels and expression of immune checkpoint molecules on CD8+ T-cells in the tumor site. The combination effectively activated OVA-specific CD8+ T-cells. Furthermore, anti-PD-L1 alone and in combination with cisplatin, but not cisplatin alone, induced interferon-gamma-producing CD4+ T-cells. Conclusion These findings provide a rationale for anti-PD-L1 plus cisplatin becoming a promising combination therapy for patients with cancer.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL.

We describe a 57-year-old woman who was diagnosed as precursor B-cell acute lymphoblastic leukemia with marked eosinophilia (ALL-eo). She presented with low grade fever and eosinophilia (absolute count 16.5 × 109/l). Most of eosinophils had hypogranular cytoplasm. Immature cells were absent in her peripheral blood. Since her platelet count was low, bone marrow examination was carried out. 57.2 % of nucleated cells were blastic cells positive for CD10, 19, and 20. Chromosomal analysis revealed a karyotype of 46,XX,t(5;14)(q31;q32). Despite induction chemotherapy, her disease progressed and she died of sepsis a month later. ALL-eo is extremely rare and the diagnosis might be delayed unless leukemic cells are seen in peripheral blood. Therefore, bone marrow should be examined as soon as possible in cases with eosinophilia not only for the differential diagnosis of eosinophilic disorders but also not to overlook ALL-eo.

In recent years, the transmission losses induced by surface roughness of conductors in printed circuit boards (PCB) have become important from the standpoint of signal integrity. To clarify this effect, current measurement methods use a microstrip line or a strip line in the PCB, which is fabricated for different values of surface roughness. However, since errors are induced in each sample because of the presence of dispersion in the manufacturing process, it is difficult to extract the effect of surface roughness from the measured data. In this paper, we propose a novel method to measure the effect by means of using capacitive coupling in which the dispersion errors can be eliminated. The results have been verified using electromagnetic simulation. This method was performed on three samples having differing values of surface roughness. It was concluded that the transmission characteristics are influenced by surface roughness at higher frequencies when the skin depth becomes comparable to the surface roughness.

Diabetes mellitus (DM) is a major cause of end-stage kidney disease (ESKD). However, the difference in renal outcomes between DM patients with non-diabetic renal disease (DM and NDRD) and those with diabetic nephropathy (DN) is controversial. The aim of the present study was to evaluate the differences among patients with DN, DM, and NDRD, and non-DM chronic kidney disease (CKD) in a prospective observational study. We extracted the data of 2484 patients from 11 nephrology care centers and categorized into three groups as described above. The primary outcome was ESKD requiring renal replacement therapy. During the median follow-up of 4.44 years, 281 patients (11.3%) developed ESKD. Renal outcomes of DM and NDRD patients were similar to those of non-DM patients (p ≥ 0.05). At CKD stage G3b, the hazard ratios (95% confidence intervals) of ESKD were 7.10 (2.46–20.49) in DN patients and 0.89 (0.19–4.24) in DM and NDRD. The annual change in the estimated glomerular filtration rate (eGFR) in DN patients was significantly larger than that in other groups at stage G3b (−9.7%/year). We found that DN patients have a higher risk for ESKD than DM and NDRD or non-DM patients. In particular, GFR rapidly declined in DN at stage G3b. DM and NDRD patients can accomplish equally beneficial renal outcomes as non-DM CKD, regardless of their similar metabolic profiles as DN. In conclusion, we should prudentially consider the risk stratification of DM whether cause or comorbidity of CKD.

We conducted a retrospective collaborative investigation of bortezomib (Bor) plus dexamethasone (Dex) therapy (BD Tx) for 88 relapsed or refractory (Rel/Ref) MM patients at six institutes. One cycle BD Tx comprised of Bor (1.3 mg/m²/day) on days 1, 4, 8 and 11, and Dex on days 1, 2, 4, 5, 8, 9, 11 and 12, every 21 days, and the mean number of BD Tx cycles was 3. The overall response rate was 66.9%, the median overall survival (OS) was 510 days, and the median progression-free survival (PFS) was 113 days. Attainment of partial response (PR) with the first course of BD Tx associated with the longer OS and PFS and late good responder, while no patient who did not achieve PR with the first cycle attained better than very good PR (VGPR) with the subsequent BD Tx. Patient age of less than 64 years old also associated with the longer OS and PFS. In addition, both an earlier disease stage and Dex dosage had a significant impact on OS, while the attainment of VGPR within 2 cycles had a significantly longer PFS. Earlier BD Tx courses may be predictive for the subsequent therapeutic pathway of Rel/Ref MM.

Erlotinib treatment in combination with gemcitabine is a standard therapy for patients with locally advanced pancreatic cancer in many countries, including the US and the EU. Since mutations of the K-ras oncogene (KRAS) occur in approximately 90% of pancreatic cancers, we examined the antitumor activity of erlotinib in combination with gemcitabine in KRAS-mutated pancreatic cancer cell lines, HPAC and Capan-1, which have the KRAS mutation G12D and G12V, respectively. We analyzed the mode of inhibition of in vitro tumor cell proliferation by means of a combination index and found that a combination treatment of erlotinib plus gemcitabine had an additive effect in the two cell lines. We then examined the effect of erlotinib and gemcitabine on the phosphorylation of epidermal growth factor receptor (EGFR). Erlotinib strongly suppressed, while gemcitabine augmented the phosphorylation of EGFR, which was completely blocked by erlotinib in the two cell lines. An in vivo tumor growth inhibition test was then performed using the HPAC tumor xenograft model. The combination therapy of erlotinib and gemcitabine resulted in a significant inhibition of tumor growth compared with erlotinib or gemcitabine monotherapy. To the best of our knowledge, this is the first study to show the combination effect of erlotinib and gemcitabine in vivo using a xenograft model of a KRAS-mutated pancreatic cancer cell line.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) has been applied to patients with diffuse large B-cell lymphoma (DLBCL); it is well established that ASCT shows significant survival benefits for chemosensitive relapse. However, half of relapsed patients are resistant to salvage chemotherapy, indicating that they are not suitable for ASCT. We retrospectively analyzed the clinical records of 47 patients with DLBCL classified as high or high-intermediate (higher) risk, according to the International Prognostic Index, who underwent upfront ASCT in first complete remission (CR1). Compared with 10 patients with similar characteristics who did not receive ASCT, event free survival at 5-year was significantly superior in ASCT group. Toxicity of ASCT was acceptable and therapy-related death was not observed. We therefore propose that upfront ASCT for higher risk DLBCL in CR1 might provide survival benefit, probably because the high-dose therapy removes minimally resided tumor.

Despite recent advances in therapeutic modalities for autoimmune disease (AID) or allogeneic organ transplant, the resultant increase in the number of long-term survivors with sustained immunologic...

We propose a method for measuring effects of surface roughness on transmission characteristics eliminating the manufacturing error. We verified the structure using electromagnetic simulation and measured three different types of surface roughness.

Objective We retrospectively investigated the efficacy and predictive factors for the treatment outcomes of bortezomib plus dexamethasone (BD) as second-line induction therapy prior to high-dose chemotherapy sup- ported by autologous stem cell transplantation (HDT/ASCT) in multiple myeloma (MM) patients. Methods Sixty-six transplant eligible MM patients treated by the Kyoto Clinical Hematology Study Group between 2006 and 2011 were investigated. Conventional induction chemotherapy, including vincristine, dox- orubicin and dexamethasone (VAD) and high-dose dexamethasone (HDD), was used as first-line induction therapy in all patients, seven (10.6%) of whom attained a very good partial response (VGPR). Of the 59 pa- tients who did not attain VGPR with VAD or HDD, 33 were given BD as second-line induction therapy prior to HDT/ASCT. Results Patients not treated with BD induction showed an overall response rate (ORR, i.e., better than par- tial response) of 85.3% after induction therapy, while the ORR of patients treated with BD induction im- proved from 42.4% after conventional induction therapy to 84.8% after BD. The overall survival (OS) and progression-free survival (PFS) of patients not treated with BD induction were not significantly influenced by the response to induction therapy. Among the patients treated with BD, failure in attaining VGPR prior to ASCT was associated with a significantly shorter PFS and it also tended to show a shorter OS, while the dis- ease stage and achievement of a complete response after HDT/ASCT had no impact on OS or PFS. Conclusion The achievement of at least VGPR with second-line BD induction therapy is a prerequisite for attaining longer OS and PFS after HDT/ASCT.

The incidence of Hodgkin lymphoma (HL) is paradoxically increasing in the combination anti-retroviral therapy (cART) era. However, there has been no nationwide survey of human immunodeficiency virus (HIV)-associated HL (HIV-HL) in Japan. We retrospectively examined the clinical characteristics and outcomes of 19 newly diagnosed HIV-HL patients at 11 HIV/AIDS and hematology regional hospitals in Japan between 1991 and 2010. At the time of HL diagnosis, 79 % of patients were receiving cART. All the patients, but one received HL diagnoses in the cART era. The median CD4+ cell count at HIV-HL diagnosis was 169/μl. Mixed-cellularity classical Hodgkin lymphoma was the most common subtype occurring in 68 % of the patients; 89 % of the patients were positive for Epstein–Barr virus. Of these 19 patients, 84 % were in advanced stages, with bone marrow involvement observed in 47 % of the patients; 58 % had extranodal sites. All the treated patients were given cART concurrent with HL therapy. The complete remission rate of the treated patients was 87 %. The median OS of the entire cohort was 17 months. These results suggest that the characteristics of HIV-HL in Japan are more aggressive than those of non-HIV-associated HL in Japan, but standard chemotherapy is effective and feasible.

Recombinant human erythropoietin (rhEPO) has been used in the EU and the United States for the treatment of anemia in cancer patients after myelosuppressive chemotherapy or radiotherapy. However, several conflicting results have been reported concerning the detrimental effect of rhEPO on survival benefit in cancer patients. In experimental studies, contradictory results were also reported in in vitro tumor cell proliferation studies and in vivo tumor growth studies using tumor cells expressing EPO-receptor (EPO-R). Therefore, we tried to clarify the effect of epoetin β, a product of rhEPO, on tumor growth in xenograft models using five EPO-R-positive human cancer cell lines, namely the MCF7 breast, 786-O renal, SCH gastric, A549 lung and SK-OV-3 ovarian cancer cell lines. Epoetin β was administered once a week for 3 weeks at doses of 1,000, 3,000 and 10,000 IU/kg in accordance with the clinical administration schedule and dosages. As a result, no enhancement of tumor growth from the administration of epoetin β was observed in any of the xenograft models throughout the experiment duration. The effect of epoetin β on the antitumor activity of bevacizumab, an anti-angiogenic agent, was additionally examined using A549 and MCF7 xenograft models, since rhEPO reportedly stimulates tumor neovascularization. Epoetin β showed no significant effect on the antitumor activity of bevacizumab in either xenograft model. These findings suggest that epoetin β is not involved in in vivo tumor growth promotion.

We demonstrate, for the first time, carbon nanotube (CNT) flip chip bumps for LSI modules. The CNT bump is composed of a bundle of multi-walled CNTs. Resilient and flexible CNT bumps make flip chip LSI modules resistant to thermal stress. Furthermore, CNT bumps have a low electrical resistance and robustness over electromigration. In the experiment, the CNT bumps were used to connect a test evaluation group (TEG) chip and a host substrate, and their electrical resistance was evaluated. We found that the electrical contacts of CNT bumps with the chip and the substrate are important. For a good electrical contact, the CNT bumps were coated with gold and fixed to the chip and substrate. The resultant CNT bump with a diameter of 170 mum and a height of 100 mum exhibited a low resistance of 2.3 Omega. We then evaluated the flexibility of CNT bumps by pressing the TEG chip and measuring the displacement. The displacement between the TEG chip and host substrate was 10-20% of the bump height, demonstrating an excellent flexibility.

Background The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects for non-small cell lung cancers (NSCLC) harboring EGFR activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs within several years. The major mechanisms of acquired resistance including 2nd mutation of T790M and amplification of MET have been identified. However, the mechanisms of other resistance remain unclear. In this study, we established novel erlotinib-resistant NSCLC cells and examined their resistant mechanisms. Methods NSCLC HCC827 cells have an EGFR Exon 19 deletion and gene amplification and are highly sensitive to erlotinib. The resistant cells were established by continuously exposing HCC827 cells to 0.1, 1 or 10 µM of erlotinib in 96 well plates for three months. The resistant mechanisms were determined by direct sequence analysis and EGFR FISH analysis. Results The fourteen and three resistant cells were established by 0.1 µM and 1 µM of erlotinib exposure, respectively. No resistant cells appeared in the wells of 10 µM of erlotinib. The IC50 values of these resistant cells were more than 25-fold higher than that of parental cells. No 2nd mutation of T790M was detected in any of the resistant cells and MET gene amplification was detected in only one resistant cells. Instead, we found that 13/17 resistant cells were dominated by EGFR not amplified cells and one of resistant cells B10 consisted of more than 99.9% of EGFR not amplified cells, and that the parental cells consisted of only 2.5% of EGFR not amplified cells and 97.5% of EGFR amplified cells. EGFR not amplified clone 4D8 isolated from parental cells and showed resistance to erlotinib comparable to the resistant cells B10. Furthermore, we found that EGFR not amplified cells were constantly emerged from EGFR amplified clone isolated from parental cells under normal cell culture condition. Conclusion Loss of amplified EGFR gene with mutation causes acquired resistance in HCC827 cells when exposed to relatively low concentration of erlotinib while high concentration of erlotinib deprives HCC827 cells of the chance of emergence of resistant cells. Disclosure All authors have declared no conflicts of interest.

Introduction: Usefulness of autologous stem cell transplantation (ASCT) for diffuse large B-cell lymphoma (DLBCL) has been established. However, relapse after ASCT even at complete remission (CR) remains to be crucial. We evaluated outcome of such patients concerning the intervals from ASCT to relapse. Patients and methods: Among 77 patients who received ASCT at CR between 1997 and 2013, 26 relapsed. Their age ranged from 39 to 74 with a median of 58. They included 23 at first CR and 3 second CR. We retrospectively analyzed clinical records to investigate survival outcome. Results: Induction chemotherapy was CHOP-like regimen with or without rituximab (R). 3 patients with second CR had received MECP as re-induction therapy. Stem cells were mobilized by high-dose etoposide and MCVC was used as a conditioning regimen. Most patients had nodal relapse, but 4 patients (15.4%) at central nervous system and one (3.8%) at testis. Patients had received 1 to 4 regimens of salvage therapy, i. e., MECP, CHASE, or DHAP. CR was achieved in 3 patients (11.5%) and PR in 2 (7.7%), resulting in overall response rate of 19.2%. With observation periods of 4 to 198 months (median 22), 1-year overall survival (OS) was 50%. Although OS was significantly poor in patients who relapsed within a year after ASCT, survival from relapse was not different. Discussion: These results suggest that relapse rate after ASCT should be reduced since highly refractory disease might emerge after myeloablative therapy. Deeper remission is required and prophylaxis of sanctuary region appears to be more widely considerable in first-line therapy.

Aim: Bevacizumab (Bev) is a humanized anti-human VEGF monoclonal antibody (Ab), and maintaining VEGF inhibition with Bev plus chemotherapy beyond progression (BBP) has had clinical benefits in patients with metastatic colorectal cancer. However the mechanism of BBP is still unknown and, although some chemotherapeutic agents have been shown to modulate the host immune response against malignancies, the role of chemotherapy in BBP is also unknown. In this study, we examined the mechanism of BBP. Methods: Murine Lewis lung carcinoma (LLC) was s.c. inoculated into C57BL/6 or CCR2-/- knockout (KO) mice. Mice were randomly allocated to control and treatment groups after tumor formation. Anti-VEGF Ab (5 mg/kg, weekly) and anti-G-CSF Ab (10 µg/day, daily) were i.p. administered, and capecitabine (Cape) (718 mg/kg, daily) was p.o. administered for 18 days. Cytokine levels were analyzed by ELISA and cytometric bead array. Myeloid-derived suppressor cells (MDSC) were identified as CD11b +/Gr1 high by flow cytometry. Microvessel density (MVD) was determined by CD31-immunostaining. Results: The LLC model showed resistance to anti-VEGF Ab as previously reported [1]. In this model, anti-VEGF Ab increased the intratumor infiltration by MDSC expressing PD-L1. In addition, a significant increase in G-CSF and CCL2 was observed in the tumors. Although anti-G-CSF Ab did not affect tumor growth, anti-G-CSF combined with anti-VEGF significantly reduced tumor growth compared with anti-VEGF alone. The number of intratumor MDSC was significantly decreased in the combination group compared with the anti-VEGF group. However, no difference in the number of intratumor MDSC induced by anti-VEGF was observed in CCR2 KO mice compared with wild type mice. Adding Cape to anti-VEGF abolished both MDSC and the intratumor G-CSF level, and significantly reduced tumor growth compared with either monotherapy. MVD in the combination group was significantly reduced compared with the anti-VEGF group. Conclusions: Add-on Cape abolished MDSC by decreasing intratumor G-CSF, suggesting that the mechanism of BBP may be that chemotherapy recovers the anti-angiogenic effect of Bev. [1] Nature Biotechnology 25, 911-20 (2007). Disclosure: T. Iwai: COI Disclosure: Research funding from Chugai Pharmaceutical Co., Ltd. All other authors have declared no conflicts of interest.

Multiple myeloma has seldom been reported to complicate tumor lysis syndrome since it is an indolent disease. We report a case with multiple myeloma who rapidly developed tumor lysis syndrome after conventional induction therapy. A 63-year-old Japanese woman presented with dyspnea on effort was diagnosed as IgA-lambda type myeloma. According to the International Staging System, her stage was defined as III because of elevated serum beta-2 microglobulin at 72mg/L. Remission induction consisted of continuous intravenous administration of vincristine of 0.4mg/day and adriamycin of 15mg/day through days 1 to 4 and infusion of dexamethasone of 40mg/day on days 1-4 was given. On day 7, she felt general fatigue and loss of appetite and her serum uric acid, creatinine, and potassium elevated to 898.1μmol/L, 9.8mg/dL, and 6.7mmol/L, respectively. Inversely, serum calcium decreased to 2.0mmol/L. Under the diagnosis of tumor lysis syndrome, she was treated with hydration and diuretics, resulted in immediate regression of her symptoms and recovery of laboratory data. Besides conventional chemotherapy, newly introduced agents have also been reported to cause tumor lysis in myeloma. While previous description has identified renal dysfunction to play a crucial role in the development of tumor lysis syndrome, myeloma often complicates impaired renal function. Since International Staging System is widely applied for myeloma patients, beta-2 microglobulin that represents renal function is thought to be examined in most of them. Taken together, elevated beta-2 microglobulin appears to easily predict a risk for tumor lysis syndrome. Thus, we postulate that prevention for excessive tumor lysis should be considered for myeloma patients with elevated beta-2 microglobulin.

Background Assessing HER2 status accurately and reliably is of great importance in optimizing treatment of HER2-positive breast or gastric cancer. In gastric cancer, for which trastuzumab treatment has been recently approved in Japan, immunohistochemistry (IHC) is the standard method for evaluating the HER2 level. In this study, we examined the impact of formalin fixation conditions on HER2 staining using xenografted tumor samples from mouse models. Methods Mice (BALB-nu/nu) were subcutaneously inoculated with two human gastric cancer cell lines, SCH (score 2+) and SNU-16 (score 1+). Xenograft tumor tissues were collected and left at room temperature for 0, 6 or 24 h before being fixed with 10% neutral buffered formalin for 24 h, 5, 7 or 10 days and then embedded in paraffin. To examine the effect of fixing solution on HER2 IHC, we used 10 and 20% neutral-buffered or non-buffered formalin or Ufix (Sakura Finetek Japan). HER2 IHC was carried out according to the Hercep test. We assessed the effect on IHC by examining the atrophy of tumor cells, autolysis of tumor tissues, immunostaining intensity and immunostaining area. Results Continuous staining of HER2 on the cell membrane with moderate intensity was observed in SCH tumors, whereas partly localized staining on the cell membrane with weak immunostaining intensity was seen in SNU-16. Leaving samples for 6 h before fixation at room temperature decreased immunostaining intensity and induced atrophy of peripheral tumor tissues in both SCH and SNU-16 specimens. Leaving the specimens for 24 h before fixation induced autolysis of tumor tissues and reduced the immunostaining intensity. On the other hand, fixation in 10% neutral buffered formalin for more than 5 days diminished the HER2-stained area in SNU-16 samples. Fixing in 20% neutral buffered or in 10 or 20% non-buffered formalin reduced the staining intensity by prolonged fixation. Ufix did not influence the staining area, but atrophied the tumor cells. Conclusion Our results suggest that the timing of fixation, including when it starts and how long it takes, will influence the HER2 staining. Non-buffered or highly concentrated formalin also might affect the IHC result. It is important to optimize the conditions of sample collection and fixation.

e18091 Background: The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects for non-small cell lung cancers (NSCLC) harboring EGFR activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs within several years. The major mechanisms of acquired resistance are acquisition of a secondary EGFR mutation T790M and amplification of MET. However, the mechanisms of other resistance remain unclear. In this study, we established novel erlotinib-resistant NSCLC cells and examined their resistant mechanism. Methods: Resistant cells were established by continuously exposing EGFR mutated NSCLC cells HCC827 to 0.1, 1 or 10 μM of erlotinib in 96 well plates for three months. The resistant mechanisms were determined by direct sequence analysis and EGFR FISH analysis. Results: Resistant cells were emerged from 14/96 and 3/96 wells by 0.1 μM and 1 μM of erlotinib exposure, respectively. No resistant cells appeared in the wells of 10 μM of erlotinib. The IC50 values of these resistant cells were more than 100-fold higher than that of parental cells. No secondary mutation of T790M was detected in any of the resistant cells. Instead, we found that 13/17 resistant cells were dominated by EGFR not amplified cells and one of resistant cells B10 consisted of more than 99.9% of them, while the parental cells consisted of 2.5% of EGFR not amplified cells. Then, we isolated EGFR not amplified clone 4D8 from parental cells and found that this clone also had a resistance to erlotinib comparable to the resistant cells B10. Metaphase FISH analysis showed that EGFR amplified cells in parental cells had a chromosome 7 containing multiple copies of EGFR, while EGFR not amplified cells in B10 did not have it. Furthermore, we found that EGFR not amplified cells were constantly emerged from EGFR amplified clone isolated from parental cells under normal cell culture condition. Conclusions: Loss of a chromosome 7 containing multiple copies of EGFR causes acquired resistance in HCC827 cells when exposed to relatively low concentration of erlotinib, while high concentration of erlotinib deprives HCC827 cells of the chance of emergence of resistant cells.

Introduction Deletions of chromosome 7 are often detected in myelodysplastic syndrome. The most commonly deleted segments are clustered at band 7q22. A critical gene is therefore suggested to be located in this region. We report a patient with myelodysplastic syndrome whose marrow cells carried an inversion of 7q22 and q36 as a sole karyotypic abnormality. How this extremely rare chromosomal aberration contributes to the pathogenesis of myelodysplastic syndrome should be clarified by accumulating clinical data of such cases. Case presentation A 74-year-old Japanese man presented with pancytopenia incidentally detected by routine medical check-up. His complete blood cell counts revealed that his white blood cells had decreased to 2100/mm3, neutrophils 940/mm3, red blood cells 320×104/mm3, hemoglobin 11.1g/dL, hematocrit 33.1%, and platelets 12.6×104/mm3. Bone marrow examination showed normal cellularity with nucleated cells of 9.4×104/mm3. The proportion of blasts was 4%. A morphological examination showed only basophilic stippling of erythroblasts which was seen as dysplasia. According to World Health Organization classification, the diagnosis was myelodysplastic syndrome-u. Karyotypic analysis showed 46,XY,inv(7)(q22q36) in all of 20 metaphases examined. Additional analysis revealed the karyotype of his lymphocytes was 46,XY. He is asymptomatic and cytopenia has slowly progressed. Conclusions To the best of our knowledge, this karyotype from a clinical sample of de novo malignancies has never been documented although the identical karyotype from secondary myelodysplastic syndrome was reported. Despite the extremely low frequency, inversion of 7q22 appears to play a crucial role for myelodysplastic syndrome in this patient.

A broadband parallel-wire meander line deflection circuit has been devised. It has relatively flat deflection characteristics in the range 4–17.5 GHz. A dispersion equation for this circuit is derived by considering the phase delay at the bend of the wire, which can estimate the experimental results fairly well.