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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.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects against non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs. In this study, we established novel erlotinib resistant NSCLC cells and examined their resistant mechanisms. Resistant cells were established in 14, 3, and 0 wells exposed to 0.1, 1, and 10μM erlotinib, respectively. The IC50 values of these cells were 47- to 1209-fold higher than that of the parent cells. No secondary T790M mutation was detected in any resistant cells. However, in 13/17 resistant cells, EGFR copy number was reduced less than approximately one-eighth of parent cells, and in one resistant cell (B10), >99.99% of the population was EGFR-unamplified cells. Most (97.5%) parent cells showed EGFR amplification, but 2.5% of the population comprised EGFR-unamplified cells. An EGFR-unamplified clone (4D8) isolated from parent cells in erlotinib-free normal medium also showed erlotinib resistance comparable to the resistant B10 cells. Loss of an EGFR-amplified chromosome 7 (EGFR-ampch7) was observed in 4D8 and B10 cells. EGFR-unamplified cells were constantly maintained as a minor population of the parent cells under normal cell culture conditions. In conclusion, loss of an EGFR-ampch7 causes acquired resistance in EGFR-mutated HCC827 cells exposed to a relatively low concentration of erlotinib, but a high concentration prevents the emergence of resistance.

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.

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.