Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Immune microenvironment of basal cell carcinoma and tumor regression following combined PD-1/LAG-3 blockade.

Systemic treatment options for patients with locally advanced or metastatic basal cell carcinoma (BCC) are limited, particularly when tumors are refractory to anti-programmed cell death protein-1 (PD-1). A better understanding of immune checkpoint expression within the BCC tumor microenvironment may inform combinatorial treatment strategies to optimize response rates. CD3, PD-1, programmed death ligand-1 (PD-L1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin domain and mucin domain 3 (TIM-3)+ cell densities within the tumor microenvironment of 34 archival, histologically aggressive BCCs were assessed. Tumor infiltrating lymphocyte (TIL) expression of PD-1, PD-L1, and LAG-3, and to a lesser degree TIM-3, correlated with increasing CD3+ T-cell densities (Pearson's r=0.89, 0.72, 0.87, and 0.63, respectively). 100% of BCCs (34/34) demonstrated LAG-3 and PD-1 expression in >1% TIL; and the correlation between PD-1 and LAG-3 densities was high (Pearson's r=0.89). LAG-3 was expressed at ~50% of the level of PD-1. Additionally, we present a patient with locally-advanced BCC who experienced stable disease during and after 45 weeks of first-line anti-PD-1 (nivolumab), followed by a partial response after the addition of anti-LAG-3 (relatlimab). Longitudinal biopsies throughout the treatment course showed a graduated increase in LAG-3 expression after anti-PD-1 therapy, lending support for coordinated immunosuppression and suggesting LAG-3 as a co-target for combination therapy to augment the clinical impact of anti-PD-(L)1.

A Case of Leukocytoclastic Vasculitis and Associated Conjunctivitis Following MMR Vaccine Administration.

Introduction: Leukocytoclastic vasculitis (LCV) is a small-vessel vasculitis characterized by immune complex deposition in the walls of dermal capillaries and venules. With the COVID-19 pandemic, more adults are receiving the MMR vaccine, as it may enhance innate immune responses against COVID-19 infection. Here, we report a case of LCV and associated conjunctivitis arising in a patient secondary to immunization with the MMR vaccine. Methods and Results: A 78-year-old man on lenalidomide therapy for multiple myeloma presented to an outpatient dermatology clinic with a two-day history of a painful rash consisting of scattered pink dermal papules on bilateral dorsal and palmar hands, as well as bilateral conjunctival erythema. Histopathological findings-which revealed an inflammatory infiltrate with papillary dermal edema, as well as nuclear dust within small blood vessel walls with extravasation of red blood cells-were most consistent with LCV. It then became known that the patient had received an MMR vaccine two weeks prior to the onset of the rash. The rash was resolved with the use of topical clobetasol ointment, and the patient's eyes were cleared as well. Conclusions: This is an interesting presentation of MMR vaccine-related LCV occurring only on the upper extremities with associated conjunctivitis. Had the patient's oncologist not known about the recent vaccination, it is likely that the treatment of his multiple myeloma would have been postponed or altered, as lenalidomide can also cause LCV.

An exploratory evaluation of predictors of duration of untreated psychosis in first-episode psychosis in lower income patients.

A longer duration of untreated psychosis (DUP) may adversely affect the course and impact of the illness. Little is known about predictors of duration of untreated psychosis in lower socioeconomic strata (SES) in urban India. We examined variables associated with DUP in 60 urban lower SES patients diagnosed with first-episode, untreated psychosis. Instruments used included the Positive and Negative Syndrome Scale (PANSS), the Global Assessment of Functioning (GAF), the UCLA Social Attainment Scale (SAS), and the Stigma Subscale from the Family Interview Schedule. The mean age of the sample was 30.5 years. The sample was 50% male. Most patients (90%) were living in a nuclear family. The median DUP was 120 days. DUP was <6 months in 31.7% of the sample and >2 years in 23.3%. A third of the sample had been taken to faith healers before being brought into medical care. DUP was not significantly associated with age, sex, religion, education, family history of mental illness, marital status, employment status, income, substance use, PANSS subscale and total scale scores, and GAF scores. Presence of an antecedent stressor, acute onset of psychosis, presence of medical co-morbidities, lower stigma scores, and higher SAS scores were significantly associated with shorter DUP.

Spatial UMAP and Image Cytometry for Topographic Immuno-oncology Biomarker Discovery.

Multiplex immunofluorescence (mIF) can detail spatial relationships and complex cell phenotypes in the tumor microenvironment (TME). However, the analysis and visualization of mIF data can be complex and time-consuming. Here, we used tumor specimens from 93 patients with metastatic melanoma to develop and validate a mIF data analysis pipeline using established flow cytometry workflows (image cytometry). Unlike flow cytometry, spatial information from the TME was conserved at single-cell resolution. A spatial uniform manifold approximation and projection (UMAP) was constructed using the image cytometry output. Spatial UMAP subtraction analysis (survivors vs. nonsurvivors at 5 years) was used to identify topographic and coexpression signatures with positive or negative prognostic impact. Cell densities and proportions identified by image cytometry showed strong correlations when compared with those obtained using gold-standard, digital pathology software (R2 > 0.8). The associated spatial UMAP highlighted "immune neighborhoods" and associated topographic immunoactive protein expression patterns. We found that PD-L1 and PD-1 expression intensity was spatially encoded-the highest PD-L1 expression intensity was observed on CD163+ cells in neighborhoods with high CD8+ cell density, and the highest PD-1 expression intensity was observed on CD8+ cells in neighborhoods with dense arrangements of tumor cells. Spatial UMAP subtraction analysis revealed numerous spatial clusters associated with clinical outcome. The variables represented in the key clusters from the unsupervised UMAP analysis were validated using established, supervised approaches. In conclusion, image cytometry and the spatial UMAPs presented herein are powerful tools for the visualization and interpretation of single-cell, spatially resolved mIF data and associated topographic biomarker development.

Durable Tumor Regression and Overall Survival in Patients With Advanced Merkel Cell Carcinoma Receiving Pembrolizumab as First-Line Therapy.

PURPOSE: Merkel cell carcinoma (MCC) is an aggressive skin cancer often caused by the Merkel cell polyomavirus. Clinical trials of programmed cell death-1 pathway inhibitors for advanced MCC (aMCC) demonstrate increased progression-free survival (PFS) compared with historical chemotherapy data. However, response durability and overall survival (OS) data are limited. PATIENTS AND METHODS: In this multicenter phase II trial (Cancer Immunotherapy Trials Network-09/Keynote-017), 50 adults naïve to systemic therapy for aMCC received pembrolizumab (2 mg/kg every 3 weeks) for up to 2 years. Radiographic responses were assessed centrally per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. RESULTS: Among 50 patients, the median age was 70.5 years, and 64% had Merkel cell polyomavirus-positive tumors. The objective response rate (ORR) to pembrolizumab was 56% (complete response [24%] plus partial response [32%]; 95% CI, 41.3% to 70.0%), with ORRs of 59% in virus-positive and 53% in virus-negative tumors. Median follow-up time was 14.9 months (range, 0.4 to 36.4+ months). Among 28 responders, median response duration was not reached (range, 5.9 to 34.5+ months). The 24-month PFS rate was 48.3%, and median PFS time was 16.8 months (95% CI, 4.6 months to not estimable). The 24-month OS rate was 68.7%, and median OS time was not reached. Although tumor viral status did not correlate with ORR, PFS, or OS, there was a trend toward improved PFS and OS in patients with programmed death ligand-1-positive tumors. Grade 3 or greater treatment-related adverse events occurred in 14 (28%) of 50 patients and led to treatment discontinuation in seven (14%) of 50 patients, including one treatment-related death. CONCLUSION: Here, we present the longest observation to date of patients with aMCC receiving first-line anti-programmed cell death-1 therapy. Pembrolizumab demonstrated durable tumor control, a generally manageable safety profile, and favorable OS compared with historical data from patients treated with first-line chemotherapy.

Correction to: 33rd Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2018).

After publication of this supplement [1, 2], it was brought to our attention that due to an error authors were missing in the following abstracts. This has now been included in this correction.

PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG-PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR-PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2- cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG-PVRL2 and TIGIT-PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244.

PD-L1, PD-1, LAG-3, and TIM-3 in Melanoma: Expression in Brain Metastases Compared to Corresponding Extracranial Tumors.

Background Metastatic melanoma to the brain carries a particularly poor prognosis that may be associated with an attenuated antitumor response in the presence of central nervous system malignancies. Thus, the development of brain metastases could theoretically accelerate cancer progression both locally and systemically. Although dysregulation of checkpoint markers, such as programmed death-ligand 1 (PD-L1), programmed cell death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), have been implicated in immune dysfunction, the exact relationship between these markers and brain tumor-mediated immune suppression remains unclear. Thus, the objective of this study was to explore whether there exists a differential expression of the above checkpoint markers in the intracranial milieu as compared to tumors in the periphery, which may shed light on the mechanism behind the diminished antitumor response. Methods We identified nine patients with extracranial melanomas and matched intracranial metastases. Formalin-fixed, paraffin-embedded slides were stained for PD-L1, PD-1, LAG-3, and TIM-3 via immunohistochemistry. Qualitative analysis was performed to assess the staining of the markers in the neoplastic and lymphocytic cells, which were the two cell lineages in each biopsy.  Results Expression of PD-1 and TIM-3 between extracranial and intracranial tumoral sites was conserved. Specifically, in lymphocytes, PD-1 expression was observed in 100% of extracranial and 100% of intracranial slides, whereas TIM-3 expression was seen in 33.33% of extracranial and 33.33% of intracranial slides. Neither marker stained tumor cells, as expected. PD-L1 showed a slight variation in staining between sites, with lymphocyte staining in 100% of extracranial and 88.89% of intracranial slides, and the same percentages per site for tumor cells. The greatest variability was observed in LAG-3 lymphocyte staining, with staining in 77.78% of extracranial and 33.33% of intracranial slides. No LAG-3 staining of tumor cells was noted, as expected. Conclusion Preliminary analysis revealed the conservation of PD-L1, PD-1, LAG-3, and TIM-3 expression intra- and extracranially. This could suggest that these markers are important in maintaining an immunosuppressive phenotype at both sites. Another possibility is that this pattern of expression is associated with patients who develop brain metastasis, as this was the only subset of patients included in this study. Interestingly, LAG-3 staining of lymphocytes appeared more prominent in extracranial over intracranial tumors. Future studies should include more samples to draw out potential patterns masked by the small sample size, as well as to compare checkpoint expression in other patient groups, such as those with non-brain metastasis or those with no metastasis at all.

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

BACKGROUND: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. METHODS: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. RESULTS: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 µm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. CONCLUSIONS: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

Mutations in Coagulation Factor VIII Are Associated with More Favorable Outcome in Patients with Cutaneous Melanoma.

Coagulation factor VIII (FVIII), von Willebrand factor (VWF), and ADAMTS13 play an important role in regulation of normal hemostasis. However, little is known about their roles in patients with malignancy, particularly with cutaneous melanoma. Whole genome sequencing data are available for 25,719 cases in 126 cancer genomic studies for analysis. All sequencing data and corresponding pathology findings were obtained from The Cancer Genome Atlas. The cBioportal bioinformatics tools were used for the data analysis. Our results demonstrated that mutations in genes encoding FVIII, VWF, and ADAMTS13 were reported in 92 of 126 cancer genomic studies and high mutation rates in these three genes were observed in patients with cutaneous melanoma from three independent studies. Moreover, high mutation rates in FVIII, VWF, and ADAMTS13 were also found in patients with diffuse large B cell lymphoma (22.9%), lung small cell carcinoma (20.7%), and colon adenocarcinoma (19.4%). Among 366 melanoma cases from TCGA provisional, the somatic mutation rates of FVIII, VWF and ADAMTS13 in tumor cells were 15%, 14%, and 5%, respectively. There was a strong tendency for coexisting mutations of FVIII, VWF, and ADAMTS13. Kaplan-Meier survival analysis demonstrated that melanoma patients with FVIII mutations had a more favorable overall survival rate than those without FVIII mutations (p=0.02). These findings suggest for the first time that the FVIII mutation burden may have a prognostic value for patients with cutaneous melanoma. Further studies are warranted to delineate the molecular mechanisms underlying the favorable prognosis associated with FVIII mutations.

Hepatitis C Virus Genie: A Web 2.0 Interpretation and Analytics Platform for the Versant Hepatitis C Virus Genotype Line Probe Assay Version 2.0.

CONTEXT: Hepatitis C virus (HCV) genotyping at our institution is performed using the Versant HCV genotype 2.0 Line Probe Assay (LiPA). The last steps of this procedure are manual, laborious, and error-prone process that involves the comparison of the banding pattern on a test strip to a physical reference table. AIM: We developed a web-based HCV genotype interpretation platform that utilizes a scanned image to generate the genotypes, thus minimizing interpretation time and reducing error. SUBJECTS AND METHODS: HCV Genie 2 utilizes a database of banding patterns in conjuncture with image analysis algorithms to determine the genotype for any number of scanned LiPA strips. HCV Genie 2 is built with client-side JavaScript; allowing the program to run in the user' browser rather than on an unknown server, essentially eliminating data and patient privacy concerns. RESULTS: HCV Genie 2 was tested over 2 months and proved identical to human expert interpretation for 148 samples (>1000 bands identified). Manual intervention was required only for two faint bands and one false-positive band; this was done utilizing the built-in-user interface. Utilizing the original method, the trained laboratory technician interpretation time for 16 samples was 13.8 (±0.96) min as compared to 5.0 (±1.09) min with HCV Genie 2, a 63.8% decrease. In addition to the time savings, the new method provides an additional validation step, which decreases the potential for errors. CONCLUSIONS: Our institution has moved exclusively to utilize the new techniques and tools described here. Both experienced technicians and the molecular pathologists at our institution prefer the workflow using HCV Genie. It is easier for the technicians to prepare and document, and the pathologists are more rapidly able to review and confirm results. The use of this tool will lead to increase the quality of patient care delivered through this test methodology by decreasing the potential for error. The algorithms developed here can be ported to similar band identification platforms, most directly to other LiPAs.

Primary Renal Ewing Sarcoma in an Adult.

Primary renal Ewing sarcoma is extremely rare and carries a dismal prognosis. It is particularly diagnostically challenging due to its ability to mimic other round cell tumors. Herein we report such a case in a 28-year-old white female. The diagnosis was confirmed by immunophenotyping, fluorescence in situ hybridization, and reverse transcription polymerase chain reaction analyses.

Extramedullary Involvement by Chronic Myelogeneous Leukemia in Five Patients With Unusual Clinicopathologic Features: A Review of the Effectiveness of Tyrosine Kinase Inhibitors.

Chronic myelogeneous leukemia (CML) is associated with BCR-ABL1 fusion gene leading to an abnormal tyrosine kinase molecule. The accepted first-line treatment is imatinib mesylate (IM). CML uncommonly occurs in the extramedullary sites at initial presentation or relapse. Here we report five adult patients with CML who developed myeloid sarcoma (MS) while on treatment with IM. A retrospective medical chart analysis was performed to identify CML patients with MS who were diagnosed and treated at the University of Alabama at Birmingham. The age ranged between 21 and 36 years (median: 28.5) with a male to female ratio of 4:1. All of the patients were diagnosed with CML in chronic phase with initial treatment including IM. The median interval period between the initial diagnosis of CML and MS was 27 months (range 7 - 60 months). The sites of extramedullary involvement included lymph nodes (n = 2), central nervous system (n = 2) and hepatobiliary organs (n = 1). All patients were treated with either induction therapy or stem cell transplant (SCT) following the diagnosis of MS. The median survival was 16 months (range 1 - 72 months). The longest survival was observed in a patient who successfully received SCT therapy. IM is frequently used as the first therapeutic choice in new diagnosed CML; however, its penetration and effectiveness in extramedullary tissue is still unclear. The current report also supports the literature with less favorable prognosis of CML in younger individuals.

Adult Pleomorphic Juxtaglomerular Cell Tumor.

A 40-year-old male with chronic hypertension since his teens presented to the emergency department following a motor vehicle collision. Computed tomography scan demonstrated an incidental 1.8-cm renal mass. Partial nephrectomy revealed a vascular tumor with predominantly monomorphic epithelioid cells arranged in sheets and trabeculae with foci of nuclear pleomorphism. Tumor cells were positive for vimentin, CD34, and c-KIT. Juxtaglomerular cell tumor is a rare, benign neoplasm typically found in young adults. Pleomorphism is uncommon and, in combination with older age at diagnosis, can lead to an inaccurate malignant diagnosis. Immunohistochemistry and clinical history helps in correctly diagnosing this benign entity.