TPS9598 Background: Melanoma (Mel) accounts for the majority of skin cancer-related deaths. Most patients (pts) with a newly diagnosed Mel have resectable disease and are potentially cured by surgery. However, regional nodal and/or distant relapses can occur after curative-intent resection. Postoperative adjuvant therapy with immune checkpoint inhibitors improves relapse-free survival (RFS) and distant metastasis-free survival (DMFS) of pts at high risk of Mel. Fianlimab (anti-LAG-3) and cemiplimab (anti-PD-1) are both high-affinity, fully human, IgG4 monoclonal antibodies (MAbs) that combined have shown high clinical activity in pts with advanced Mel in a phase 1 study. Additionally, combination of relatlimab (anti-LAG-3) and nivolumab (anti-PD-1) have shown superiority over nivolumab for PFS in advanced Mel. These observations provide a rationale for use of fianlimab and cemiplimab combination in high-risk adjuvant Mel. Methods: Our study (NCT05608291) is a three-way, double-blind, phase 3 trial to compare fianlimab + cemiplimab to pembrolizumab in the adjuvant therapy (Rx) of high-risk, resected Mel. The primary objective is RFS, and the secondary objectives are overall survival, safety, pharmacology, and immunogenicity. This international trial will be conducted at 200 sites. Pt eligibilities: (1) ≥12 years of age; (2) Stage IIc, III or IV (all M-stages) and histologically confirmed Mel, completely resected ≤12 weeks prior to randomization; (3) no prior systemic anti-cancer Rx or radiation Rx for Mel in the previous 5 years; (4) no evidence of metastatic disease on staging investigations; and (5) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult pts), Karnofsky PS >70 (pts >16 years) or Lansky PS >70 (pts <16 years). Study arms (all Rx every 3 weeks intravenously for one year): A. fianlimab (1600 mg) + cemiplimab (350 mg); B. fianlimab (400 mg) + cemiplimab (350 mg); C. pembrolizumab (200 mg) + saline/dextrose placebo. The placebo controlled trial will enroll about 1530 pts, randomized 1:1:1 to Arms A:B:C, treated for up to 1 year. The trial will stratify by disease stage (stage IIIA vs IIC-IIIB-IIIC vs IIID-IV [M1a/b] vs IV [M1c/d]), and geography (North America vs Europe vs Rest of World). The primary endpoint is investigator-assessed RFS. The secondary endpoints include efficacy (overall survival, DMFS, melanoma-specific survival), safety [treatment-emergent adverse events (TEAEs), interruption or discontinuation of drugs due to TEAEs], pharmacokinetic (concentrations of fianlimab and cemiplimab in serum over time), immunogenicity (anti-drug Abs and neutralizing Abs in serum against fianlimab or cemiplimab), and patient reported outcomes. The first analysis will be performed when 242 RFS events have been observed. Clinical trial information: NCT05608291 .

TPS9614 Background: Basal cell carcinoma (BCC) is the most common form of non-melanoma skin cancer in the United States. Surgical excision is the standard treatment, with < 1% of cases progressing to locally advanced or metastatic disease. Hedgehog pathway inhibitors (HHIs) are the first-line therapy for advanced BCC (aBCC); the US Food and Drug Administration and European Medicines Agency have approved the use of cemiplimab (a programmed cell death-1 inhibitor) in advanced BCC (aBCC) patients previously treated with (or are inappropriate for) HHI. Limited real-world data exist on the clinical characteristics, disease management and progression, and survivorship of patients with aBCC. The ongoing C.A.S.E. study aims to evaluate the efficacy, safety, disease evolution, survivorship, and patient reported outcomes (PRO) in patients treated with cemiplimab in the real-world setting. Methods: This trial in progress (NCT03836105) aims to describe the effectiveness and safety of cemiplimab 350 mg administered every 3 weeks for treatment of patients with aBCC in real-world clinical settings. Up to 100 adult patients with aBCC who are prescribed commercially available cemiplimab from ~65 study sites in the United States will be included. The duration of follow-up will be 24 months. Endpoints for this study relate to real-world efficacy, including overall survival; progression-free survival; objective response rate, (partial or complete response); and disease control rate, defined as the percentage who do not progress for ≥ 6 months; Time to response, duration of response, time to treatment failure, and disease-specific death will also be assessed. Real-world safety outcomes will also be captured, including immune-related adverse events, infusion-related reactions, and serious adverse events. Patient selection criteria and treatment patterns will be analyzed using descriptive statistics. This study also aims to describe the patient experience of real-world treatment with cemiplimab. PROs including global quality of life, functioning, and symptoms will be captured at baseline and follow-up visits via the EORTC QLQ-C30 and the Skin Care Index. Recruitment for this trial is ongoing. Clinical trial information: NCT03836105 .

9583 Background: Recent studies have shown 5-yr recurrence rates for Stage IIB and IIC melanoma of up to 46%. These high-risk patients currently have few options for adjuvant therapy to prevent this inevitable recurrence, with the only FDA approved therapy being high-dose interferon-alfa, which is quite toxic. However, there are now immunotherapies (anti-PD1) and targeted therapies (anti-BRAF and anti-MEK combinations) which are approved as adjuvants for Stage III patients, some of whom will have a lower baseline recurrence risk than those with Stage IIB/IIC melanoma. We sought to determine if adjuvant PD1 inhibition with nivolumab (N) would improve the recurrence free survival (RFS) compared to historical RFS rates. Methods: Our study (NCT03405155) is a single-arm, open label, multi-center, phase 2 clinical trial evaluating RFS at 24 months in patients with Stage IIB/IIC melanoma on treatment with N at 480 mg IV every 4 weeks for 12 cycles. Overall survival is a secondary endpoint. Associated translational research includes circulating tumor cell DNA and immune correlates. Results: Twenty three patients with Stage IIB and three patients with Stage IIC melanoma were enrolled onto the study and received at least one dose of N. At data cutoff, 22 patients remain in follow up, as four patients withdrew consent at different time points in the study – one patient after one dose who wished to discontinue, one due to concern for COVID and need for repeat visits, one due to insurance issues, and one due to recurrence and wish to discontinue (which was captured in study data and RFS calculations). Seventeen patients have been on the clinical trial for at least two years with nine patients having finished treatment but with less than two years follow-up; the median follow-up is currently 21.9 months. Two patients demonstrated melanoma recurrence, one after receiving cycle six of N and another one year after completing treatment, resulting in a 87.8% RFS (90% CI (64.2%-96.3%) at 2 years, compared to the historical RFS at 2 years of 70%. No N related serious adverse events (SAEs) were observed, with only 2% Grade 3 AEs observed (varied and unrelated to treatment) and all others were Grade 1-2, including 21% GI, 18% cutaneous, and 10% musculoskeletal, respiratory, and fatigue, each; overall, 2% of these Grade 1-2 AEs were treatment related. Conclusions: Our preliminary results show a trend towards improved RFS in patients with Stage IIB/IIC melanoma treated with nivolumab. The cohort has not reached a minimum follow up of at least 2 years for RFS; patients are continuing to be monitored. On study, we observed the expected adverse events, without evidence of new toxicities. Data maturation will reveal the full effect of adjuvant N on disease relapse and overall survival and distant metastasis-free survival in stage IIB/IIC melanoma patients. Clinical trial information: NCT03405155.

Background: CMP-001 comprises a CpG-A oligodeoxynucleotide packaged within a virus-like particle. It is designed to activate tumor-associated plasmacytoid dendritic cells via TLR9 inducing an interferon-rich tumor microenvironment and anti-tumor CD8+ T cell responses. Materials and Methods: CMP-001-001 is an ongoing phase Ib trial evaluating intratumoral (IT) CMP-001 in combination with pembrolizumab (administered per label) in subjects with advanced melanoma resistant (either did not respond or progressed) on prior anti-PD-1 monotherapy or in combination. During dose escalation, subjects were enrolled to cohorts of ≥ 3 subjects at CMP-001 doses of 1, 3, 5, 7.5, and 10 mg in two dosing schedules (weekly for 7w, followed by q3w; or weekly for 2w, followed by q3w). CMP-001 was administered IT into an accessible lesion(s), and response assessed in all target lesions (injected and non-injected) by RECIST v1.1. Study therapy was continued until progression, toxicity, investigator decision or withdrawal of consent. Baseline and on-therapy serum was collected for cytokine analysis. Immunohistochemical and RNA-Seq analysis was performed on available pre- and post-treatment tumor biopsies. Results: As of December 31, 2017, 68 subjects have been treated (44 in Escalation and 24 in Expansion). Safety data from 63 subjects demonstrated a manageable acute toxicity profile consisting predominately of fever, N/V, headache, hypotension and rigors. Grade 3/4 related AEs reported in ≥1 subject; hypotension (n=7), anemia (n=2), chills (n=2), hypertension (n=2) and fever (n=2). The Objective Response Rates (ORR) across all dose cohorts on weekly (n=40) and q3week schedules (n=13) were 22.5% (9/40; 95 % CI 11-39%) and 7.7%% (1/13; 95% CI 0-36%) respectively. For subjects dosed weekly at 3 and 5 mg, the ORR was 33.3% (6/18 95% CI 13-59%). Of the 10 responders, 1 progressed (w36), 2 withdrew consent (w13, w25), 7 remain on study with 2 subjects maintaining their response though w72. Regression of non-injected tumors occurred in cutaneous, nodal, hepatic, and splenic metastases. CMP-001 induced TLR9 activation with a median 5.9 fold increase in serum CXCL10 (range of 0.9 - 276.3; mean fold increase of 21.8 with SD=48.8; n=39). Immunohistochemical and RNA-Seq analysis of tumor biopsies revealed increases in tumor-infiltrating CD8 T cells (>5 fold), PD-L1 expression (>3 fold increase in H score), and transcriptional signature of inflammation in 2/4 subjects with analyzable pre-and post-treatment samples. Conclusions: CMP-001 in combination with pembrolizumab resulted in objective, durable tumor responses with tolerable toxicities in subjects with advanced melanoma resistant to prior anti-PD-1 therapy. CMP-001 dosing at 5 mg/weekly has been selected for further evaluation in the ongoing dose expansion phase of this study. Citation Format: Mohammed Milhem, Rene Gonzales, Theresa Medina, John M. Kirkwood, Elizabeth Buchbinder, Inderjit Mehmi, Jiaxin Niu, Montaser Shaheen, Ryan Weight, Kim Margolin, Jason Luke, Aaron Morris, David Mauro, Arthur M. Krieg, Antoni Ribas. Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT144.

e15069 Background: Immune checkpoint inhibitors have become an integral part of treatment for a variety of malignancies. Given the favorable side effect profile compared to chemotherapy, there is increased interest in using immunotherapy in senior adults. Unfortunately, there is limited and conflicting data exploring the effect of age on checkpoint blockade efficacy and toxicity. We hypothesize that due to increased immune-senescence, older patients will experience less treatment limiting immune toxicity. Methods: A total of 170 patients were identified as receiving anti CTLA-4 or -1/PDL-1 antibodies between 2012 and 2017 at TJUH. Data was analyzed by age cohorts: age < 65, age 65-74, and age > 75. Chi-square analysis was used to compare the various reasons for therapy discontinuation between age groups. Results: 47% of patients were less than age 65; 37% were between age 65 and 75 and 16% of patients were older than age 75. Baseline characteristics between the groups were similar. Median CCI and RMH score was similar across the groups. Interestingly, the rate of immune toxicity requiring therapy discontinuation was higher among patients > 75 compared to < 65 though not significantly different (p = .098). Conversely, rates of disease progression while on immunotherapy were significantly higher in patients < 65 compared to those > 75 years (p = value here 0.037). Conclusions: Despite evidence for immuno-senescence in the elderly, rates of immune toxicity did not differ significantly by age. Further studies are needed to explore interplay of aging, the immune system and check point inhibitors. Reasons for Discontinuing Checkpoint Blockade, n (%) Age Group (yrs) < 65 65-75 > 75 Number of Patients 80 (47.1) 63 (37.0) 27 (15.9) Number of disease sites; mean (median) 2.91 (3) 2.82 (3) 3.08 (3) Charleston Comorbidity Index at Immunotherapy Initiation; mean (median) 0.76 (1) 0.80 (1) 1.41 (1) Royal Marsden Score at Immunotherapy Initiation; mean (median) 7.22 (8) 8.85 (9) 8.22 (9) Doses of Immunotherapy; mean (median) 7.6 (4) 7.6 (4) 8.7 (4.5) Disease progression Rate 32 (40.0) 33 (52.4) 6 (22.2) Immunotoxicity Rate 10 (12.5) 13 (20.6) 7 (25.9) Death Rate 9 (11.3) 6 (9.5) 4 (18.5) Never discontinued treatment 21 (26.3) 6 (9.5) 5 (18.5)

9592 Background: Despite successful treatment of primary uveal melanomas, up to 50% of patients subsequently develop systemic metastasis, with the liver involved in up to 90% of patients. At our institution, recognition of the poor prognosis associated with liver metastasis has led to the use of various liver-directed treatment modalities including transarterial chemoembolization (TACE) with BCNU, drug-eluting beads with doxorubicin (DEBDOX), immunoembolization (IE) with GM-CSF, and radioembolization with Yttrium 90 radioactive microspheres. The purpose of this study is to compare overall survival between uveal melanoma patients with hepatic metastasis before and after the shift of initial treatment from systemic to liver-directed approaches. Methods: A retrospective single-institution chart review was performed on consecutive series of uveal melanoma patients with hepatic metastasis who were treated at Thomas Jefferson University between 1971–1993 (Cohort 1, n = 98) and 2000–2017 (Cohort 2, n = 634). The following data was collected from medical records: primary tumor stage and genetic abnormalities, primary eye treatment, date to hepatic and extra-hepatic metastasis, types of liver-directed and systemic treatments utilized, and date of death. Time from development of hepatic metastasis to death (OS-Liver) and time from initial treatment of primary uveal melanoma to death (OS-Eye) in individual cohorts were measured and analyzed. Results: 81% of cohort 1 patients received systemic chemotherapy as their initial treatment for liver metastasis, while 91% of cohort 2 patients (n = 574) initially received liver-directed treatments including IE (n = 296), BCNU TACE (n = 147), DEBDOX (n = 45), radioembolization (n = 37), and other liver-directed treatments (n = 49). OS-Liver in cohort 1 and cohort 2 was 4.8 months and 16.4 months, respectively (P < 0.001). More importantly, OS-Eye in cohort 2 (5.1 years) is much longer than that of cohort 1 (3.3 years) (P < 0.001). Conclusions: Liver-directed treatments provided significant survival benefit for uveal melanoma patients with hepatic metastasis.

e15068Background: Immune Checkpoint Blockade (ICB) has demonstrated efficacy across a variety of tumor types. However, treatment is often complicated by immune related adverse events (irAEs), leadi...

e15095 Background: Immune checkpoint blockade is being used with increasing frequency across a variety of tumor types. Corticosteroids are used in diverse clinical scenarios and can have immune-modulatory effects. The administration of steroids with immune checkpoint blockade in combination with cytotoxic chemotherapy has not diminished treatment efficacy in select patient populations1. However, the effect of steroids on the development of immune-related adverse events (irAEs) across tumor types is unknown. We hypothesized that pretreatment with corticosteroids reduces treatment-limiting irAEs. Methods: A single institution registry of patients (n = 163) receiving immune checkpoint blockade, including anti-CTLA-4 antibody (n = 51) or anti-PD-1 antibody (n = 112), was reviewed. Various primary tumor types were included (33% melanoma, 31% non-small cell lung, 36% other). Patients were determined to have discontinued treatment because of irAEs versus any other cause (disease progression, infection, comorbidity, or death). Results: None of the 17 patients (0%) who were receiving corticosteroids prior to starting immunotherapy experienced treatment-limiting irAEs (average dose 34mg/day prednisone or equivalent, only one patient taking < 10mg/day prednisone). This is compared to 29 of 146 patients (19.9%) who were not taking steroids at the start of treatment (p = 0.045). Interestingly, pretreatment steroids were not associated with an increase in disease progression or death. Conclusions: Incidence of treatment-limiting immune-related adverse events was significantly decreased, regardless of tumor type, in patients receiving corticosteroids prior to initiation of immunotherapy. An associated decrease in treatment efficacy was not seen. Table: Incidence of treatment-limiting adverse events in patients undergoing immunotherapy Treatment-Limiting Adverse Events - n (%) Immune Infection or Comorbidity Disease Progression or Death Ongoing Treatment Total On Steroids When Immunotherapy Initiated? Yes 0 (0) 7 (41.1) 8 (47.1) 2 (11.8) 17 (100) No 29 (19.9) 19 (13.0) 71 (48.6) 27 (18.5) 146 (100) Total 29 (17.8) 26 (16.0) 79 (48.4) 29 (17.8) 163 (100)

Title: Suppression of human T cell activation by glycerol monolaurate derivatives Background: Glycerol Monolaurate is composed of a 12 carbon chain fatty acid with an ester linkage to a glycerol head group. Interestingly, GML inhibits the growth of bacteria, fungi, and enveloped viruses. It is used commercially in several products as a preservative and an antimicrobial agent; however the effect of GML on mammalian cells is not yet fully established. Previous work in this lab has shown GML inhibits T cell signaling by disrupting cell membrane lipid rafts and localization of Arp 2/3 complex subunits. Methods: GML derivatives were tested to determine what components of GML are responsible for its T cell inhibiting activity. These compounds differ from GML in carbon chain length, head group, linker, and position of the laurate group. Activation was measured via LAT clustering, cytokine production, and calcium signaling. Results: Derivatives with a carbon chain less than 12 carbons did not inhibit T cell activation. In addition, the linker and head group polarity were also found to contribute to GML’s ability to inhibit cytokine production, however changing the position of the laurate group did not affect GML’s activity. Conclusion: None of the GML analogs inhibited T cell activity better than GML. However, some compounds that did not disrupt cell membrane organization still inhibited cytokine release. This suggests GML’s effects are more complex than those seen by the lipid raft disruption alone. Overall, these data contribute to the understanding of a novel class of immune modulating lipids and suggest novel therapeutic applications of GML including targeting immune cancers and altering immune cell function during cancer treatment. THE ROLE OF PROSTAGLANDINS IN COLLECTIVE, INVASIVE CELL MIGRATION Emily Fox1, Tina Tootle1 1Anatomy and Cell Biology Dept., University of Iowa Collective cell migration – the coordinated movement of tightly or loosely associated cells – is important for both normal development and tumor invasion. While prostaglandins (PGs), short-range lipid signaling molecules, regulate cell migration, and are known to be up regulated in many cancerous tissues, their mechanisms of action are poorly understood in both single and multicellular migration contexts. To address this knowledge gap we use the collective, invasive, epithelial migration that occurs during Drosophila oogenesis. The Drosophila ovary contains chains of developing follicles composed of 15 germline derived nurse cells and 1 oocyte surrounded by a layer of somatic epithelial cells. During Stage 9 of oogenesis, a cluster of 6-8 of these somatic cells delaminate from the outer epithelium and migrate invasively between the nurse cells to the oocyte border; this migration is termed border cell migration. To study the roles of PGs in border cell migration, we utilize genetic mutations in pxt, the Drosophila cyclooxygenase-like enzyme, which is responsible for all PG synthesis. Using quantitative analyses, I find that loss of Pxt causes aberrant border cell migration. Loss of Pxt results in both a significant delay in border cell migration and an increase in cluster length compared to wild-type controls. We hypothesize that both the delay and alteration in cluster morphology are due to changes in among the border cells and/or between the border cells and the surrounding nurse cells. While E-Cadherin appears to be unaffected by the loss of Pxt, integrin levels on the interface between the border cells and the nurse cells is reduced. As integrin-based adhesion is essential for correctly timed border cell migration and cluster cohesion, our data supports the model that PGs regulate integrins to control border cell migration and cluster morphology. Future work will further investigate this model as well as the role of PGs in the border cell cluster vs the nurse cells. Our studies on PG signaling during border cell migration provide insights into the conserved mechanisms by which PGs regulate collective, invasive cell migrations. Indeed, high levels of PGs and integrins are independently associated with cancer migration and metastasis. Assessing the molecular heterogeneity of cutaneous T cell lymphoma using single cell RNA-seq

AIM To compare PD-L1 expression between metastatic uveal melanoma (MUM) and metastatic cutaneous melanoma (MCM). MATERIALS & METHODS A total of 295 MCM and 78 MUM specimens were analyzed for tumor cell PD-L1 expression. Additionally, 91 MCM and 45 MUM specimens were analyzed for PD-1 expression on tumor-infiltrating lymphocytes. RESULTS A total of 77/295 (26.1%) MCM specimens expressed PD-L1 as compared to 4/78 (5.1%) MUM specimens (p < 0.0001). PD-1 expression on tumor-infiltrating lymphocytes was greater in MCM (73.6%; 67/91) than in MUM (51.1%; 23/45), respectively (p = 0.009). CONCLUSION Significant differences exist in PD-L1 expression between MCM and MUM. The lower PD-L1 expression in MUM may provide a rationale for failure of PD-1 inhibitor therapy and suggests that immune evasion in this disease may occur via alternative mechanisms.

The combination of immune checkpoint inhibitors ipilimumab and nivolumab has been recently been FDA approved for first line treatment of unresectable and metastatic BRAF wild type melanoma. The approval came following the impressive results of the CheckMate 067, where the combination of ipilimumab and nivolumab appeared to outperform each as a single agent in regards to response rate and progression free survival. Though we await final overall survival data, the combination will likely be adapted by many oncologists and integrated into the ever changing melanoma treatment algorithm. In this article we aim to summarize the data leading up to the recent FDA approval and publication by Larkin et al. that presents the results from the CheckMate 067 trial. We will also further explore the feasibility, challenges, and applicability of combination immune checkpoint inhibitor therapy.

9569 Background: Uveal melanoma is the most common primary intraocular malignant tumor in adults. Up to 50% of uveal melanoma patients die of metastasis, usually to the liver. Cell free DNA (cfDNA) provides a commercially available, non-invasive mechanism for monitoring disease activity in malignancy. Recently, studies have investigated cfDNA in uveal melanoma and have demonstrated an association with hepatic metastasis, metastatic volume as well as PFS and OS. It remains unclear if cfDNA can predict recurrent metastatic disease in high-risk uveal melanoma patients. Methods: We conductedan exploratory study of cfDNA following treatment for primary intraocular uveal melanoma. Three cohorts were investigated: high-risk patients ( > 50% estimated recurrence rate) with no clinical evidence of metastatic disease (cohort 1), patients with newly developed metastatic disease of the liver by surveillance MRI (cohort 2), patients with previously established metastatic disease (cohort 3). cfDNA profiles were provided by Guardant360 complete exon sequencing and analyzed for the presence of GNAQ/GNA11 mutations and MYC amplification which represent driver mutations present in > 80% of uveal melanomas. cfDNA was evaluated within 1 week of surveillance imaging studies. Results: Cohort 1 revealed no G-protein/MYC abnormalities by cfDNA (0/32). 34.4% (11/32) of patients in cohort 1 had mutations unrelated to G-protein/MYC. Cohort 2 showed a 10% (1/10) G-protein/MYC detection rate. Cohort 3 showed a G-protein MYC detection rate of 75% (18/24) which approaches the expected mutation rate in uveal melanoma. Interestingly, cfDNA did not detect G-protein/MYC abnormalities in hepatic tumors less than 2.0cm in diameter, regardless of cohort. Conclusions: MRI is a more sensitive screening test than cfDNA for detection of hepatic metastasis in uveal melanoma. cfDNA negative for G-protein/MYC alterations correlates with the absence of clinically detectable disease (100% specificity) and a positive result in the adjuvant setting should prompt further evaluation. This study suggests a detection threshold for cfDNA of > 2.0cm hepatic tumor diameter, which may have implications for the utilization of cfDNA across tumor types.

1. Cristofanilli, M, et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004; 351(8): 781-91. 2. Bidard FC, et al., Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int J Cancer. 2014 Mar 1; 134(5):1207-13. 3. Weight RM, et al. Photoacoustic detection of metastatic melanoma cells in the human circulatory system. Opt Lett . 2006 Oct 15; 31(20):2998-3000. 4. O’Brien CM, et al. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow. J Biomed Opt . 2012 Jun; 17(6):612-21. • Circulating Uveal Melanoma Cells (CUMCs) were successfully quantified by the photoacoustic method including single cell detection. • Recovery rates of pigmented uveal melanoma cells suspended in a neutral density solution approached 25%. • 60% CUMCs are lost during PBMC isolation. Superior isolation techniques should be investigated to increase CUMC recovery. • Recovery rates for CUMCs in whole blood averaged 10% of expected cell yield (23/216 noise adjusted cell detection). • The Photoacoustic Method offers a viable platform for the detection of CUMCs. • Studies analyzing CUMCs from patients with metastatic disease are ongoing.

e14536 Background: Checkpoint inhibitors have been shown to improve outcomes in patients with metastatic cutaneous melanoma. Thyroid-related laboratory abnormalities are one of the most common abnormalities seen with these treatments. We hypothesized that thyroid-related laboratory abnormalities predict future treatment-limiting immune-related adverse events (irAEs). Methods: An exploratory study was conducted in melanoma patients (n = 85) who have received anti-CTLA-4 antibody alone (n = 26), anti-PD-1 antibody alone (n = 26) or both (n = 23) at Thomas Jefferson University. Data were collected at baseline and at the time of individual treatments by a retrospective review of electronic medical records. irAEs were categorized into the following: hypophysitis, thyroid dysfunction, lipase elevation, pneumonitis, colitis, transaminitis, acute kidney injury, and diabetes. Results: Incidence and time-to-onset in days of irAEs is shown in table. Number of patients experiencing 0, 1, ≥ 2 irAEs was 35%, 26% and 39% respectively. It is of note that 85.7% (18/21) patients with thyroid-related laboratory abnormalities subsequently developed a serious irAE that required discontinuation of treatment (pneumonitis n = 1, colitis n = 4, hepatitis n = 7, AKI n = 1, DM n = 1, hypophysitis n = 2, thyroid disease n = 2) and this is much higher in incidence compared to those without thyroid-related laboratory abnormalities (P < 0.05). Conclusions: Incidence of individual irAEs was in line with previously published rates. Thyroid-related laboratory abnormalities had a statistically significant correlation (85.7%) with subsequent development of a treatment-limiting irAE. Hypophysitis Overt thy roid dysfunction Chemical thy roid dysfunction Lipase elevation Pneumonitis Colitis Transaminitis AKI DM CTLA- 4 % 12.2 12.3 50 4.1 8.2 22 59.2 12.2 0 Onset 62 87 63 107 79 43 75 63 N/A PD-1 % 5.1 10.3 22.2 7.6 12.7 18 66 22.6 2.6 Onset 109 69 122 38 105 66 58 49 98 Both % 8.7 26.1 30.4 8.7 17.4 26 69.6 0 4.3 Onset 196 118 51 181 175 137 131 N/A 172

e14540 Background: Checkpoint inhibitors such as anti-CTLA4 and anti-PD1 antibodies have changed the standard care for advanced melanoma. However, there has been little reported on the effectiveness in non-melanoma skin cancers. Here we report on three cases where patients were treated with checkpoint inhibitors for other indications and had dramatic improvement of their pre-cancerous lesions and non-melanoma skin cancers. Methods: Observations regarding pre-cancerous and non-melanoma skin lesions were made during treatment with checkpoint inhibitors. Results:Case 1: A 72-year old man presented with numerous basal cell carcinomas (BCC), squamous cell carcinomas (SCC), and a Stage IIB primary cutaneous melanoma (T3bN0M0). His primary melanoma was treated surgically. At 9 months, he developed a local recurrence and distant metastases to the mediastinum and bone. He was started on combination ipilimumab and nivolumab. After 2 months, his melanoma regressed and all existing cutaneous BCC and SCC were resolved. One year later, there have been no new skin carcinomas. Case 2:A 67-year old man with history of SCC of the lung initially presented with cutaneous metastasis. At the time, he also had a number of primary cutaneous SCCs that were biopsy proven. He was started on nivolumab and had improvement of his metastatic lung carcinoma and resolution of numerous primary SCCs. Case 3:A 86-year old woman presented with a Stage IIA (T3aN0M0) melanoma of the right shin that was treated surgically. Four years later, she developed metastatic disease to the right pelvis. At her metastatic presentation, she was also noted to have extensive solar and senile elastosis with diffuse actinic keratoses (AK). She received two doses of ipilimumab followed by two doses of pembrolizumab, at which time there was complete resolution of the AKs. Conclusions: Our observation indicates that checkpoint inhibitor therapy may be effective for the treatment of cancerous and pre-cancerous non-melanoma skin lesions including AK, BCC, and SCC. This could be a potential new indication for checkpoint inhibitors to prevent invasive and time-consuming measures such as repeated surgeries, radiation and to produce much better cosmetic outcomes.

Abstract Circulating tumor cells (CTCs) have been shown to be a prognostic marker in breast cancer1. We hypothesize that circulating melanoma cell (CMC) detection could be utilized in the management of uveal melanoma, including early intervention. Prior methodologies for circulating uveal melanoma cell (CUMC) detection have been fraught with poor sensitivity, limiting their clinical utility2. Development of an improved method is necessary to establish the clinical utility of CUMC monitoring. Photoacoustics, also referred to as laser-induced ultrasound, is a novel platform for the detection and capture of CMCs. Photoacoustics uses short duration pulsed light to create ultrasonic acoustic waves in an optically absorbing medium, in this case melanin within melanoma3. As light is absorbed by irradiated chromophores, the optical energy gets converted into kinetic thermal energy trapped within the chromophore and subsequent thermal expansion ensues. Transient thermoelastic expansion of the absorbent cell results in the propagation of ultrasonic acoustic waves which can be detected and analyzed using a piezoelectric response mechanism. In addition, detected CMCs can be isolated by a two-phase flow cell separation technique4. Due to the low cost and melanoma specific capabilities of photoacoustics, we evaluated this technology for the purpose of CUMC detection. Methods: Cells from uveal melanoma cell line UM002B, established at Thomas Jefferson, were titrated to various cell concentrations and analyzed in a neutral density solution utilizing the photoacoustic method. Uveal melanoma cells of differing concentrations were spiked into isolated healthy donor peripheral blood mononuclear cells (PBMCs) and healthy whole blood samples. PBMC isolates were analyzed for CUMCs. Results: CUMCs were successfully quantified by the photoacoustic method including single cell detection. Recovery rates of cultured cells in a neutral density solution approached 25%. Recovery rates for CUMCs in whole blood averaged 10% of expected cell yield (56/540 cells detected) with a higher detection rate at lower cell concentrations. Photoacoustics offers a viable method for the detection of CUMCs with an accuracy that meets or exceeds previously reported CUMC yields. Studies analyzing CUMCs from patients with metastatic disease are ongoing. 1. Cristofanilli, M, et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med, 2004. 351(8): 781-91. 2. Bidard FC, et al., Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int J Cancer. 2014 Mar 1;134(5):1207-13. 3. Weight RM, et al. Photoacoustic detection of metastatic melanoma cells in the human circulatory system. Opt Lett. 2006 Oct 15;31(20):2998-3000. 4. O’Brien CM, et al. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow. J Biomed Opt. 2012 Jun;17(6):061221. Citation Format: Ryan M. Weight, Shingo Sato, Masahiro Ohara, Mizue Terai, Michael Mastrangelo, Marlana Orloff, Benjamin Goldschmidt, John Viator, Takami Sato. Detection of circulating tumor cells in high-risk primary uveal melanoma by the photoacoustic method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 471.

Abstract Background: Circulating tumor cells (CTCs) represent a surrogate biomarker for hematogenous metastases. The detection of CTCs has gained increasing interest for prediction of clinical outcome. However, it remains to be determined whether uveal melanoma cells circulating in a peripheral vein predict systemic recurrence and poorer overall prognosis. Uveal melanoma, the most common primary cancer of the eye in adults, is unique in that the uveal tract is devoid of lymphatics. Since systemic recurrence develops exclusively via hematogenous spread, patients with uveal melanomas present a special opportunity to explore the prognostic potential of CTCs. Although the lung is the first organ through which venous drainage from the affected eye passes and indeed the only organ through which all venous blood must pass, 80-95% of systemic metastases are found first in the liver without development of lung metastases. To address these issues, we investigated the numbers of CTCs in paired arterial (femoral) and venous (antecubital) blood specimens obtained from uveal melanoma patients with hepatic metastases. Methods: CTCs in blood specimens were measured in 17 uveal melanoma patients with multiple hepatic metastases, including 10 patients with liver-only metastases and 7 patients with hepatic and extra-hepatic metastases. Peripheral arterial and venous blood specimens were collected at the same time prior to liver directed treatment. CTCs were analyzed using CellTracks Circulating melanoma Cell Kit by CellSearch System. The clinical information and sources of blood specimens were blinded when CTCs were analyzed. Result: CTCs were detectable from all 17 arterial blood specimens (100%) (median 5, range 1 to 168). In contrast, much smaller numbers of CTCs were detectable in 52.9% (9/17) of venous blood specimens (median 1, range 0 to 8) from the same patients. In terms of degree of tumor burden, patients who have hepatic as well as extra-hepatic metastasis showed higher numbers of arterial CTCs (median 12, range 5 to 168), compared to patients who have liver-only metastasis (median 4, range 1 to 11). More importantly, there is no significant correlation between numbers of arterial CTCs and the degree of tumor volume in the liver in patients who have liver-only metastases. Conclusions: Using this technology, the detection of uveal melanoma cells in peripheral blood is feasible. Peripheral capillary beds effectively filter CTCs from venous blood; thus venous blood might not be the best source for measurement of CTCs in patients with metastatic uveal melanoma. The paucity of clinically evident non-hepatic metastases despite the effective filtration by peripheral capillary beds remains unexplained and may be a demonstration of the “seed and soil” hypothesis. CTCs are a non-invasive source of uveal melanoma cells for evaluating tumor biology. Further investigation is warranted. Citation Format: Mizue Terai, Zhaomei Mu, David Eschelman, Carin Gonsalves, Ken Kageyama, Michael J. Mastrangelo, Marlana Orloff, Ryan Weight, Massimo Cristofanilli, Takami Sato. Detection of circulating melanoma cells in paired arterial and venous specimens from uveal melanoma patients with hepatic metastatic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 384. doi:10.1158/1538-7445.AM2015-384

Background: CTCs provide prognostic information and their application is under investigation in multiple tumor types. Of the multiple variables inherent in any such process, none is more important to outcome than the appropriateness of the sample source. To address this question, we investigated CTCs in paired peripheral venous and arterial blood specimens obtained from stage IV uveal melanoma patients. Methods: Blood specimens were obtained from both common femoral arteries and antecubital veins in 17 uveal melanoma patients with multiple hepatic metastases for CTC measurements. Finding: CTCs were detectable with greater frequency (100%) and in larger numbers (median 5, range 1 to 168) in all arterial blood specimens than in venous samples (52.9%; median 1, range 0 to 8). Patients with hepatic as well as extra-hepatic metastasis showed higher number of arterial CTCs, compared to patients with liver-only metastasis (p = 0.003). There was no significant association between the number of arterial CTCs and the tumor burden within the liver in patients who had liver-only metastases. Interpretation: Our data indicate that arterial blood specimens might be a better source of circulating uveal melanoma cells. Although less conveniently processed, perhaps arterial blood should be evaluated as sample source for measurement of CTCs.

Detection of circulating tumor cells (CTC's) in human blood and lymph systems has the potential to aid clinical decision making in the treatment of cancer. The presence of CTC's may signify the onset of metastasis, indicate relapse, or may be used to monitor disease progression. A photoacoustic flowmetry system was designed and tested for detecting circulating melanoma cells (CMC's) by exploiting the broadband absorption spectrum of melanin within CMC's. The device was tested on cultured melanoma cells in saline suspension and in a Stage IV melanoma patient. The device showed a detection threshold of a single melanotic melanoma cell from culture. Transient photoacoustic events were detected in a sample derived from a Stage IV melanoma patient that corresponded to particles passing through the laser beam path, indicating the presence of single melanoma cells in the human circulatory system.