Markovic, Svetomir N.

AbstractBackgroundMerkel cell carcinoma (MCC) is a rare, aggressive cutaneous malignancy that usually occurs in the head/neck or extremities. However, there are reports of MCC developing in the lymph nodes or parotid gland without evidence of a primary cutaneous lesion.MethodsWe reviewed 415 patients with biopsy‐proven MCC. Patients with MCC of unknown primary (n = 37, 9%, MCCUP) made up the study cohort. The primary endpoints of the study were rate of recurrence, disease‐free survival, and overall survival.ResultsPatients with MCCUP presented with tumors in lymph nodes (n = 34) or parotid gland (n = 3). Nodal disease was most commonly detected in the inguinal/external iliac (n = 15) or axillary (n = 14) regions. The mean age at diagnosis was 70 years and 24% were female. Patients presented with distant metastases in 24.3% of cases. Patients with stage IIIA disease treated with regional lymph node dissection (RLND) had a lower risk of disease recurrence (hazard ratio 0.26, p = 0.046). Recurrence‐free survival was 59.3% at 5 years. Disease‐specific survival was 63.3% at 5 years.ConclusionPatients with MCCUP have a high risk of recurrence and mortality. The optimal treatment for MCCUP has yet to be elucidated, although therapeutic RLND appears beneficial for these patients.

AbstractPurpose: Absolute lymphocyte count recovery at day 15 (ALC-15) post-autologous stem cell transplantation (ASCT) is a powerful prognostic indicator for survival for multiple hematologic malignancies and metastatic breast cancer. The relationship of ALC-15 with clinical outcomes in primary systemic amyloidosis is unknown.Experimental Design: We evaluated 145 consecutive patients with primary systemic amyloidosis who underwent ASCT at the Mayo Clinic from 1996 to 2003. The ALC-15 threshold was set at 500 cells/μL based on our previous observations.Results: The median patient follow-up was 22 months (range, 3-87 months). Higher hematologic complete response was observed in patients with an ALC-15 ≥ 500 cells/μL compared with patients with an ALC-15 < 500 cells/μL (41% versus 21%, P < 0.0008, respectively). The median overall survival and progression-free survival times were significantly better for the 59 patients that achieved an ALC-15 ≥ 500 cells/μL compared with 86 patients with ALC-15 < 500 cells/μL (not reached versus 53 months, P < 0.0003 and not reached versus 27 months, P < 0.0001, respectively). Multivariate analysis showed ALC-15 to be an independent prognostic factor for overall survival and progression-free survival.Conclusions: ALC-15 ≥ 500 cells/μL is associated with significantly improved clinical outcomes following ASCT in patients with primary systemic amyloidosis.

Background Patients with metastatic melanoma rely on PD-(L)1 immunotherapy, but only one-third of patients experience treatment response and all initial responders eventually develop resistance. Tumour-derived extracellular vesicles expressing Programmed death ligand 1 (evPD-L1) and soluble Programmed death ligand 1 (sPD-L1) in peripheral blood of patients with melanoma limit PD-(L)1 immunotherapy and correlate with poor survival. Therapeutic plasma exchange (TPE) removes immunosuppressive evPD-L1 and sPD-L1. We hypothesise that TPE may rescue and restore antimelanoma immunity. Methods In this two-arm study, 60 patients with metastatic melanoma progressing on checkpoint inhibition will be accrued. All patients will undergo radiotherapy on days 1–5 (at least one measurable lesion will not be irradiated) and ongoing checkpoint inhibition on day 8 and every 2–3 weeks per standard of care. Patients with baseline sPD-L1 level of ≥1.7 ng/mL and adequate clinical capacity will be enrolled in the TPE intervention arm and will undergo TPE on days 5–7, in addition to standard of care radiotherapy and immunotherapy. Other patients will remain in the standard of care arm. The primary endpoint of the study is to evaluate safety. Secondary endpoints include kinetics of sPD-L1 and evPD-L1 and clinical response by RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Study registered at ClinicalTrials.gov ( NCT04581382 ). Ethics and dissemination This trial has been approved by the Mayo Clinic Institutional Review Board. It will assess the safety and feasibility of TPE in improving outcomes for PD-(L)1 inhibitor immunotherapy in melanoma. Data will be maintained on a secure database with deidentified patient information. Data will be shared on publication in a peer-reviewed journal without the aid of professional writers. If successful, this trial will lay the ground for phase II studies that will include cancer treated with PD-(L)1 inhibitors which may benefit from TPE such as renal, bladder and lung cancers. Trial registration number NCT04581382 .

AbstractOur phase III trial reported that autograft‐absolute lymphocyte count (A‐ALC) improved survival post‐autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) for a short‐term follow‐up of 2 years. We evaluated retrospectively in our phase III trial patients that the A‐ALC still confers survival benefit with a longer follow‐up. With a median follow‐up of 127.6 months, patients infused with an A‐ALC ≥ 0.5 × 109 cells/kg experienced better overall survival (HR = 0.392, 95% confidence of interval [CI]: 0.224–0.687, p < 0.001) and progression‐free survival (HR = 0.413, 95% CI: 0.253–0.677), p < 0.0004). This study supports that A‐ALC provides long‐term survival benefit post APBHSCT.

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy. Significance: Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

Immune checkpoint inhibitors (ICIs) can cause pituitary dysfunction due to hypophysitis. We aimed to characterize ICI-induced hypophysitis and examine its association with overall survival in this single-center retrospective cohort study of adult patients with cancer who received an ICI from January 1, 2012 through December 31, 2016. A total of 896 patients were identified who received ipilimumab alone (n=120); ipilimumab and nivolumab (n=50); ipilimumab before or after pembrolizumab (n=70); pembrolizumab alone (n=406); and nivolumab alone (n=250). Twenty-six patients (2.9%) developed hypophysitis after a median of 2.3 months. Median age at the start of ICI was 57.9 years and 54% were men. Hypophysitis occurred in 7.9% of patients receiving ipilimumab alone or in combination or sequence with a programmed cell death protein 1 inhibitor; 1.7% after pembrolizumab alone, never after nivolumab alone. Secondary adrenal insufficiency occurred in all hypophysitis cases. Use of ipilimumab alone or in combination was associated with pituitary enlargement on imaging and mass effects more frequently than pembrolizumab alone. Occurrence of hypophysitis was associated with improved overall survival by univariate analysis (median 50.7 vs 16.5 months; p=0.015) but this association was not observed in multivariable landmark survival analysis (HR for mortality 0.75; 95% CI 0.38 to 1.30; p=0.34) after adjusting for age, sex and malignancy type. To conclude, hypophysitis occurred most frequently after ipilimumab and manifested as anterior hypopituitarism affecting the corticotrophs more commonly than thyrotrophs and gonadotrophs. Mass effects and pituitary enlargement occurred more frequently in ipilimumab-induced hypophysitis. The association of hypophysitis with overall survival needs further investigation.

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Abstract The autograft absolute lymphocyte count (A-ALC) ≥ 0.5 x 10 9 cells/kg is a survival prognostic factor for lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). However, the A-ALC ≥ 0.5 x 10 9 cells/kg has not been tested as prognostic factor against double hit/triple hit lymphomas (DHL/THL). Thus, we set up to investigate if A-ALC ≥ 0.5 x 10 9 cells/kg is a prognostic factor for overall survival (OS) and progression-free survival (PFS) for DHL/THL post-APBHSCT. From January 2012 until December 2020, we identified 77 DHL/THL patients treated with APBHSCT. All patients required to have the diagnosis of DHL/THL by FISH for rearrangements of MYC, BCL2, and BCL6. Of the 77 patients, 62 patients were classified as DHL and 15 patients as THL. Of the DHL, 82 % (52/68) had the MYC/BCL2 and 16% (10/62) the MYC/BCL6 rearrangements. Dividing the cohort into two groups (A-ALC ≥ 0.5 x 10 9 cells/kg and A-ALC &lt; 0.5 x 10 9 cells/kg), both groups were balanced in regard to de novo versus transformed histology (p = 0.3), cell of origin (p = 0.6), DHL and THL (p = 0.6), MYC/BCL2; MYC/BCL6; and MYC/BCL2/BCL6 rearrangements (p = 0.6), first line chemotherapy (p = 0.2), salvage chemotherapy (p = 0.5), disease status prior to APBHSCT (CR1, CR, and PR) (p = 0.07), stem cell mobilization (Plerixafor versus not) (p = 0.6), and infusion of CD34 count (p = 0.2). With a median follow-up of 20.4 months (range, 0.4-94.5 months) for the entire cohort and with a median follow-up of 33.5 months (range, 3.5-94.5 months) for the patients that remained alive, DHL/THL patients infused with A-ALC ≥ 0.5 x 10 9 cells/kg experienced superior OS (HR = 0.251, 95%CI 0.117-0.539, P &lt; 0.0004) and PFS (HR = 0.347, 95%CI 0.160-0.753, P &lt; 0.007). The 5-year OS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 73% (95% confidence interval [CI], 52%-87%) and for the A-ALC &lt; 0.5 x10 9 cells/kg group was 18% (95% CI, 7%-39%) (Figure 1A). The 5-year PFS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 73% (95% CI, 59%-85%) and for the A-ALC &lt; 0.5 x10 9 cells/kg group was 13% (95% CI, 5%-33%) (Figure 1B). Multivariate analysis showed that A-ALC was an independent predictor for OS (HR =0.178, 95%CI 0.052-0.614, p &lt;0.005) and PFS (HR = 0.400, 95%CI 0.189-0.850, p &lt;0.02). Our study showed that A-ALC is a prognostic factor for survival in DHL/THL patients undergoing APBHSCT. Our current practice for all lymphoma patients is not only to collect enough stem cell for hematologic engraftment, but also A-ALC ≥ 0.5 x 10 9 cells/kg to improve clinical outcomes post-APBHSCT. Figure 1 Figure 1. Disclosures Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Paludo: Karyopharm: Research Funding.

Abstract Our group published a double blind, randomized phase III study (Porrata et al, BBMT 2016; 22: 1017-1023) showing that the infusion of autograft absolute lymphocyte count (A-ALC) ≥ 0.5 x 10 9 cells/kg is a survival prognostic factor for lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). However, a limitation of the study was the short-term follow-up duration of two years post-APBHSCT. To assess if the A-ALC still provide survival benefit we looked at the 121 lymphoma patients enrolled in the phase III from December 10, 2007 until October 12, 2010 survival outcomes with a longer follow-up duration of up to 13 years post-APBHSCT. Since the accrual for the study, 52 patients had died of recurrence lymphoma, 4 patients of therapy-related acute myelogenous leukemia, 3 patients of unknown causes, 1 patient of acute respiratory distress syndrome, 1 patient of anaplastic astrocytoma, and 1 patient of septic shock. The current median follow-up is 86 months (range: 2.1-158.1 months) for the entire cohort and 127.8 months (range: 5.9-158.1 months) for patients still alive. Patients infused with an A-ALC ≥ 0.5 x 10 9 cells/kg continue to experience superior overall survival (OS) (HR = 0.392, 95%CI 0.224-0.687, P &lt; 0.001) and progression-free survival (PFS) (HR = 0.413, 95%CI 0.253-0.677, P &lt; 0.0004). The 10-year OS rates for the A-ALC ≥ 0.5 x10 9 cells/kg patients was 70% (95% confidence interval [CI], 55%-81%) and for the A-ALC &lt; 0.5 x10 9 cells/kg patients was 36% (95% CI, 25%-49%) (Figure 1A). The 10-year PFS rates for the A-ALC ≥ 0.5 x10 9 cells/kg group was 57% (95% CI, 43%-70%) and for the A-ALC &lt; 0.5 x10 9 cells/kg group was 28% (95% CI, 19%-40%) (Figure 1B). Autograft lymphocyte subset analysis including autograft-CD3 (A-CD3), autograft CD4 (A-CD4), autograft CD8 (A-CD8), and autograft natural killer cells (A-NK), showed that the infusion of A-NK was a predictor for OS (HR = 0.518, 95%CI 0.292-0.919, P &lt; 0.02) and PFS (HR = 0.393, 95%CI 0.230-0.670, P &lt; 0.0006). The 10-year OS rates for the A-NK ≥ 0.09 x10 9 cells/kg patients was 67% (95% confidence interval [CI], 50%-80%) and for the A-NK &lt; 0.09 x10 9 cells/kg patients was 42% (95% CI, 31%-54%) (Figure 1C). The 10-year PFS rates for the A-NK ≥ 0.09 x10 9 cells/kg group was 59% (95% CI, 43%-73%) and for the A-NK &lt; 0.09 x10 9 cells/kg group was 32% (95% CI, 23%-44%) (Figure 1D). Both the A-ALC and A-NK were independent predictor for OS [ A-ALC: HR = 0.367, 95%CI 0.195-0.690, P &lt; 0.001) and A-NK: HR = 0.411, 95%CI 0.275-0.892, P &lt; 0.01] and for PFS [ A-ALC: HR = 0.477, 95%CI 0.271-0.840, P &lt; 0.01) and A-NK: HR = 0.490, 95%CI 0.266-0.903, P &lt; 0.02]. With a longer follow-up of 13 years, this study shows that the infusion of A-ALC continues to provide better clinical outcomes to lymphoma patients undergoing APBHSCT. These study findings support our standard practice changed of not only collecting CD34 stem cell for hematologic engraftment, but also A-ALC for lymphoma patients undergoing APBHSCT to improve clinical outcomes. Further, autograft immune effector studies are warranted such as A-NK to develop a more immunocompetent targeted autologous graft verus tumor effect. Figure 1 Figure 1. Disclosures Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Paludo: Karyopharm: Research Funding.