Priore, Giuseppe Del

Abstract Background: Micronuclei (MN) are a result of biological DNA repair mechanisms which form due to internal chromosomal aberrations indicating sub-clonal cancer populations with higher cell survivability and drug therapy resistance. MN are often observed as small fragments of nucleic acids excised from a primary nucleus in Circulating Stomal Cells (CStCs) in response to DNA damage. CStCs with damaged DNA undergoing repair mechanisms, such as those that form MN, are likely to have upregulated expression of programmed cell death ligand (PD-L1) which can be abnormally high on cancer cells that have high mutational burdens. We evaluated CStCs in metastatic breast cancer (mBC) patients for presence of MN and the cell's PD-L1 expression, to determine its prognostic significance to clinical outcomes. Methods: We enumerated MN formation in CStCs in a prospective pilot study using n=76 mBC patients starting new lines of treatment. Whole peripheral blood (7.5mL) was procured and filtered for CStCs and then stained for PD-L1. DAPI was used to identify MN, defined by small (<3 µm) DAPI+ circular formations within the cytoplasm, separate from the primary nucleus. We compared number of MN to PD-L1 expression of all cells, and the MN presence to all available clinical variables. Patients’ progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST v1.1 over two-years. Results: MN were identified in 62% (n=46/76) of CStCs and MN positive CStCs had a significantly higher PD-L1 expression than MN negative CStCs (p=0.0082). A linear relationship between increasing MN number and higher PDL-1 expression within cells was identified, R=0.9411. Further, presence of MN with in CStCs was significantly prognostic for worse PFS (HR=2.62 95%CI 1.50-4.59, p=0.0012) and worse OS (HR= 3.07 95%CI 1.56-6.03, p = 0.002) over 24 months. Conclusion: MN formations in mBC are observable biomarkers of an underlying repair mechanism which suggest the survivability of sub-clonal cancer populations associated with more aggressive cancer subtypes with worsening progression rates. Further studies to evaluate the effect of PD-1 immunotherapies in MN positive patients is ongoing. Citation Format: Dimpal M. Kasabwala, Massimo Cristofanilli, R. Katherine Alpaugh, Saranya Chumsri, Carolina Reduzzi, Cha-Mei Tang, Rena G. Lapidus, Giuseppe Del Priore, William V. Williams, Daniel L. Adams. Micronuclei in circulating stromal cells correlates with PD-L1 expression and predicts progression in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 304.

Abstract Background: SV-BR-1-GM is an irradiated allogeneic cell line derived from a hormone receptor-negative (HR-) HER2-positive (HER2+) breast cancer (BC), now engineered to express HLA class I and II antigens, secrete GM-CSF and function as an antigen-presenting cell. Here we report post-hoc exploratory data for metastatic BC (MBC) patients treated with the SV-BR-1-GM regimen (SV) alone (NCT03066947) and in combination (CO) with immune checkpoint inhibitors (ICIs) (NCT03328026). Methods: SV includes cyclophosphamide 300 mg/m2 i.v. 48-72 hours prior to SV-BR-1-GM (20 × 106 cells) intradermally followed by interferon-alpha-2b at the SV-BR-1-GM inoculation sites on days 2 and 4. SV was given every 2 weeks for 3 cycles and then monthly in NCT03066947, and every 3 weeks in combination with PD-1 inhibitors in NCT03328026. Treatment was continued until disease progression or unacceptable toxicities. Results: A total of 34 refractory MBC patients (pts) were treated, including 24 with SV, and 14 with CO, including 4 who crossed over from SV to CO plus one who restarted the SV after a protocol specified administrative interruption in treatment. Median prior regimens were 5; 55% of pts had hormone receptor-positive (HR+) BC, 42% had HER2+ BC, and 16% were triple-negative BC (TNBC). Objective response rates (ORR) were 4% for monotherapy and 7% for combination. Clinical benefit rate (CBR) was 21% for monotherapy and 29% for combination. Responses and clinical benefits were seen across most BC subtypes (see table). Notably, CBR among 10 pts with HR+ BC (any HER2) who received the combination was 50% (5/10). The duration of response (DOR) among 4 patients treated with monotherapy was 49-223 days and DOR among 6 patients treated with combination was 72-292 days. Median PFS was 2.8 months for monotherapy and 4.2 months for combination. Median OS was 7.0 months for monotherapy, and 12.0 months for combination. Of 28 pts with available prior treatment data, 12 (43%) had PFS on study treatment exceeded PFS on prior treatment, of these 8/12 had >1 HLA match and 5/8 had ≥ 2 loci HLA-matched with SV-BR-1-GM. PFS ratio improvement was independent of the prior number of lines of therapy or BC subtypes. The SV-BR-1GM regimen was well tolerated with the most common treatment-related adverse events (AEs) being injection site reaction in 67%. There was no dose-limiting toxicity, special interest AEs such as cytokine storm, and no grade 5 observed. There were 2 non-treatment-related grade 4 AEs: worsening pleural effusion and altered mental status. Significant improvement in PFS was observed in patients with matched HLA and in association with the delayed-type hypersensitivity skin test, peripheral blood circulating tumor cells, and cancer-associated macrophages. Conclusions: SV-BR-1-GM demonstrated promising activity in patients with MBC. Treatment was well tolerated with no concerning AEs. The PFS ratio compares favorably with prior penultimate standards of care, more notably in patients with matched HLA. Clinical benefits were observed across multiple subtypes of BC, particularly in patients with HR+ disease receiving combination therapy. Phase II clinical trial is currently ongoing to evaluate the efficacy of SV-BR-1-GM in combination with ICIs. Clinical benefit and PFS on SV-BR-1-GM SV-BR-1-GM Whole Cell Therapeutic Vaccine in Heavily Pre-Treated Metastatic Breast Cancer Results Stratified by HER2 and HLA Matching Citation Format: Saranya Chumsri, William Williams, Mingjin Chang, Miguel Lopez-Lago, Charles Wiseman, Jarrod Holmes, Chaitali Nangia, Karim Mohammed, Minal Barve, Shaker Dakhil, Bonnie Guerin, Giuseppe Del Priore, Carmen Calfa. Allogeneic, Antigen-Presenting, GM-CSF-secreting, SV-BR-1-GM Whole Cell Therapeutic Vaccine in Advanced Metastatic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-12.

Abstract Introduction: D/L-alpha-metyrosine (SM-88, racemetyrosine) is a clinical stage chemically modified racemic amino acid therapy with broad anticancer activity in patients across 15 different indications. While amino acid metabolism has been leveraged in oncology imaging for decades, SM-88 offers a novel therapeutic approach to selectively disrupt cancer cells. This study explored phenotypic and mechanistic effects of exposing cancer cells to D/L-alpha-metyrosine as well as potential immunomodulation of the in vivo tumor microenvironment related to tyrosine hydroxylase inhibition and catecholamine depletion. Methods: In vitro experiments were conducted in a range of human and mouse cancer cell lines, including MCF-7, 4T1, HCT116, CAPAN2, PAN02, PANC01. The effect of SM-88 methyl-ester (SM-88 ME) alone on viability, apoptosis, migration/invasion, protein synthesis inhibition, autophagy, cell cycle arrest, and ROS generation was assessed. In vivo experiments included xenograph studies of HCT116 with monotherapy consisting of either IP injections up to 200 mg/kg/day of SM-88 ME, or orally administered SM-88 up to 324 mg/kg/day. Orthotopic xenographs of 4T1 and PAN02 models using monotherapy doses up to 200 mg/kg/day are ongoing. Ongoing immunohistochemistry and flow cytometry is focused on correlation with LAT1 expression and efficacy, alteration in macrophage polarization (M1/M2 ratio), Tregs, and various T cell populations in the tumor microenvironment. Results: Exposure to SM-88 ME exhibited a dose dependent increase in ROS, as well as decreases in caspase 3/7 activation. An increase in ROS following exposure to SM-88 ME was also observed in PAN02 cells. Similar decreases in caspase 3/7 activation were observed in CAPAN2, HCT116, and MCF7 cells. Interestingly, across a variety of cell lines including PAN02, PANC1, and 4T1 cell morphology changes suggested the formation of autophagic vacuoles. Viability across all cell lines was reduced in a dose and time dependent manner. In a mouse HCT116 SC xenograft model, mice receiving 324 mg/kg/day SM-88 ME showed significantly reduced tumor size compared to control mice. Additional in vitro and in vivo experiments are ongoing. Conclusions: The importance of amino acid metabolism in cancer has gained greater awareness over the past decade, however potential therapeutic approaches in this area remain limited. SM-88 has been dosed in over 180 cancer patients with encouraging efficacy and safety findings to date. Early data suggest increases in ROS generation and decreases in apoptosis may occur in certain cancer types (PAN02 pancreatic cancer), suggesting a shift away from apoptosis and towards autophagy. Potential immune modulatory effects of SM-88, including alterations in macrophage polarization related to tyrosine hydroxylase inhibition and subsequent reductions in catecholamine production are being examined. Citation Format: Alexander Vandell, Jonathan Eckard, Steve Hoffman, Giuseppe Del Priore, Martin Fernandez-Zapico. In vitro and in vivo anticancer effects of D/L-alpha-metyrosine (SM-88), a novel metabolism-based therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5998.

Abstract Introduction: TYME-18 is a combination of an ambiphilic steroid acid, tauroursodeoxycholic acid (TUDCA) and surfactants (N-methyl pyrrolidone, polidocanol, and linoleic acid) administered intratumorally to induce regression without injuring adjacent tissue. Methods: Balb/C mice were inoculated with CT-26 cancer (106 cells) until a tumor was established to approximately 100 mm3. An initial feasibility semi-quantitative assessment was performed with 10 animals (table 1). A larger confirmatory experiment testing the individual components of TYME-18 was also conducted (tables 2 & 3). Mice received intra-tumoral injections of TYME-18 with surfactants; or surfactants without TUDCA, or vehicle with measurements 3 times/wk. Intra-tumoral injections were administered 6 times at 3-day intervals over a total of 4 weeks. Animals were sacrificed when tumors reached 2000 mm3 or at the end of the study (16 weeks). Table 1.HISTOPATHOLOGIC FINDINGSFinal tumor sizes & intratumoral necrosis. TYME-18 vs Control. CT-26 model. (Fisher's Exact Test p=0.001)GroupAnimal IDTumor SizeIntratumoral NecrosisControlG1A-1>1 cmModerateG1A-2>1 cmMildG1B-2>1 cmMildG1B-3>1 cmModerateG1B-4>1 cmModerate-TreatedG2A-1<1 – 5 mmSevereG2A-3<1 – 5 mmSevereG2B-1<1 – 5 mmSevereG2B-3<1 – 5 mmSevereG2B-55 – 6 mmSevere- Table 2.Days Post Implementation average tumor volume mm3 (ANOVA P<0.0001)0369121520Vehicle83.1157.9263.4401.4613.9849.41368.9Surfactant Alone76.4131.1166.3209.6274.3345.7642.8TYME-1880.528.228.934.235.854.457.2- Table 3.Tumor Free Mice at Study End: Day 112 (Fisher's Exact Test p < 0.0001)Treatment# of Mice# Tumor Free Mice% Tumor Free MiceVehicle2000Surfactant Alone1218.33TYME-18121191.6 Results: TYME-18 demonstrated a significant difference between treated and untreated animals (see Tables 1, 2, 3). A minor reduction in tumor cell mass was observed in response to the surfactant, with the greatest response (ANOVA p=.001) observed in the TYME-18 (surfactant + TUDCA) (table 2). Discussion: The local administration of an ambiphilic steroid acid along with surfactants, impeded the ability of tumors to increase in size. Both the surfactants and the ambiphilc steroid acid contributed to this effect with an apparent synergistic effect. Aside from the physical chemical acid effect on tissue, ambiphilic steroid acids are now recognized as important regulators of energy metabolism. Their effects on FXR, LXR, PPAR-α, PPAR-γ are known to regulate important changes in energy metabolism including insulin utilization, the sensitivity of the insulin receptor, the transcription of the genes that mediate glucose and lipid metabolism as well as various immune effects; all of which could be useful in the treatment of cancer. Currently, TYME-18 is under investigation as a simple and nontoxic treatment for tumors for which systemic therapy is not indicated. Citation Format: John Rothman, Steve Hoffman, Allyson Ocean, Jonathan Eckard, Giuseppe Del Priore, Martin Fernandez-Zapico. In vivo mouse model data demonstrating reduction in tumor cell proliferation following intratumoral administration of TYME 18 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2764.

Abstract Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a >50% decrease in tumor growth at three weeks of treatment (p<0.05). Ongoing preclinical experiments are testing the full pharmacodynamic effects of SM-88 on pancreatic cancer cells, as well as potential effects on the tumor microenvironment due to catecholamine interruption. In results from the ongoing phase 2 trial of SM-88 in heavily pretreated metastatic pancreatic cancer patients, SM-88 has shown monotherapy antitumor activity based on RECIST 1.1 criteria. Additionally, 16/24 patients demonstrated a reduction in circulating tumor cells, with the median nadir decline of 63%. LAT1 has been reported to be important in EMT transition and mesenchymal cell survival (Holldorsson S, Cancer letters 2017); examination of the impact of SM-88 on this important emerging biomarker is ongoing. Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B15.

277 Background: SM88 is an oral tyrosine isomer ( TI) and repurposed drugs (CYP3a4 inducer, mTOR inhibitor, oxidative stress catalyst). This regimen has reported no drug related grade 3, 4 or 5 adverse events (JCO 2013 31: e22095) and potential clinical activity in PC (JCO 2018 36: TPS 4156). PC patients have risk factors for malabsorption and a cachexia syndrome rendering some oral therapies potential unreliable. We present PK data from the completely enrolled first stage of an ongoing dose finding phase II in PC. Methods: Open label randomized TI (control – 230 mg bid vs. experimental - 430 mg bid) along with the lowest approved doses of the other agents. Samples were drawn from four patients at pre-dose and up to six h post dosing on cycle day one for two cycles after a single dose of all four components. Results: Mean age: 71.3 (59 – 80), BMI = 25.3 (21.9 – 28.8) and median three prior chemo lines, two post-pancreas surgery and one s/p RT. There was no evidence that the combination altered the expected PKs at C2. There were no serious drug related adverse events. Favorable RECIST, PERCIST or biomarker activity was documented at both TI dose levels. The PK results of the repurposed drugs were consistent with the published data for these agents, despite the combination use and lower than typical label doses; data will be provided. These results were consistent with previous animal and healthier patient PK of SM88. Conclusions: Single dose C1 and C2 TI PKs are consistent with preclinical predictions (ESMO 2016. Ann Oncol (2016) 27 (supp_6): 1605P) and those previously reported in a healthier population of cancer patients (JCO 2018 36: TPS 4156). Based on these results and PK ones from the repurposed drugs, additional data is being collected in order to define the ongoing phase II expansion cohort dose. SM-88 appears to have predictable PK even in heavily pretreated PC patient with prior radiotherapy and surgery. Clinical trial information: NCT03512756. [Table: see text]

Abstract Purpose: We sought to test the hypothesis that the presence of telomerase activity in peritoneal washings of patients treated for ovarian carcinoma is a sensitive and specific indicator of the presence of residual disease. We hypothesized that this test, if added to second-look procedure protocols, could help determine whether residual disease is present or not in patients who have completed their adjuvant chemotherapy for ovarian carcinoma. Experimental Design: Peritoneal washings were obtained from 100 consecutive patients undergoing a second-look procedure after treatment for ovarian carcinoma (cases) and from 100 patients undergoing surgery for benign gynecological conditions (controls). The washings were assayed for telomerase activity using the telomerase repeat amplification protocol. The results were compared to the histological and cytological findings. Results: Among our 100 cases, 82 (82%) had either positive second-look procedures or expressed telomerase in their peritoneal washings. Fifty-three (53%) had positive second-look procedures, whereas 66 (66%) tested positive for telomerase. Twenty-nine of the 47 patients (62%) with negative second-look procedures tested positive for telomerase. Of the 53 patients with positive second-look procedures, 37 (70%) tested positive for telomerase. None of the 100 controls (0%) expressed telomerase in their peritoneal washings. Conclusions: Telomerase activity in peritoneal washings of patients treated for ovarian carcinoma and undergoing a second-look procedure may provide a means of increasing the sensitivity of such procedures for the detection of residual disease while maintaining a high level of specificity.