Citations

Abstract The human IGHV4-34 gene encodes antibodies which are intrinsically autoreactive when the VH domain is unmutated. Therefore, B cells expressing IGHV4-34 B-cell receptor immunoglobulins (BcR IG) are normally under close scrutiny in order to avoid unwanted autoreactivity, especially against DNA. The IGHV4-34 gene is frequently utilized in chronic lymphocytic leukemia (CLL), where, typically, it shows a high load of somatic hypermutation (SHM). We have previously reported distinctive SHM patterns amongst IGHV4-34 CLL, especially for subsets with stereotyped BcR IG. However, although a large number of cases (~2000) was previously studied, since even the largest subsets account for only ~3% of CLL, meaningful conclusions could not be reached for smaller subsets. Here we revisit this issue in a series of 16,528 CLL cases and focus on IGHV4-34 expressing subsets: #4 (IGHV4-34/IGHD5-18/IGHJ6 | 156 cases, 0.9%); #11 (IGHV4-34/IGHD3-10/IGHJ4 | 16 cases, 0.1%); #16 (IGHV4-34/IGHD2-15/IGHJ6 | 41 cases, 0.25%); #29 (IGHV4-34/IGHD: unassignable/IGHJ3 | 39 cases, 0.24%); and #201 (IGHV4-34/IGHD: unassignable/IGHJ3 | 43 cases 0.26%). Focusing on codons 27-104 within the VH domain (from CDR1-IMGT to FR3-IMGT), we calculated the sequence distance between subsets and the corresponding IGHV4-34 germline sequence based on a pairwise qualitative and quantitative comparison of the respective amino acid composition. The minimum distance calculated, and hence the greatest identity, was observed between subsets #4 and #16, both concerning IgG-switched cases (IgG-CLL), which is notable given the overall rarity of IgG-CLL. In contrast, the maximum distance, implying the least identity, was between subsets #16 and #201, the latter concerning IgM/D-CLL. Extreme variations between subsets were noted in codons spanning the entire VH domain. This result is consistent with our finding of a subset-biased distribution of mutations over the VH domain. More specifically, while subsets #11, #16, #29 and #201 had a lower frequency of mutations within VH CDR1 compared to VH CDR2, the exact opposite was seen in subset #4, with 40% of mutations in VH CDR1 versus 27% in VH CDR2. In addition, subsets #4, #11, #16 and #29 had a similar distribution of mutations in VH FR2 and VH FR3, in contrast to subset #201 that showed a preference for VH FR3 over VH FR2. Consequently, we noted that certain positions were targeted in a subset-specific manner e.g. codon 28 in VH CDR1 was heavily targeted in subsets #4 (68.6%) and #16 (87.8%), with most cases carrying an acidic amino acid (AA) introduced by SHM, glycine to glutamic acid, G>E: 51.3% for subset #4 and 78% for subset #16. The high prevalence of acidic AA introduced by SHM in these subsets is notable considering the electropositive nature of their VH CDR3 (especially of subset #4), strongly recalling edited anti-DNA antibodies. Interestingly, the G>E change was identified at a much lower frequency in other IGHV4-34 subsets: 18.75% for subset #11; 2.6% for subset #29; 7% for subset #201, all of which carried electronegative VH CDR3. Further, we noted that certain positions were heavily targeted in all subsets e.g. 56-86% targeting for SHM at codon 92 in VH FR3 where serine is encoded by the agc triplet, the ”hottest of hotspots”. This result could be viewed as sequence- rather than subset-dependent and linked to the molecular features of this codon, which is supported by the low targeting of codon 93 (0-6%), also encoding serine by the tct triplet. Other positions were targeted in all subsets but at vastly different frequencies e.g. codon 64 was targeted in 37.8% in subset #4 rising to 100% in subset #29. Finally, positions heavily targeted by SHM in certain subsets were unmutated in other subsets e.g. codon 36 in VH CDR1 remained unmutated in subset #16, in contrast 76.9% of subset #29 were mutated at this position resulting in an AA change. In conclusion, we document different spectra of SHM and AA changes between stereotyped IGHV4-34 CLL subsets. The finding of subset-biased, recurrent AA changes at certain codons indicates that the respective progenitor cells may have responded in a specific manner to the selecting antigen(s), despite expressing the same IGHV gene, indicating a functional purpose for these modifications. This is exemplified by the molecular characteristics of the recurrent AA changes in subset #4, thereby offering interesting pathogenetic hints. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 739 The combination of a calcineurin inhibitor and methotrexate has been the standard of care in graft-vs.-host disease (GVHD) prophylaxis for over 25 years, with resultant rates of grade II-IV acute GVHD between 30–50%. The mTOR inhibitor, sirolimus, has demonstrated promise in a number of Phase II trials as an immunosuppressant used for GVHD prophylaxis. The BMT CTN, sponsored by the NHLBI and NCI, conducted a multicenter, randomized controlled trial comparing the combination of tacrolimus and sirolimus (Tac/Sir) with tacrolimus and methotrexate (Tac/Mtx) as GVHD prophylaxis after matched, related donor (MRD) hematopoietic stem cell transplantation (HSCT). Methods: Eligible patients were between ages 2 – 60 years, and had acute leukemia in remission, myelodysplasia or chronic myeloid leukemia in chronic or accelerated phase. All had adequate organ function, and a 6/6 HLA-A, B, DRB1 matched sibling donor. 304 patients were randomly assigned to either Tac/Sir (n = 151) or Tac/Mtx (n = 153) as GVHD prophylaxis after TBI-based conditioning and MRD HSCT. An intent-to-treat analysis was performed on the primary endpoint of Grade II-IV GVHD-free survival 114 days from randomization. Ten subjects who received busulfan-based conditioning and were previously reported were excluded from analysis. Three subjects who did not undergo HSCT are included in the primary analysis, but not secondary analyses. Results: Treatment groups were well balanced. The median age of participants was 44 years (range 13 – 59) and 83% had acute leukemia. Neutrophil and platelet engraftment were both faster in the Tac/Sir group (14 vs. 16 days, p < 0.001; 16 vs. 19 days, p = 0.03, respectively), but this did not affect the time to first hospital discharge (20 vs. 21 days, p = 0.37). The incidence of grade II-IV and grade III-IV acute GVHD at 100 days were lower in the Tac/Sir group (26 vs. 34%, p = 0.17; 8 vs. 15%, p = 0.05). Day 100 treatment-related mortality was no different between groups (7 vs. 7%, p = 0.43). The primary endpoint of 114-day acute GVHD-free survival was not statistically different between groups (67 vs. 62%, p = 0.38, Figure). The cumulative incidence of relapse at 2 years from transplantation was not different between groups (27 vs. 30%, p = 0.81). The competing-risk cumulative incidence of chronic GVHD was higher in the Tac/Sir arm (54 vs. 43%, p =0.044). Overall toxicities were not different between groups, with two notable exceptions. The peak and average OMAS oral mucositis scores were lower in the Tac/Sir arm (peak 0.70 vs. 0.96, p < 0.001; average 0.31 vs. 0.47, p < 0.001), however, there was an increased rate of the endothelial injury syndromes, veno-occlusive disease (11 vs. 4%, p = 0.03), and thrombotic microangiopathy (5 vs. 1%, p = 0.05) in the Tac/Sir arm. Causes of death were not different between groups. At 2 years from transplantation, disease-free (DFS) and overall survival (OS) were not different between study arms (DFS 53 vs. 53%, p = 0.76; OS 60 vs. 61%, p = 0.44). Conclusions: No difference in 114-day acute GVHD-free survival was noted between treatment arms. Compared with Tac/Mtx in MRD HSCT, Tac/Sir is associated with more rapid engraftment, less severe acute GVHD and oral mucositis, excess chronic GVHD and endothelial injury syndromes, and similar long-term outcomes. Understanding the trade-offs between regimens, Tac/Sir can be used as an alternative to Tac/Mtx in MRD HSCT. Disclosures: Cutler: Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding. Off Label Use: Sirolimus - Prevention of GVHD Tacrolimus - Prevention of GVHD. Waller:Outsuka: Research Funding.

Abstract This study aims to evaluate the efficacy of Nile tilapia skin as a xenograft for the treatment of partial-thickness burn wounds in children. This is an open-label, monocentric, randomized phase II pilot study conducted in Fortaleza, Brazil. The study population consisted of 30 children between the ages of 2 and 12 years with superficial “partial-thickness” burns admitted less than 72 hours from the thermal injury. In the test group, the tilapia skin was applied. In the control group, a thin layer of silver sulfadiazine cream 1% was applied. Tilapia skin showed good adherence to the wound bed, reducing the number of dressing changes required, the amount of anesthetics used, and providing benefits for the patients and also for healthcare professionals, by reducing the overall work load. The number of days to complete burn wound healing, the total amount of analgesics required throughout the treatment, burn improvement on the day of dressing removal, and pain throughout the treatment were similar to the conventional treatment with silver sulfadiazine. Thus, tilapia skin can be considered an effective and low-cost extra resource in the therapeutic arsenal of pediatric superficial partial thickness burns.

Abstract Background: Axi-cel is an FDA-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after ≥ 2 prior therapies. In Phase 2 of ZUMA-1, the objective response rate was 83% (58% complete responses [CRs]) (Locke et al. Lancet Oncol. 2018). Previous work showed that pre-existing expression of immune-related genes (Immunosign® 21 with NanoString®) may be associated with response to axi-cel (Rossi et al. AACR 2018. #LB-016). This expanded post-hoc analysis includes novel analysis associating T cell density with efficacy. Methods: Pts received a target dose of 2.0 × 106 CAR T cells/kg in ZUMA-1 (NCT02348216). Pretreatment (before conditioning) tumor tissues were analyzed by immunohistochemistry for density of T cell subsets (CD3+, CD8+). A prespecified bioinformatics algorithm was used to generate a numerical index (Immunoscore®; Galon et al. J Pathol. 2014) and analyses cutoffs. Higher Immunoscore® represents increased intra-tumor T cell infiltration. TME was evaluated for Immunosign® 21 and for additional myeloid- and dendritic cell-related genes. The association between Immunoscore®, CD3+, or CD8+ T cell density and CR was evaluated, as was the association of Immunoscore® with Immunosign® 21. Results: Pretreatment tumor biopsy samples from 25 pts were analyzed (data cutoff Aug 11, 2017). Of pts with high Immunoscore®, 77% (7/9) had CR and 11% (1/9) were nonresponders. Of those with low Immunoscore®, 38% (6/16) were nonresponders and 50% (8/16) had CR. The pts in this category who achieved CR had a lower median pretreatment tumor burden by sum of product diameters. Overall, higher pretreatment Immunoscore® was associated with achievement of CR (P = .048). Pretreatment intra-tumor densities of CD3+ and CD8+ T cells separately quantified, were also positively associated with achievement of CR (P = .02 and .036 by t test). In addition, higher pretreatment Immunosign® 21 was associated with clinical efficacy. Values for Immunosign® 21 and Immunoscore® demonstrated 82% concordance (95% CI, 65-93; r2 = 0.451). Finally, TME gene expression analysis indicated a pan-inflammatory profile, including myeloid- and dendritic cell-related gene expression, in pts who achieved CR with higher Immunosign® 21 and Immunoscore®. Conclusions: These results link pre-existing T cell involved lymphoma TME with clinical outcome in patients post axi-cel. They also suggest that factors intrinsic to tumor biology may influence CAR T cell efficacy through the immune microenvironment. In addition, this treatment modality may overcome an unfavorable TME in a subset of patients with low disease burden. Finally, these results support CAR T cell treatment optimizations designed to overcome an immune detrimental TME. John Rossi and Jérôme Galon contributed equally. Citation Format: John M. Rossi, Jérôme Galon, Edmund Chang, Regis Perbost, Nathalie Scholler, Sarah Turcan, Corinne Danan, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Francesca Milletti, William Y. Go, Adrian Bot. Pretreatment immunoscore and an inflamed tumor microenvironment (TME) are associated with efficacy in patients (Pts) with refractory large B cell lymphoma treated with axicabtagene ciloleucel (Axi-Cel) in ZUMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT153.

Background Endoscopic skull‐base surgery (ESBS) is employed in the management of diverse skull‐base pathologies. Paralleling the increased utilization of ESBS, the literature in this field has expanded rapidly. However, the rarity of these diseases, the inherent challenges of surgical studies, and the continued learning curve in ESBS have resulted in significant variability in the quality of the literature. To consolidate and critically appraise the available literature, experts in skull‐base surgery have produced the International Consensus Statement on Endoscopic Skull‐Base Surgery (ICAR:ESBS). Methods Using previously described methodology, topics spanning the breadth of ESBS were identified and assigned a literature review, evidence‐based review or evidence‐based review with recommendations format. Subsequently, each topic was written and then reviewed by skull‐base surgeons in both neurosurgery and otolaryngology. Following this iterative review process, the ICAR:ESBS document was synthesized and reviewed by all authors for consensus. Results The ICAR:ESBS document addresses the role of ESBS in primary cerebrospinal fluid (CSF) rhinorrhea, intradural tumors, benign skull‐base and orbital pathology, sinonasal malignancies, and clival lesions. Additionally, specific challenges in ESBS including endoscopic reconstruction and complication management were evaluated. Conclusion A critical review of the literature in ESBS demonstrates at least the equivalency of ESBS with alternative approaches in pathologies such as CSF rhinorrhea and pituitary adenoma as well as improved reconstructive techniques in reducing CSF leaks. Evidence‐based recommendations are limited in other pathologies and these significant knowledge gaps call upon the skull‐base community to embrace these opportunities and collaboratively address these shortcomings.

Purpose Despite increasing emphasis in medical school education on quality and systems improvement, many medical schools lack sufficient faculty with expertise to teach systems improvement. Using the pedagogical content knowledge framework, this study explores how faculty engage students in systems improvement work and faculty perceptions of the outcomes for the health system and students. Method In May–June 2017, the authors interviewed 12 of 13 invited faculty with experience in teaching and engaging first-year medical students in systems improvement work, the course of students’ systems improvement work over time, the impact of students’ projects on health systems, and students’ learning and attitudes about systems improvement. The authors conducted qualitative analysis iteratively with data collection to sufficiency. Results Six emergent themes characterized faculty’s approach to guiding students in systems improvement work: faculty–student relationship, faculty role, student role, faculty–student shared responsibility for projects, faculty and student content knowledge, and project outcomes. The faculty–student relationship was foundational for successful systems improvement work. Faculty roles included project selection, project management, and health systems interactions. Students engaged in systems improvement as their faculty leveraged their knowledge and skills and created meaningful student roles. Faculty and students shared responsibility and colearned systems improvement content knowledge. Faculty defined successful outcomes as students’ learning about the systems improvement process and interprofessional collaboration. Conclusions Findings highlight the critical importance of pedagogical content knowledge to engage early learners in systems improvement work, understand their learning interests and needs, and manage their projects longitudinally.