CD103 and periplakin are potential biomarkers for response of metastatic melanoma to pembrolizumab.

This study was designed to screen for preliminary evidence of predictive markers of melanoma response to PD-1 blockade. We hypothesized that the following immune markers would be positive predictors of response: increased densities of CD103 + CD8 + T cells or Th1 lineage T-bet + T cells, high expression of CXCL9-11 and presence of tertiary lymphoid structures. Conversely, we hypothesized that the high expression of barrier molecules would be a negative predictor of response. Patients with advanced melanoma treated with pembrolizumab were identified, and clinical response as well as overall survival data were collected. Tumor samples were evaluated by multiplex immunofluorescence histology. All statistical analyses were performed in R Studio and Microsoft Excel using the Mann-Whitney U test, chi-square test, Spearman's rank correlation and Kaplan-Meier survival curves. Sixty-five advanced melanoma patients were identified, of whom 46 met inclusion criteria and were included in this study. Increased densities ( P = 0.04) and proportions ( P = 0.02) of CD8 + T cells expressing CD103 + were associated with complete response (CR) to pembrolizumab. Improved survival was associated with increased proportions of CD8 + cells expressing CD103 ( P = 0.0085) as well as decreased density of periplakin + cells ( P = 0.012) and periplakin + SOX10 + cells ( P = 0.0012). The density and proportion of CD8 + T cells expressing CD103 + positively correlated with PD-L1 expression, though PD-L1 expression was not significantly correlated with outcomes. This screening study found that increased density and proportion of CD8 + T cells expressing CD103 and decreased density of periplakin were associated with positive outcomes in patients with melanoma metastases treated with pembrolizumab and may warrant further study.

Proliferating CD8+ T Cell Infiltrates Are Associated with Improved Survival in Glioblastoma.

BACKGROUND: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. METHODS: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. RESULTS: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). CONCLUSIONS: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.

Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Structures.

The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.

Formation and phenotypic characterization of CD49a, CD49b and CD103 expressing CD8 T cell populations in human metastatic melanoma.

Integrins α1ß1 (CD49a), α2ß1 (CD49b) and αEß7 (CD103) mediate retention of lymphocytes in peripheral tissues, and their expression is upregulated on tumor infiltrating lymphocytes (TIL) compared to circulating lymphocytes. Little is known about what induces expression of these retention integrins (RI) nor whether RI define subsets in the tumor microenvironment (TME) with a specific phenotype. Human metastatic melanoma-derived CD8 TIL could be grouped into five subpopulations based on RI expression patterns: RIneg, CD49a+ only, CD49a+CD49b+, CD49a+CD103+, or positive for all three RI. A significantly larger fraction of the CD49a+ only subpopulation expressed multiple effector cytokines, whereas CD49a+CD103+ and CD49a+CD49b+ cells expressed IFNγ only. RIneg and CD49a+CD49b+CD103+ CD8 TIL subsets expressed significantly less effector cytokines overall. Interestingly, however, CD49a+CD49b+CD103+ CD8 expressed lowest CD127, and highest levels of perforin and exhaustion markers PD-1 and Tim3, suggesting selective exhaustion rather than conversion to memory. To gain insight into RI expression induction, normal donor PBMC were cultured with T cell receptor (TCR) stimulation and/or cytokines. TCR stimulation alone induced two RI+ cell populations: CD49a single positive and CD49a+CD49b+ cells. TNFα and IL-2 each were capable of inducing these populations. Addition of TGFß to TCR stimulation generated two additional populations; CD49a+CD49bnegCD103+ and CD49a+CD49b+CD103+. Taken together, our findings identify opportunities to modulate RI expression in the TME by cytokine therapies and to generate subsets with a specific RI repertoire in the interest of augmenting immune therapies for cancer or for modulating other immune-related diseases such as autoimmune diseases.

Lymphoid aggregates in desmoplastic melanoma have features of tertiary lymphoid structures.

Desmoplastic melanomas (DM) have unique and challenging clinical presentations and histomorphology. A characteristic feature is the presence of scattered lymphoid aggregates. However, the nature of these aggregates is not defined. We hypothesized that they may be tertiary lymphoid structures (TLS), and may be associated with programmed death ligand 1 (PD-L1) expression. We searched our tissue database for 'pure' DMs and for scars as control tissues, collected clinical information, and reviewed H&E histology. We performed multispectral imaging after staining for CD8, CD20, PNAd, FoxP3, CD83, and Ki67, and assessed PD-L1 expression by immunohistochemistry. Pure DM samples were evaluable in 11 patients. All had desmoplastic stroma and lymphoid aggregates on H&E. The lymphoid aggregates of eight of the 11 (72%) DM samples and only three of the 11 scars contained features of TLS, defined as distinct clusters of B cells and CD8 T cells, CD83 dendritic cells in T-cell zones, and PNAd vasculature resembling high endothelial venules. PD-L1 was expressed by at least 1% of melanoma cells in six and by at least 5% of immune cells in 10 of the 11 DM samples. We found that most lymphoid aggregates in DM are organized, classical TLS. PD-L1 expression was detected in most cases and was highest in two cases of DM with TLS. However, low PD-L1 expression in some cases suggests that some DM cells may be unresponsive to interferon-γ. TLS support antigen presentation and T-cell responses in chronic inflammation and cancer. Their presence in DM likely reflects an adaptive immune response, which may be enhanced with immune therapies.

Predicting surgical skill acquisition in preclinical medical students.

BACKGROUND: The purpose of this study was to identify factors that predict medical student success in acquiring invasive procedural skills. We hypothesized that students with interest in surgery and with prior procedural experience would have higher rates of success. METHODS: Preclinical students were enrolled in a simulation course comprised of suturing, intubation, and central venous catheterization. Students completed surveys to describe demographics, specialty interest area, prior experience, and confidence. Using linear regression, variables predictive of proficiency were identified. RESULTS: Forty-five participants completed the course. Under univariate analysis, composite pretest score was inversely associated with confidence (P = .039). Under multivariable analysis, female gender was associated with higher pretest suturing score (P = .016). Male gender (P = .029) and high confidence (P = .021) were associated with greater improvement in suturing. CONCLUSIONS: Among novices, higher confidence can predict lower baseline technical proficiency. Although females had higher pretest suturing scores, high confidence and male gender were associated with the greatest degree of improvement.

Cumulative sum: An individualized proficiency metric for laparoscopic fundamentals.

BACKGROUND: A reliable metric of technical proficiency is indispensable to the training of fellows and residents. The purpose of this study was to determine whether cumulative sum (Cusum) has predictive validity in laparoscopic training. We hypothesized that Cusum would be a better predictor of technical ability in fundamentals of laparoscopic surgery (FLS) than traditional practice volume metrics. METHODS: Twenty medical students were recruited to practice three FLS tasks: peg transfer, circle cut, and intracorporeal knot tie. Up to 7hours of self-directed practice was allotted to each participant. Practice attempts were scored by standard FLS criteria and monitored via Cusum. Each participant's terminal Cusum performance was analyzed retrospectively. Posttests were conducted by faculty blinded to practice performance. RESULTS: Eighteen participants completed the study (90%). Median adjusted posttest scores were 102.3, 84.1, and 78.6 for peg transfer, circle cut, and knot tie, respectively. For the knot tie task, participants who exceeded the Cusum decision interval during their final practice attempts performed significantly higher on posttesting (81.2 vs 71.5, p=0.015). Knot tie terminal Cusum score was positively associated with posttest performance after adjusting for practice volume (p=0.031). Total practice volume and practice time were not significantly associated with posttest performance for any FLS task. CONCLUSION: Cusum score is a more valid representation of FLS proficiency than practice volume or practice time. Incorporating Cusum in a clinical setting may promote more efficient allocation of time resources and operative volume.

Adaptive simulation training using cumulative sum: a randomized prospective trial.

BACKGROUND: Cumulative sum (Cusum) is a novel tool that can facilitate adaptive, individualized training curricula. The purpose of this study was to use Cusum to streamline simulation-based training. METHODS: Preclinical medical students were randomized to Cusum or control arms and practiced suturing, intubation, and central venous catheterization in simulation. Control participants practiced between 8 and 9 hours each. Cusum participants practiced until Cusum proficient in all tasks. Group comparisons of blinded post-test evaluations were performed using Wilcoxon rank sum. RESULTS: Forty-eight participants completed the study. Average post-test composite score was 92.1% for Cusum and 93.5% for control (P = .71). Cusum participants practiced 19% fewer hours than control group participants (7.12 vs 8.75 hours, P < .001). Cusum detected proficiency relapses during practice among 7 (29%) participants for suturing and 10 (40%) for intubation. CONCLUSIONS: In this comparison between adaptive and volume-based curricula in surgical training, Cusum promoted more efficient time utilization while maintaining excellent results.

Assistant instructors facilitate simulation for medical students.

BACKGROUND: A procedural training protocol for medical students must be cognizant of faculty opportunity costs, which may preclude individually supervised practice. Meanwhile, sporadic exposure in large group settings yields suboptimal proficiency. The purpose of this study is to assess the effectiveness of undergraduate assistant instructors in providing one-on-one simulation-based instruction for basic invasive techniques. MATERIALS AND METHODS: Investigators designed proficiency-based checklists by faculty consensus for three simulation tasks as follows: orotracheal intubation, central venous catheterization, and suturing. Four undergraduate students were trained as instructors. Interrater agreement between instructors using the task checklists ranged from 0.754-0.866. Instructors conducted up to seven flexibly scheduled, one-on-one practice sessions with clinically inexperienced medical student participants. Sessions comprised repetitive task attempts with an evaluation after every attempt. On completion of the training protocol, participants underwent evaluation by an experienced surgeon blinded to practice session performance. Study participants were surveyed to assess satisfaction. RESULTS: Twenty-nine participants completed the study. Median total practice time was 8.75 h (interquartile range 7.12-8.75). Posttest pass rates were 93% (26/28), 71% (20/28), and 68% (19/28) for suturing, intubation, and central venous catheterization, respectively. Ninety-seven percent (27/28) of participants were satisfied with their experience, and 62% (18/29) advocated for protocol adoption into the standard preclinical curriculum. Estimated cost saved using student instructors, based on departmental collections for surgical faculty, was $43,760. CONCLUSIONS: Clinically inexperienced assistants may be trained as instructors for basic simulation tasks with excellent interrater reliability. Deploying these assistant instructors makes effective, one-on-one technical training for preclinical medical students financially feasible.

Verification of accurate technical insight: a prerequisite for self-directed surgical training.

Simulation-based surgical skills training during preclinical education is a persistent challenge due to time constraints of trainees and instructors alike. Self-directed practice is resource-efficient and flexible; however, insight into technical proficiency among trainees is often lacking. The purpose of this study is to prospectively assess the accuracy of self-assessments among medical students learning basic surgical suturing. Over seven weekly practice sessions, preclinical medical students performed serial repetitions of a simulation-based suturing task under one-on-one observation by one of four trainers. Following each task repetition, self- and trainer-assessments (SA-TA) were performed using a 36-point weighted checklist of technical standards developed a priori by expert consensus. Upon study completion, agreement between SA and TA was measured using weighted Cohen's kappa coefficients. Twenty-nine medical students each performed a median of 25 suture task repetitions (IQR 21.5-28). Self-assessments tended to overestimate proficiency during the first tertile of practice attempts. Agreement between SA and TA improved with experience, such that the weighted kappa statistics for the two-handed and instrument ties were >0.81 after 18-21 task attempts. Inexperienced trainees frequently overestimate technical proficiency through self-assessments. However, this bias diminishes with repetitive practice. Only after trainees have attained the capacity to accurately self-assess can effective self-directed learning take place.