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Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States, with less than half of patients living >5 years following diagnosis. The NCCN Guidelines for Ovarian Cancer provide recommendations for the diagnosis, evaluation, treatment, and follow-up for patients with ovarian, fallopian tube, and primary peritoneal cancers. These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines, including revised guidance on alternative chemotherapy regimens for patients with advanced age and/or comorbidities, a new algorithm for recurrent low-grade serous carcinoma based on developing research and novel therapeutic agents, and updated language regarding tumor molecular analysis applications in ovarian cancer.

BACKGROUND: RNA-based predictive biomarkers are not assessed in the clinical diagnostic setting. Potential biomarkers include long noncoding RNAs (lncRNAs), a class of molecules emerging as critical regulators of diverse cellular functions, including therapy resistance. Advances in sequencing technologies have made whole transcriptome profiling (RNA-seq) possible in formalin fixed, paraffin embedded (FFPE) tissue. In this study, we assessed the feasibility of detecting lncRNAs in clinical FFPE specimens, using clinical grade RNA-seq technology offered at a CLIA-certified laboratory. Our second aim was to investigate expression of lncRNAs contributing to endocrine resistance in a cohort of metastatic breast cancer patients. METHODS: RNA-seq was performed on clinical FFPE specimens as a physician-ordered diagnostic test in a CLIA-certified laboratory. RNA-seq libraries were constructed using ribosomal RNA depletion method and > 100 M reads were sequenced for each library. We checked for contaminating sequences using fastqScreen against multiple genomes, including human and E. coli among others. To assess the quality of the sequencing libraries, we used the nf-core RNASeq pipeline. Trimmed reads were mapped to the GRCh38 reference and abundance of transcripts from the Gencode comprehensive annotation (v33) was estimated. Each library was assessed for quality control (QC). Mapped reads were merged into a matrix of counts per gene for each sample. Differential expression was assessed between metastatic breast cancers with and without endocrine resistance. RESULTS: We obtained twenty-five cases (23 patients) of ER-positive, HER2 negative metastatic breast carcinoma that had undergone whole transcriptome sequencing at a CLIA-certified molecular laboratory. Of these, 6 (24%) cases had failed QC: 5 patients’ specimens had low RNA content (all core biopsies); 1 patient had total RNA content comprised of less than 75% human reads (lumpectomy). The mean age for specimens that failed sequencing QC was significantly different from those that passed (637.2 days vs 193.7 days, t-test, p = 0.04). After QC, a total of 19 cases were analyzed for differentially expressed lncRNA profiles. Of these, 5 cases were clinically annotated as hormone sensitive; 14 cases had acquired secondary hormone resistance. Thirty-six lncRNAs were identified as differentially expressed (7 antisense; 25 intergenic; 3 intronic; 1 sense-overlapping). Of these, lncRNAs which were found to be over-expressed in endocrine resistant tumors compared to endocrine sensitive tumors are involved in various oncogenic pathways such as cell cycle regulation (Linc0051, MIR205HG, ST8SOA6-AS1), tumor progression (LINC00885, LINC00319), and chemoresistance (SIRLNT). LncRNAs which were downregulated in endocrine resistant tumors compared to endocrine sensitive tumors are involved in stem cell state (CCDC144NL.AS1) and endocrine resistance (MIR2052HG). CONCLUSION: It is feasible to detect lncRNA biomarkers in clinical FFPE tissue specimens, using a laboratory-developed RNA sequencing pipeline in a CLIA-certified genomic laboratory. FFPE-RNA-seq pipeline can potentially expand the molecular information that could be gleamed from clinical FFPE specimens to answer pertinent clinical questions, such as therapy resistance. Table 1.Summary data of metastatic ER+ breast cancer evaluated in this studyFailed RNA QC Success RNA QCNumber of samples (%)6 (24%)19 (76%)Mean specimen age (range)27 days (11-44)194 days (3-1331)Number of specimens which are biopsies (%)5 (83%)17 (89%)Number of specimens derived from bone source (%)2 (33%)1 (5%)Number of specimens derived from visceral organs (%)2 (33%)12 (63%)Number of specimens derived from soft tissue or lymphoid sources (%)2 (33%)6 (32%)Acquired endocrine resistance5 (83%)14 (74%)Endocrine sensitive1 (17%)5 (26%) Citation Format: Christina H. Wei, Denis O'Meally, Michele Kirschenbaum, Allen Mao, Chetan Kancharla, Kevin V. Morris, Daphne Stewart. Feasibility of detecting long noncoding RNAs in clinical FFPE specimens using clinical grade RNA-sequencing pipeline [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-05.

Methods: Women with metastatic breast cancer and their partners completed couples’ tailored biopsychosocial screening and alignment in perception of prognosis immediately before consultation with a Medical Oncologist. In addition, couples were offered a standardized couples’ session before the medical consultation, individual couples’ counseling, and a strengths-based group intervention. As a component of biopsychosocial screening, each patient and her partner were asked individually their understanding of prognosis. They were asked their perception of likelihood of cure with supporting text and percentages provided: 76-100%; 51-75%; 26-50%, or 0-25%. Results: To date 254 women were considered eligible for this program. Complete data for both partners is available on 205. All the patients had metastatic breast cancer prior to their Medical Oncology appointment. The average age of the patient was 54 years (Range 25-84) and 55 years (Range 26-84) for the partner. In the perception of prognosis, 48.7% of patients and their partner were aligned and 51.3% were misaligned. The patient was more likely to have considered their prognosis worse in 59% and the partner 41%. The most commonly endorsed distress items for the patient were: Worry about the future 61%; Side effects of treatment 60%; Fatigue 59%; How my family will cope 58%; and Sleeping 49%. Distress for the partner included: Feeling anxious or fearful 49%; Wanting to best help my partner 37% and Sleeping 37%. Both the patient and partner sought assistance with Understanding treatment options 73.6%; Feeling anxious or fearful 62.5%, Worry about the future 57.3% Fatigue 56.3%, and Pain 56.3%- Partner practical distress was significantly higher for those couples who were not in alignment, p Citation Format: Joanne Mortimer, James Waisman, Yuan, Sayeh Lavasani, Daphne Stewart, Mina Sedrak, Niki Patel, Courtney Bitz, Karen Clark, Marianne Razavi, Matthew J Loscalzo. Integrating the patient and partner distress and perceptions about prognosis in women with metastatic breast cancer guides the medical oncology consultation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-11-12.

Background: Breast tumors with HER2/CEP17 fluorescent in situ hybridization (FISH) ratio < 2 and HER2 copy number ≥ 6, defined as Group 3 FISH pattern by the 2018 ASCO/CAP HER2 testing guidelines, are clinically rare. Their biologic and molecular characteristics are under-characterized. They require only a concomitant HER2 immunohistochemistry score of at least 2+ to merit HER2 “positive” status by the ASCO/CAP guidelines. We seek to characterize the genomic and tumor microenvironment landscape of breast tumors with this unique HER2 FISH pattern. Our second aim is to assess the clinicopathologic features with emphasis on HER2-targeted therapy response.Method: Breast cancers with Group 3 FISH pattern were evaluated by the following methods: 1) High-resolution genome-wide copy number alterations by molecular inversion probe (MIP) array; 2) molecular profiling of tumor immune microenvironment, tumor signaling pathways, and PAM50-based intrinsic subtypes by Nanostring nCounter Breast Cancer 360 Panel; 3) tumor infiltrating lymphocytes (TIL) histologic quantitation, and 4) clinical chart review. Classically amplified HER2 breast tumors (Group 1 FISH pattern; ratio ≥ 2 and HER2 copy number ≥ 2) were used as comparison. Results: Thirty-five (1.3%) cases were identified from 2731 consecutive clinical cases that underwent HER2 FISH testing from 2014 to 2019. Of those, thirteen consecutive cases (spanning 2014 - 2017) with sufficient genomic material were analyzed using MIP array. Group 3 tumors had a more complex karyotype and greater chromosomal instability, compared to classically amplified HER2 breast tumors. None of the Group 3 tumors showed HER2 locus amplification at 17q12. Instead, most showed gain of the 17q arm. Six of the Group 3 tumors were profiled by Nanostring nCounter. Compared to HER2 classically amplified tumors, Group 3 tumors were more immune cold, enriched in ER signaling and TGF-beta signaling pathways. In contrast, HER2 classically amplified tumors were enriched in immune infiltration, cytokine and chemokine signaling, PI3K and MAPK signaling, epithelial-mesenchymal transition signaling, and proliferation (P < 0.5 for all). PAM50 analysis showed that classically amplified tumors were more enriched for HER2-subtype (2/4; 50%), while the majority of the Group 3 tumors were enriched for Luminal B-subtype (5/6; 83%). TIL percentage was statistically higher in HER2 classically amplified tumors compare to Group 3 tumors (avg 53% vs 3%; P = 0.02). Clinicopathologic correlation revealed a high rate of ER positivity and high tumor grade in Group 3 tumors. Group 3 FISH pattern can occur as de novo or in the context of FISH status change following therapy. In the 17 evaluable patients for HER2-targeted treatment efficacy, none of the eight patients who received HER2-targeted neoadjuvant therapy achieved complete pathologic response. Nine of ten patients who received TDM-1 in the metastatic setting progressed with minimal treatment response. Significantly, most of these patients (16/17; 94%) were considered overall HER2 positive by the latest ASCO/CAP guideline. Conclusion: Breast tumors with Group 3 HER2 FISH pattern are molecularly and clinically dissimilar from classically amplified HER2 positive breast tumors. HER2-targeted therapy did not appear efficacious in either the neoadjuvant or metastatic/recurrent settings. The lack of apparent efficacy of HER2-targeted therapy, in the context of their HER2 positive status by the current HER2 guideline assessment, warrants further investigation of this HER2 FISH subtype. Citation Format: Christina H Wei, Lixin Yang, Daphne Stewart, Victoria Bedell, Daniel Schmolze, Sophia Apple, Joyce L. Murata-Collins, Raju Pillai, Joanne E. Mortimer. Genomic and clinical characterization of breast tumors with unusual HER2 FISH pattern (ratio < 2, HER2 copy number ≥ 6): Are they mostly HER2 “positive?” [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-05.

Background This trial evaluated the safety and efficacy of ipatasertib in combination with carboplatin, carboplatin/paclitaxel, or capecitabine/atezolizumab in patients with metastatic triple–negative breast cancer (mTNBC). Methods Eligibility criteria were mTNBC, RECIST 1.1 measurable disease, no prior use of platinum for metastatic disease (Arms A and B), and no prior exposure to immune checkpoint inhibitor (Arm C). Primary endpoints were safety and RP2D. Secondary endpoints were progression–free survival (PFS), response rate, and overall survival. Results RP2D for Arm A (n = 10) was ipatasertib 300 mg daily, carboplatin AUC2, and paclitaxel 80 mg m−2 days 1, 8, and 15 every 28 days. RP2D for Arm B (n = 12) was ipatasertib 400 mg daily and carboplatin AUC2 days 1, 8, and 15 every 28 days. RP2D for Arm C (n = 6) was likely ipatasertib 300 mg 21 days on 7 days off, capecitabine 750 mg m−2, twice a day, 7 days on 7 days off, and atezolizumab 840 mg days 1 and 15 every 28 days. The most common (≥10%) grade 3-4 AEs at RP2D for Arm A (N = 7 at RP2D) were neutropenia (29%), diarrhea (14%), oral mucositis (14%), and neuropathy (14%); Arm B had diarrhea (17%) and lymphopenia (25%); and Arm C had anemia, fatigue, cognitive disturbance, and maculopapular rash (17% each). Overall responses at RP2D were 29% Arm A, 25% Arm B, and 33% Arm C. PFS was 4.8, 3.9, and 8.2 months for patients on Arms A, B, and C, respectively. Conclusions Continuous dosing of ipatasertib with chemotherapy was safe and well-tolerated. Further study is warranted in understanding the role of AKT inhibition in treatment of TNBCs. Trial registration NCT03853707.

Glucocorticoids, which are administered with chemotherapy, cause hyperglycemia. Glycemic variability among breast cancer patients without diabetes is not well known. A retrospective cohort study was conducted involving early-stage breast cancer patients without diabetes who received dexamethasone prior to neoadjuvant or adjuvant taxane chemotherapy between August 2017–December 2019. Random blood glucose levels were analyzed, and steroid-induced hyperglycemia (SIH) was defined as a random glucose level of >140 mg/dL. A multivariate proportional hazards model was used to identify the risk factors of SIH. Out of 100 patients, the median age was 53 years (IQR: 45–63.5). A total of 45% of patients were non-Hispanic White, 28% Hispanic, 19% Asian, and 5% African American. The incidence of SIH was 67%, and glycemic fluctuations were highest in those with glucose levels of >200 mg/dL. Non-Hispanic White patients represented a significant predictor for time to SIH, with a hazard ratio of 2.5 (95% CI: 1.04, 5.95, p = 0.039). SIH was transient in over 90% of the patients, and only seven patients remained hyperglycemic after glucocorticoid and chemotherapy completion. Pretaxane dexamethasone-induced hyperglycemia was observed in 67% of the patients, with the greatest glycemic lability in those patients with blood glucose levels of >200 mg/dL. The non-Hispanic White patients had a higher risk of developing SIH.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States, with less than half of patients livinggt;5 years following diagnosis. The NCCN Guidelines for Ovarian Cancer provide recommendations for the diagnosis, evaluation, treatment, and follow-up for patients with ovarian, fallopian tube, and primary peritoneal cancers. These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines, including revised guidance on alternative chemotherapy regimens for patients with advanced age and/or comorbidities, a new algorithm for recurrent low-grade serous carcinoma based on developing research and novel therapeutic agents, and updated language regarding tumor molecular analysis applications in ovarian cancer.

This study investigated the safety and antitumor activity of aromatase inhibitors (AI) with immune checkpoint inhibitor (ICI) pembrolizumab in patients with hormone receptor positive (HR+) human epidermal growth factor receptor 2-negative (HER2−) metastatic breast cancer (MBC) in a phase II study with a safety lead-in (NCT 02648477). Patients received pembrolizumab plus AI up to 2 years or until confirmed progression or unacceptable toxicity. Key eligibility criteria were HR+ HER2− MBC; RECIST v1.1 measurable disease; adequate organ function; and ECOG 0-1. Primary endpoints were safety and overall response rate. A 3-at-risk design was used for the safety lead-in with a targeted accrual of 20 patients. Grade 2 adverse events (AEs) included 35% fatigue, 20% rash, and 10% hot flashes. Grade 3 immune-related AEs (irAEs) related to pembrolizumab included 5% elevated AST/ALT, 5% rash, and 5% lymphopenia. Two (10%) patients had partial responses, three (15%) had stable disease, and 15 (75%) had progression of disease. Median progression-free survival was 1.8 months (95% CI 1.6, 2.6), median overall survival was 17.2 months (95% CI 9.4, NA), and median follow-up time was 40.1 months (range 31.3–46.8 months). The combination was well tolerated, but clinical activity was comparable to AI alone.

Purpose: Glucocorticoids administered with chemotherapy cause hyperglycemia even in the non-diabetes setting. The prevalence of hyperglycemia and glycemic variability among breast cancer patients without diabetes are not well known, especially across different race/ethnicities. Methods: A retrospective cohort study was conducted involving early-stage breast cancer patients without diabetes who have received dexamethasone prior to (neo) taxane adjuvant chemotherapy between August 2017-December 2019. Random blood glucose levels were extracted, and steroid-induced hyperglycemia (SIH) was defined as a random glucose level of >140 mg/dL. Multivariate proportional hazards model was used to identify risk factors for SIH.Results: Of 100 total patients, the median age was 53 years (IQR: 45-63.5). Forty-five percent were non-Hispanic white, 28% Hispanic, 19% Asian, and 5% African American. The incidence of SIH was 67%, and glycemic fluctuations were highest in those with glucose levels >200 mg/dL. Non-Hispanic white remained as a significant predictor for time to SIH with a hazard ratio of 2.5 (95% CI: 1.04, 5.95, p=0.039). SIH was transient in over 90% patients, and only 7 patients remained hyperglycemic after glucocorticoid and chemotherapy completion.Conclusions: Pre-taxane dexamethasone-induced hyperglycemia in 67% of patients with the greatest glycemic lability in women with blood glucose level > 200 mg/dL. Non-Hispanic white had a higher risk of developing SIH. SIH patients should be monitored using a continuous glucose monitoring or self-monitoring of blood glucose devices. Diagnostic tests for diabetes such as HbA1c, oral glucose tolerance test, fasting plasma glucose, and endocrinology consultation should also be recommended.

BackgroundIn this phase II clinical trial, we evaluated the efficacy of the nonanthracycline combination of carboplatin and nab-paclitaxel in early stage triple-negative breast cancer (TNBC).Patients and MethodsPatients with newly diagnosed stage II–III TNBC (n = 69) were treated with neoadjuvant carboplatin (area under the curve 6) every 28 days for four cycles plus nab-paclitaxel (100 mg/m2) weekly for 16 weeks. Pathological complete response (pCR) and residual cancer burden (RCB) were analyzed with germline mutation status, tumor-infiltrating lymphocytes (TILs), TNBC molecular subtype, and GeparSixto immune signature (GSIS).ResultsSixty-seven patients were evaluable for safety and response. Fifty-three (79%) patients experienced grade 3/4 adverse events, including grade 3 anemia (43%), neutropenia (39%), leukopenia (15%), thrombocytopenia (12%), fatigue (7%), peripheral neuropathy (7%), neutropenia (16%), and leukopenia (1%). Twenty-four patients (35%) had at least one dose delay, and 50 patients (72%) required dose reduction. Sixty-three (94%) patients completed scheduled treatment. The responses were as follows: 32 of 67 patients (48%) had pCR (RCB 0), 10 of 67 (15%) had RCB I, 19 of 67 (28%) had RCB II, 5 of 67 (7%) had RCB III, and 1 of 67 (2%) progressed and had no surgery. Univariate analysis showed that immune-hot GSIS and DNA repair defect (DRD) were associated with higher pCR with odds ratios of 4.62 (p = .005) and 4.76 (p = .03), respectively, and with RCB 0/I versus RCB II/III with odds ratio 4.80 (p = .01). Immune-hot GSIS was highly correlated with DRD status (p = .03), TIL level (p < .001), and TNBC molecular subtype (p < .001). After adjusting for age, race, stage, and grade, GSIS remained associated with higher pCR and RCB class 0/I versus II/III with odds ratios 7.19 (95% confidence interval [CI], 2.01–25.68; p = .002) and 8.95 (95% CI, 2.09–38.23; p = .003), respectively.ConclusionThe combination of carboplatin and nab-paclitaxel for early stage high-risk TNBC showed manageable toxicity and encouraging antitumor activity. Immune-hot GSIS is associated with higher pCR rate and RCB class 0/1. This study provides an additional rationale for using nonanthracycline platinum-based therapy for future neoadjuvant trials in early stage TNBCs. Clinical trial identification number: NCT01525966Implications for PracticePlatinum is an important neoadjuvant chemotherapy agent for treatment of early stage triple-negative breast cancer (TNBC). In this study, carboplatin and nab-paclitaxel were well tolerated and highly effective in TNBC, resulting in pathological complete response of 48%. In univariate and multivariate analyses adjusting for age, race, tumor stage and grade, “immune-hot” GeparSixto immune signature (GSIS) and DNA repair defect (DRD) were associated with higher pathological complete response (pCR) and residual cancer burden class 0/1. The association of immune-hot GSIS with higher pCR holds promise for de-escalating neoadjuvant chemotherapy for patients with early stage TNBC. Although GSIS is not routinely used in clinic, further development of this immune signature into a clinically applicable assay is indicated.

Background: CF33-hNIS-anti-PD-L1 is a novel chimeric orthopoxvirus shown to have anti-cancer activity in triple negative breast cancer (TNBC) xenografts. For clinical tracking of the oncolytic virus (OV), human sodium iodide symporter (hNIS) transgene was inserted into the virus. hNIS gene expression allows cells to take up iodine and be visible by non-invasive imaging techniques. Animal studies showed that tumor cells infected with CF33-hNIS-anti-PD-L1 express functional hNIS and are visible by single-photon emission computed tomography (SPECT) or positron emission tomography (PET). The current report describes imaging results from an ongoing first-in-human trial. Methods: This is a phase I, single center, single arm clinical trial evaluating the safety and tolerability of CF33-hNIS-anti-PD-L1 intratumoral (IT) injections in patients with metastatic TNBC. Key eligibility criteria include patients with unresectable or metastatic TNBC; progressed on at least 2 prior chemotherapies; ECOG 0-2; RECIST 1.1 measurable disease; and at least one tumor amenable to repeated IT injections. Eligible patients receive CF33-hNIS-anti-PD-L1 IT at 1 of 8 assigned dose levels (from 1 × 105 PFU to 3 x 108 PFU) on Days 1 and 15 of each 28-day cycle for a total of 3 cycles of treatment. The primary objective is to evaluate the safety and tolerability of CF33-hNIS-anti-PD-L1. Secondary objectives are to determine optimal biological dose, recommended phase II dose, and response rates. Exploratory objectives include assessing feasibility of non-invasive hNIS imaging as a method of tracking viral infection and replication. SPECT whole body imaging was performed at Cycle 1 Day 8 (C1D8). Results: From October 2021 to October 2022, 8 patients were enrolled in this ongoing study and received at least 1 dose of CF33-hNIS-anti-PD-L1 injection at one of the first 3 dose levels (1 x 105, 3 x 105, or 1 x 106 PFU). All 8 patients underwent SPECT imaging at C1D8 with 6/8 patients (75%) having uptake at the site of injection on the SPECT imaging study. Four of 4 patients (100%) with injection sites at metastatic subcutaneous nodules, intramuscular masses, or axillary lymph nodes and 2/4 patients (50%) with injection sites at matted dermal metastatic lesions had uptake on SPECT. Conclusion: SPECT imaging successfully showed enhancement at the injected lesions in 75% of patients treated with CF33-hNIS-anti-PD-L1, even at current low doses, suggesting local viral replication and hNIS expression. Further analysis will evaluate the correlation of SPECT imaging results with pathologic immune cell infiltrate, viral staining, and tumor response. This is the first known report of hNIS-based imaging to track oncolytic poxvirus replication in humans and gives promise that this technology may be used for noninvasive tracking of systemically administered OV and other therapies. Citation Format: Jamie Green Rand, Dave Yamauchi, Shyambabu Chaurasiya, Jianying Zhang, Raju Pillai, Colt Egelston, Jonathan Kessler, Badri Modi, Leslie Chong, Amanda Seiz, Giovanni Selvaggi, Nick Ede, Mireya Murga, Norma Martinez, Wichanee Borisuthirattana, Hans Meisen, Susan Yost, James Waisman, Daphne Stewart, Joanne Mortimer, Yuman Fong, Yuan Yuan. hNIS imaging data from a first-in-human trial of the oncolytic virus CF33-hNIS-antiPD-L1 in patients with triple negative 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 6788.

Background: Despite recent FDA approvals of immune checkpoint inhibitor pembrolizumab and drug-antibody conjugate in the treatment of metastatic triple negative breast cancer (mTNBC), the overall survival benefit of these therapies remains modest. Oncolytic virotherapy (OV) utilizes genetically modified viruses to infect and kill cancer cells while sparing healthy cells. CF33-hNIS-anti-PD-L1 (CHECKvacc) is a novel chimeric orthopoxvirus with robust anti-cancer activity in TNBC xenografts. Cells infected with CHECKvacc were shown to express functional human sodium-iodide symporter (hNIS) and anti-PD-L1 proteins. hNIS gene transfer allows tracking of virus by 99mTc single-photon emission computed tomography (SPECT). Our preliminary animal studies demonstrated that tumor cells infected with CHECKvacc successfully secrete functional hNIS and anti-PD-L1. CHECKvacc administered by intratumoral injection appears safe and is generally well-tolerated. CHECKvacc detects and effectively kills TNBC at doses several magnitudes lower than other OVs in xenograft models. Methods: This study is a first-in-human phase I, single center, single arm clinical trial evaluating the safety and tolerability of CHECKvacc intratumoral injection in patients with mTNBC. Key eligibility criteria include patients with unresectable or metastatic TNBC; progressed on at least 2 prior chemotherapies including an immune checkpoint inhibitor-containing regimen; ECOG 0-1; RECIST 1.1 measurable disease; and at least one tumor amenable to repeated intratumoral injections. Eligible patients receive CHECKvacc intratumorally at one of 8 assigned dose levels (1 × 105 PFU, 3 × 105 PFU, 1 × 106 PFU, 3× 106 PFU, 1 × 107 PFU, 3 × 107 PFU, 1 × 108 PFU, 3 x 108 PFU) on Days 1 and 15 of each 28-day cycle for a total of 3 cycles of treatment. The primary objective is to evaluate the safety and tolerability of CHECKvacc by CTCAE v5.0. Secondary objectives are to determine optimal biological dose, recommended phase II dose (RP2D), and response rate by RECIST1.1. The first 3 subjects of dose level 1 were enrolled sequentially for safety monitoring. Once the initial 3 subjects were treated sequentially, the study followed the Phase I Queue 3+3 (IQ 3+3) design which expands a dose level up to 8 subjects after a single DLT has been observed. Enrollment to the final RP2D may be expanded to include up to 12 patients for efficacy assessment. The estimated targeted accrual is 33 patients (minimum) to 78 patients (maximum). Correlative aims include assessing viral kinetics, viral plaque assay, 99mTc SPECT imaging for virus tracking, peripheral blood and tumor tissue for antiviral immune activation, and tumor microenvironment changes in association with response to therapy. Results: From October 2021 to June 2022, 6 patients were enrolled and received at least 1 dose of CHECKvacc injection at dose level 1 (1 x 10^5 pfu) and 2 (3 x 10^5 pfu). The intratumoral CHECKVacc injections were well tolerated. No DLTs were observed. No treatment emergent AEs (TEAEs) have been reported for the 6 patients so far. 99mTc SPECT imaging for virus tracking is on-going. Baseline and on treatment tumor biopsies were submitted for spatial immune profiling. Peripheral blood at baseline, on treatment and EOT were subjected for flow cytometry analysis. Conclusion: CF33hNIS-antiPD-L1 administered by intratumoral injection in patients with mTNBC is safe and well tolerated at the dose levels tested. Clinical trial information: NCT05081492 Citation Format: Yuan Yuan, Jamie G. Rand, Jianying Zhang, Jonathan Kessler, Badri Modi, Raju Pillai, Colt A. Egelston, Shyambabu Chaurasiya, Mireya Murga, Aileen Tang, Norma Martinez, Hans Meisen, Dave Yamauchi, Susan E. Yost, Leslie Chong, Amanda Seiz, Bonnie Nixon, Nick Ede, James Waisman, Daphne Stewart, Mina S. Sedrak, Yuman Fong. Phase I study of intratumoral administration of CF33-hNIS-antiPD-L1 (CHECKvacc) in patients with metastatic triple negative 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 OT3-03-01.

Background: The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is standard-of-care for patients with hormone receptor positive (HR+) HER2- metastatic breast cancer (MBC). Immune modulatory effects of CDK4/6i are well documented preclinically but poorly understood in the clinical setting. Our previous study combining letrozole, palbociclib and pembrolizumab in patients with HR+ MBC (NCT02778685) showed a promising complete response rate of 31%. Dynamic changes in peripheral blood mononuclear cell (PBMC) subpopulations indicated that palbociclib may increase CD8+ TEMRA (terminally differentiated effector memory cells) and CD4+ TEM (effector memory cells) and enable immune activation. The current cohort 3 was designed to study the immune modulatory effect of palbociclib as an immune-priming agent with a biomarker enriched design. Methods: Women with ECOG 0-1, HR+ HER2- MBC, RECIST 1.1 measurable disease, no prior therapy for MBC were enrolled. Patients with endocrine therapy, including aromatase inhibitor +/- ovarian suppression or fulvestrant, were eligible. A palbociclib + ET lead-in design was used, starting on day -28 followed by combination therapy with pembrolizumab added on C1D1. Peripheral blood and tumor biopsy at baseline and on-treatment were collected to allow in-depth analysis of biomarkers predicting response to the combination. The primary endpoint was to evaluate if the palbociclib potentiated immune responses as a “priming” agent through PBMC analysis and on treatment tumor biopsy. Secondary endpoints included other immune cell subsets and changes that follow the combination with pembrolizumab. With 25 patients, assuming a standard deviation of 0.51 in the relative change in classic monocytes in PBMCs, there is 90% power to detect a relative change of log(C1D1/baseline) of 34.5% with a type I error (two-sided) of 0.05. Results: Between August 2020 and April 2022, 16 patients were enrolled in cohort 3. Currently 11 patients have adverse event (AEs) and 16 patients have response data. Median age was 57 years (39-72). 8/11 (73%) were non-Hispanic white, 1/11 (9%) Hispanic, 1/11 (9%) Asian, and 1/11 (9%) African American. 87% patients had grade 3 AEs, and 30% had grade 4 AEs. Grade 3 AEs were 9/11 (82%) neutropenia, 5/11 (45%) leukopenia, 1/11 (9%) elevated LFTs, and 1/11 (9%) each lymphopenia, hot flashes, febrile neutropenia, and pneumonitis. Grade 4 AEs were 1/11 (9%) lymphopenia. 8/16 (50%) patients achieved a partial response (PR), 5/16 (31%) had stable disease (SD), and 1/16 (6%) had progression of disease (PD) by RECIST 1.1. Additionally, 2/16 (13%) patients were too early to determine best overall response. Response rate (CR+PR) was 50%. PBMCs and tumor microenvironment profiling are ongoing. Conclusion: The combination of palbociclib, pembrolizumab and ET is well tolerated, and a response rate of 50% was identified in HR+ MBC patients who received this combination as front-line therapy. Dynamic changes in peripheral blood mononuclear cells and tumor microenvironment profiling are ongoing. Citation Format: Yuan Yuan, Colt A. Egelston, Weihua Guo, Susan E. Yost, Paul H. Frankel, Christopher Ruel, Mireya Murga, Aileen Tang, Norma Martinez, Daniel Schmolze, Daphne Stewart, James Waisman, Kelly Yap, Joanne Mortimer, Niki Tank. Phase II trial of palbociclib plus endocrine therapy followed by combination of pembrolizumab, palbociclib and endocrine therapy in patients with hormone receptor positive 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 OT2-01-06.

Purpose We update a patient series that reported a high incidence of infection with Gram-positive cocci in women treated with the combination of pertuzumab and trastuzumab and further characterize this clinical problem. Patients Treating physicians and advanced practice partners identified women who developed infections while on treatment with pertuzumab and trastuzumab alone or in combination with chemotherapy and enrolled them onto this registry trial. Results Between March, 2014 and May, 2017, 48 patients with HER2-positive breast cancers were reported to have 59 individual infections. The median age was 48 years. Twenty-four patients received neoadjuvant therapy, 17 were treated for metastatic disease, and 7 were treated in the adjuvant setting. Pertuzumab and trastuzumab were combined with carboplatin and docetaxel in 24 (49%) patients, docetaxel in 10 (21%), nab-paclitaxel in 12 (24%), and without other agents in 2 (4%). Granulocyte growth factors were administered in 24 (49%) patients and no patients were documented to be neutropenic. Folliculitis developed in 25 (52%) patients and was counted as a single infection. Abscesses developed at a number of sites in 24 (49%) patients, including a septic knee requiring total knee replacement. Paronychia occurred in 7 (15%) patients, and 5 (10%) developed cellulitis. When cultures were obtained, Gram-positive cocci were consistently identified. Hypogammaglobulinemia was documented in 14 (36%) of the 33 patients tested. Conclusions Our data continue to support an increased risk of infections with Gram-positive cocci as a potentially serious adverse event in women treated with pertuzumab and trastuzumab.

Antibody drug conjugates (ADC) are a novel class of cancer therapeutics, delivering cytotoxic therapy directly to cancer cells, and show promise in the management of platinum-resistant ovarian cancer. Herein we summarize the ADC landscape currently in clinical study.Mirvetuximab Soravtansine, IMGN853, is an ADC targeting the folate receptor alpha (FRα) and has demonstrated promising single agent activity and a favorable toxicity profile in FRα-positive, platinum-resistant, epithelial ovarian cancer (EOC). The antitumor effect is seen primarily in less heavily pretreated EOC patients with moderate-to-high FRα tumor expression. A phase III study, randomizing patients to either IMGN853 or the physician's choice of single-agent chemotherapy has completed accrual. Additional ADC are being evaluated in ovarian cancer including agents that target NaPiB2, Trop2, mesothelin, and MUC16 are in phase 1 clinical trials.ADC bind antigens overexpressed on cancer cells and provide site-selective drug delivery, with the goal to increase therapeutic efficacy of cytotoxics while decreasing the off-target toxicity of the payloads. With appropriate antigen selection and adequate, measurable antigen threshold targets, these new agents may provide an improved strategy for overcoming resistance to standard chemotherapy in ovarian cancer.

BackgroundHyperglycemia is recognized as a common adverse event for patients receiving alpelisib but has been little studied outside of clinical trials. We report the frequency of alpelisib-associated hyperglycemia in a real-world setting and evaluate proposed risk factors.Patients and MethodsWe retrospectively identified patients with PIK3CA-mutated, hormone receptor-positive, metastatic breast cancer who initiated treatment with alpelisib plus fulvestrant between August 2019 and December 2021. Ordinal logistic regression evaluated 5 characteristics (diabetes, prediabetes, body mass index [BMI], age, and Asian ancestry) as independent risk factors for ALP-associated hyperglycemia grades 2-4. Risk of error from multiple hypothesis testing was controlled using the false discovery rate method.ResultsThe study included n = 92 subjects, all but 1 female, mean age 59.9 (+11.9) years with 50% non-Hispanic White, 15% Hispanic/Latino, 13% Asian, 9% African/Black, and 13% other/unknown. In total 34% of patients had diabetes, 10% had pre-diabetes, and 56% had normoglycemia. Thirty-six percent were obese, 32% were overweight, 25% were normal weight, and 7% were lean. Frequency of grades 1-4 hyperglycemia in current subjects (64.1%) was similar to hyperglycemia reported in the SOLAR-1 trial (63.7%). Our subjects’ risk of grades 2-4 hyperglycemia was independently increased by pre-existing diabetes (Odds ratio 3.75, 95% CI, 1.40-10.01), pre-diabetes (6.22, 1.12-34.47), Asian ancestry (7.10, 1.75-28.84), and each unit of BMI above 20 (1.17, 1.07-1.28).ConclusionWhile receiving alpelisib, patients of Asian ancestry, as well as patients with pre-existing hyperglycemia and/or BMI above 20, should be closely monitored for hyperglycemia. The mechanism underlying the current association of alpelisib-associated hyperglycemia with Asian ancestry is independent of BMI and merits further study. The high incidence of hyperglycemia resulted in a change in practice to include consultation with a diabetes nurse educator or endocrinologist at the start of alpelisib.

Background Immunogenic cell death has been suggested as a mechanism for HIPEC, through the release of heat shock proteins, and resulting in activation of tumor-specific T cells. Methods This is a subgroup analysis of a Phase I trial using HIPEC with cisplatin 75 mg/m2 at time of optimal cytoreduction. Metastatic tumors from nine ovarian cancer patients were collected intraoperatively before and after HIPEC. Differentially expressed genes and pathways of pre- and post-HIPEC tumors were analyzed via whole-transcriptome sequencing (WTS). Immunofluorescent (IF) staining and multispectral imaging were used to determine composition and PD-1 expression of CD8+ and conventional CD4+ tumor-infiltrating lymphocytes (TILs), using antibodies against CD8, FOXP3, PD-1, and pancytokeratin (to distinguish cancer islands from stroma). Kaplan-Meier and log-rank tests compared the overall and progression free survivals (PFS) of patients with low versus high%PD-1 changes, based on dichotomization with respect to the median%PD-1 change. Results Heatshock proteins comprised the top differentially upregulated genes in HIPEC-treated tumors. Gene set enrichment analysis revealed significant upregulation of immune pathways, including antigen processing/presentation. IF staining demonstrated increased%PD-1 in cancer islands of CD8+ and CD4+ TILs after HIPEC. In stroma, the largest%PD-1 change occurs in an exceptional responder (PFS, OS 5 years), while the lowest%PD-1 change occurred in a poor responder. Kaplan-Meier curves demonstrate superior PFS in patients with high stromal PD-1 changes in TILs after HIPEC. Conclusions Increased PD-1 expression after HIPEC suggests early HIPEC-induced T cell activation, and is associated with improved survival, implicating a potential future role for PD-1 inhibitors following HIPEC in ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common histology of ovarian cancer defined as epithelial cancer derived from the ovaries, fallopian tubes, or primary peritoneum. It is the fifth most common cause of cancer-related death in women in the United States. Because of a lack of effective screening and non-specific symptoms, EOC is typically diagnosed at an advanced stage (FIGO stage III or IV) and approximately one third of patients have malignant ascites at initial presentation. The treatment of ovarian cancer consists of a combination of cytoreductive surgery and systemic chemotherapy. Despite the advances with new cytotoxic and targeted therapies, the five-year survival rate for all-stage EOC in the United States is 48.6%. Delivery of up-to-date guideline care and multidisciplinary team efforts are important drivers of overall survival. In this paper, we review our frontline management of EOC that relies on a multi-disciplinary approach drawing on clinical expertise and collaboration combined with community practice and cutting edge clinical and translational research. By optimizing partnerships through team medicine and clinical research, we combine our cancer center clinical expertise, community practice partnership, and clinical and translational research to understand the biology of this deadly disease, advance therapy and connect our patients with the optimal treatment that offers the best possible outcomes.

The development of new HER2-directed therapies has resulted in a significant prolongation of survival for women with metastatic HER2-positive breast cancer. Discoveries in the laboratory inform clinical trials which are the basis for improving the standard of care and are also the backbone for quality improvement. Clinical trials can be completed more rapidly by expanding trial enrollment to community sites. In this article we review some of the challenges in treating metastatic breast cancer with HER2-directed therapies and our strategies for incorporating our community partners into the research network.

Cancer is a disease associated with aging. As the US population ages, the number of older adults with cancer is projected to dramatically increase. Despite this, older adults remain vastly underrepresented in research that sets the standards for cancer treatments and, consequently, clinicians struggle with how to interpret data from clinical trials and apply them to older adults in practice. A combination of system, clinician, and patient barriers bar opportunities for trial participation for many older patients, and strategies are needed to address these barriers at multiple fronts, five of which are offered here. This review highlights the need to (1) broaden eligibility criteria, (2) measure relevant end points, (3) expand standard trial designs, (4) increase resources (e.g., institutional support, interdisciplinary care, and telehealth), and (5) develop targeted interventions (e.g., behavioral interventions to promote patient enrollment). Implementing these solutions requires a substantial investment in engaging and collaborating with community-based practices, where the majority of older patients with cancer receive their care. Multifaceted strategies are needed to ensure that older patients with cancer, across diverse healthcare settings, receive the highest-quality, evidence-based care.

Use of 64Cu-DOTA trastuzumab Positron Emission Tomography to predict pathologic complete response in locally advanced HER2 positive breast cancer. Background: We have demonstrated that 64Cu-DOTA trastuzumab is an effective PET imaging agent of HER2 positive breast cancer and correlated with HER2 status by IHC. Methods: Patients with locally advanced breast cancer were eligible if they had biopsy confirmed HER2 + disease, locally advanced disease and were to be treated with neoadjuvant therapy. Pretreatment staging included a breast MRI, 18F-FDG-PET/CT and 64Cu-trastuzumab-PET/CT (64CuT-PET). Prior to injection of 64Cu-trastuzumab, patients received 50 mg of cold trastuzumab per our previously established protocol. 64CuT-PET was obtained at 21-25 h over fields of view chosen in reference to MRI. Uptake in lesions was measured in terms of body wt-normalized SUV. Lesions with intensity > adjacent tissue were considered positive on PET. Breast MRI was obtained after 4 cycles of chemotherapy and preoperatively. Response to therapy was determined on the surgical specimen. Results: Eighteen women have completed study and are evaluable for treatment response. The median age was 56 years (Range 32-62); 5 were ER- and PR-; 13 ER+/- PR+. One patient had increased 64CuT-PET uptake at T4 which was not appreciated on conventional imaging and subsequent biopsy confirmed metastatic disease. She is included in the analysis as she ultimately underwent surgical management of the breast. Complete pathologic response was seen in 14/18 patients - 11/14 ER+/- PR + and 3/4 ER-/PR-. Four patient had residual disease at surgery; 3 of 4 were HER2 negative and 1 was HER2+. Uptake on 64CuT-PET correlated with increased uptake on 18F-FDG-PET. The ratio of 64CuT-PET: 18F-FDG-PET ≥ 0.7 was associated with complete pathologic response at surgery.Conclusions: The ratio of 64CuT-PET to 18F-FDG-PET ≥ 0.7 was associated with complete pathologic response in women undergoing neoadjuvant chemotherapy for locally advanced HER2+ breast cancer. Residual disease after HER2-directed therapy was frequently HER2 negative. Citation Format: Joanne Mortimer, Russell Rockne, Jessica Liu, Yuan Yuan, Daphne Stewart, James Waisman, Paul Frankel, Laura Kruper, Vikram Adhikarla. Use of 64Cu-DOTA trastuzumab positron emission tomography to predict pathologic complete response in locally advanced HER2 positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-03-22.

Background: Older adults are less likely to be included in clinical trials leading to the approval of novel cancer treatments. The Institute of Medicine and ASCO have identified therapeutic phase II trials as a key research priority to increase the evidence base for older adults with cancer. While targeted therapies may represent a less toxic option for older patients, few trials have studied their tolerability and efficacy in older adults. Here, we present a phase II study (NCT01273610) of the combination of trastuzumab and lapatinib in older patients with HER2+ metastatic breast cancer (MBC), incorporating geriatric oncology principles in the study design. Methods: Patients age ≥ 60 years with MBC and any number of prior chemotherapy (CT) lines received trastuzumab (either 4mg/kg loading dose followed by 2mg/kg weekly or 8mg/kg followed by 6mg/kg q/3 weeks) plus lapatinib 1000 mg/m2 daily in 21-day cycles. Patients completed a pre-treatment geriatric assessment including measures of function, comorbidity, cognition, nutrition, and psychosocial status. A toxicity risk score developed for older adults receiving cytotoxic CT was calculated for each patient (Hurria et al. JCO 2011 & 2016). Relationships between tolerability (dose reductions and grade (G) ≥ 3 toxicity attributed to treatment) and risk score analyzed using a log2 transformation were assessed using generalized linear models, Student's t tests, and Fisher's exact test. Response rate (RR) and progression free survival (PFS) were evaluated. Results: 40 patients (mean age 72 [60-92]) were accrued from 04/11 to 05/15. 25% (n = 10) were ≥ 75 years of age. 65% of patients (n = 26) had HR+ tumors and 35% (n = 14) were receiving ≥ 3rd line treatment. Median number of cycles was 4 (0-28). RR was 23% (n = 9, 95% CI 11-38%; 1 complete, 8 partial). 23% (n = 9) achieved stable disease. PFS was 2.7 months (95% CI 2.5-12). Based on the toxicity risk score, 21% (n = 8), 54% (n = 21), and 26% (n = 10) were at low, intermediate, and high risk. 70% (n = 28) of patients had G ≥ 2 toxicities and 20% (n = 8) G ≥ 3 toxicities. G 2 and 3 diarrhea occurred in 28% (n = 11) and 5% (n = 2) respectively. 5% (n = 2) were hospitalized due to treatment-related toxicity. No G ≥ 3 cardiac toxicities were observed. 23% of patients (n = 9) had treatment delays, and 43% (n = 17) required a lapatinib dose reduction. The mean toxicity risk score was higher in patients who required dose reductions (Student's t: p = 0.02). No statistically significant relationship was found between toxicity risk scores and the presence of G ≥ 3 treatment toxicity (logistic regression: OR = 3.08, 95% CI [0.54, 21.2], p = 0.22). Conclusions: Among older patients with MBC (79% at intermediate or high risk of G ≥ 3 cytotoxic CT toxicity), trastuzumab and lapatinib were well tolerated, with only 20% experiencing G3 toxicities. The toxicity risk score was not found to be significantly related with treatment toxicity, which may be explained by the very low incidence of G3 events. Patients with a low toxicity risk score were not likely to require a lapatinib dose reduction. Citation Format: O'Connor T, Soto-Perez-de-Celis E, Blanchard S, Chapman A, Kimmick G, Muss H, Luu T, Waisman JR, Li D, Mortimer J, Yuan Y, Somlo G, Stewart D, Katheria V, Levi A, Hurria A. Tolerability of the combination of lapatinib and trastuzumab in older patients with HER2 positive metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-08.

Objectives: To demonstrate safety and tolerability of HIPEC in ovarian cancer. Methods: A phase I, single-institution feasibility study was designed to evaluate the safety and feasibility of HIPEC at time of optimal cytoreductive surgery (CRS) in ovarian cancer patients, followed by optional normothermic intraperitoneal chemotherapy, at City of Hope National Comprehensive Cancer Center (Duarte, CA). Primary endpoint of the trial was to evaluate the safety and feasibility of HIPEC at time of optimal cytoreductive surgery (CRS). Progression-free survival was a secondary end-point. Exploratory endpoints included the assessment of molecular markers in tumors. Eligibility criteria included epithelial ovarian cancer (stage III or IV), primary or recurrent, ECOG 0-1, and appropriate surgical candidates. 35 patients were enrolled between 2015 and 2019, who underwent optimal CRS (gross residual disease less than 1 cm), and received HIPEC intra-operatively with cisplatin at 75 mg/m2 for 60 minutes immediately following optimal CRS. Patients were stratified as either 1) primary disease (n=19), defined as having received HIPEC at time of interval CRS following neoadjuvant chemotherapy for primary EOC; 2) recurrent disease (n=16), defined as having received HIPEC at time of interval or upfront CRS for recurrent (salvage) EOC. Adjuvant chemotherapy was given at the discretion of the treating oncologist following HIPEC. Results: Median age was 58 years (36-74 years), with a mean BMI of 26. Primary (interval) patients represented 54% of patients, with pretreatment clinical stages III (53%) and IV (47%). Recurrent (salvage) patients represented 45.7%, with platinum-sensitive (75%) and platinum-resistant (25%). The most common histological subtype was high grade serous (69%). All primary (interval) patients underwent neoadjuvant chemotherapy (3-4 cycles) prior to interval CRS and HIPEC. The majority of recurrent patients (81%) did not receive neo-adjuvant chemotherapy. Thirty-three patients underwent closed technique and two underwent laparoscopic technique. Total mean operating time was 8.4 hours (4.6-15.3). Mean length of stay was 10 days (3-30). All patients underwent optimal cytoreduction (69% with complete R0 resection). Peritoneal carcinomatosis index (PCI) ranged 2 to 30 (median of 8). Patients with PCI /= 8 whose PFS was 12.6 (p=0.04). Time to initiation of adjuvant chemotherapy in recurrent patients was 8.4 weeks. 22.9% of the ovarian cancer cohort was gBRCA mutated. Median PFS for primary patients was 12.6 months (95% CI 6.4; NR); with median follow up of 22.1 months (95% CI 12.6, 31.5). Median PFS for recurrent patients was 18.8 months (95% CI 7.2; 28.7), with median follow up of 36.3 months (95% CI 21.1, 51.5). There were no grade 4 or 5 AEs. Anemia was the most common Grade 3 AE (43%), followed by liver dysfunction and electrolyte abnormalities. The most common AE of any grades were abdominal pain (86%), anemia (77%), and nausea (66%). Two patients (6%) had grade 3 acute kidney injury and one patient had grade 3 chronic kidney disease. Sodium thiosulfate was introduced to the trial in January 2018, following which there were no grade 1-4 acute or chronic kidney injuries. Download : Download high-res image (61KB) Download : Download full-size image Conclusions: HIPEC in ovarian cancer was well tolerated, without any grade 4 or 5 AEs. Renal toxicities were mitigated with sodium thiosulfate. Patients with a low PCI ( /= 8.

1078 Background: Ipatasertib (ipat) is an AKT inhibitor which has shown efficacy in combination with paclitaxel and atezolizumab in patients with triple negative breast cancer (TNBC). In previous trials, ipat was given 21 days on 7 days off due to gastrointestinal toxicities. The current trial was designed to test the safety and efficacy of ipat continuous dosing in combination with carboplatin (carbo) or carboplatin/paclitaxel (carbo/taxol). The trial was later amended to include an additional arm using ipat 21 days on 7 days off with capecitabine/atezolizumab (cape/atezo) to explore the safety of the combination. Methods: Patients with metastatic TNBC and up to 2 lines of prior chemotherapy were enrolled to receive the following: Arm A, ipat 400 mg daily, carbo AUC 2 and taxol 80 mg/m2 IV days 1, 8, 15, every 28 days; Arm B, ipat 400 mg daily, carbo AUC 2 IV days 1, 8, 15, every 28 days; Arm C, ipat 300 mg daily 21 days on 7 days off, cape 750 mg/m2 7 days on 7 days off, atezo 840 mg IV every 28 days. Ipat continuous dosing was used for Arms A and B. Ipat 21 days on 7 days off dosing was used for Arm C. The primary endpoint is safety and recommended phase II dose (RP2D). Secondary endpoints are response rate (RR) and overall survival (OS). Results: Twenty-three patients with median age 49 (29-75) were enrolled from 04/2019 to 12/2020, with 9 in Arm A, 10 in Arm B, and 4 in Arm C. A total of 15/23 (65%) had dose delay and 10/23 (43%) had dose modification. 3/4 (75%) of patients in Arm A had dose limiting toxicities (DLT) including diarrhea and gastric pain, which led to de-escalation to dose -1 with ipat (300 mg daily). 5 more patients were treated at dose -1 of Arm A with only 1 DLT (maculo-papular rash). No DLTs were observed in Arm B. Of the 4 patients treated in Arm C, 1 had DLT (maculo-papular rash). The RP2D for Arms A and B are: ipat 300 mg/carbo AUC2/taxol 80 mg/m2; ipat 400 mg/carbo AUC2. RP2D for Arm C has not been determined and accrual is ongoing. There were no clinically significant G4 toxicities in Arm A; G3 toxicities included 4/9 (44%) diarrhea, 1/9 (11%) hypertension, 1/9 (11%) stomach pain, and 1/9 (11%) neutropenia. For Arm B, G3 toxicities included 2/10 (20%) diarrhea, 1/10 (10%) anemia, 1/10 (10%) maculo-papular rash, and 1/10 (10%) hyperglycemia. For Arm C, there was 1/4 (25%) G3 maculo-papular rash. Best overall responses for Arm A were: 2/9 (22%) PR, 4/9 (44%) SD, and 3/9 (33%) PD. Best responses for Arm B were 2/10 (20%) PR, 6/10 (60%) SD, and 2/10 (20%) PD. For Arm C, best responses were 3/4 (75%) SD, and 1 not evaluable (repeat biopsy showed HER2+ disease). With a median follow up of 8.1 months, the median PFS was 4.0 months (95% CI [2.6, 5.3]). Conclusions: Continuous dosing of ipatasertib in combination with carbo or carbo/taxol is well-tolerated with modest efficacy. Clinical trial information: NCT03853707 .

Background: The PI3K-ATK pathway is one of the most common cancer drivers in breast cancer, and the AKT inhibitor ipatasertib (ipat) has shown great efficacy in patients (pts) with metastatic triple negative breast cancer (mTNBC). The current phase I trial is designed to test the safety and efficacy of the following ipat combinations: ipat + carboplatin (C) + paclitaxel (T); ipat + C; ipat + capecitabine (cape) + atezolizumab (atezo). Trial Design: This is a Phase I open-label study for pts with mTNBC. Eligible pts receive one of three regimens: A) weekly carbo AUC 2 plus taxol 80 mg/m2 days 1, 8, 15 and daily ipat 300 mg every 28 days; B) weekly carbo AUC 2, days 1, 8, 15 and daily ipat 400 mg every 28 days; C) cape 750 mg bid 1 week on 1 week off, ipat 300 mg daily and atezo 840 mg iv days 1, 15 every 28 days. Eligibility Criteria: Eligible patients must have histologically confirmed mTNBC (ER/PR ≤ 10%, HER2- per ASCO/CAP); RECIST 1.1 measurable disease; 0-2 lines of chemotherapy prior for mTNBC; AEs recovered to ≤ Gr 2 per CTCAE 5.0; adequate bone marrow, hepatic and renal function. Prior exposure to AKT targeted therapy is excluded.Specific Aims: Primary objectives are to evaluate the safety and tolerability of the combinations and determine the recommended Phase II dose (RP2D) of the combinations. Secondary objectives are to evaluate response rate, clinical benefit rate, progression free survival, and overall survival. Statistical Design: For the safety-lead in, a “3 at risk design” will be utilized to assess toxicity for the combination therapy. The DLT period is 1-cycle (28 days). Each participant will remain on the dosing level according to the escalation dose level they were enrolled in, and intra-dose level escalations will not be allowed, even if the MTD is defined at a higher dose level. Rules for escalation are as follows: if escalating from Level 1, two dose levels will be open, Level 2A, and Level 2B. Only if both 2A and 2B result in a decision to escalate will dose level 3 for this triplet be open. When both 2A and 2B are both open, slots will be given to the arm with the most open slots (starting with 2A if there are ties). When a maximum tolerable dose level has been defined by the dose escalation portion of the study, and the recommended phase 2 dose (RP2D not to exceed the MTD) has been selected, additional patients will be accrued to confirm the tolerability of the regimen. For Arm C, at least 12 patients will be treated at the RP2D to confirm tolerability. Additional patients can be accrued if the total number of patients accrued does not exceed 21 patients (e.g. if the RP2D is dose level 1, with 2A and 2B not well-tolerated based on 3 patients on each 2A and 2B, the total at RP2D could be 15). If one agent is discontinued due to toxicity, then the participant may continue to receive the remaining single agent or doublet agent therapy on protocol. With 12 patients, any specific severe toxicity with 20% incidence will be observed with 93% probability. Citation Format: Yuan Yuan, Susan E. Yost, Jin Sun Lee, Paul H. Frankel, Christopher Ruel, Mireya Murga, Aileen Tang, Norma Martinez, James Waisman, Niki Patel, Mina Sedrak, Daphne Stewart, Sayeh Lavasani, Joanne Mortimer. Phase I study combining ipatasertib with chemotherapy and atezolizumab in patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-24-01.

Objectives: We have maintained a local registry of skin and nail infections in patients receiving pertuzumab and trastuzumab as treatment for HER2 positive breast cancer. Over the past 16 months, we have continued to observe an increase in infectious complications in patients receiving the combination of pertuzumab and trastuzumab with or without chemotherapy. We expand a series of prospectively identified patients who developed infections while on these regimens. Methods: We became concerned about an increased incidence of infections shortly after the FDA approval of pertuzumab, and created an IRB approved registry of these patients. Results: Twenty-eight women were identified to have 32 separate infections (often at more than one site); 9 after cycle 1; 6 after cycle 2, 9 after cycle 3 and 8 after 4 or more cycles. The median age was 51 (Range 25-67); 14 received pertuzumab, trastuzumab, carboplatin, and docetaxel (PTCH), 5 pertuzumab, trastuzumab, and docetaxel, 7 pertuzumab, trastuzumab, and nab-paclitaxel, and 2 pertuzumab and trastuzumab. Folliculitis of the scalp, abdomen, and/or buttocks was observed in 19 patients, abscesses in 8 patients (4 of whom required incision and drainage) and cellulitis in 2. Severe paronychial infections involving one to 16 digits were observed in 4; 2 pt required surgical removal of 2 nails. Quantitative immunoglobulins were found to be low in 8 of 17 women tested; 2 patient had low total protein but did not have an assessment of quantitative immunoglobulins. All patients were initially treated with oral antibiotics, and 6 required hospitalization. Cultures were obtained in 10 patients; Staphylococcus aureus was identified in 4, methicillin resistant Staphylococcus aureus (MRSA) in 5, Enterococcus faecalis in 1. A 57 year old pt receiving neoadjuvant PTCH died on cycle 2 day 7. Autopsy was consistent with sepsis and gram positive cocci were identified. A 62 year old became septic and developed renal failure. Skin biopsies were performed in 3 patients and are consistent with changes associated with EGFR inhibition. Conclusions: We believe these infections are a result of combining pertuzumab with trastuzumab as 2 pts received no concurrent chemotherapy. An awareness of this complication is critical as some infections may be life-threatening. We have initiated patient education to ensure awareness of this potential complication. Citation Format: Mortimer JE, Yuan Y, Jung J, Kruper L, Stewart D, Chung S, Yu WK, Mendelsohn M, D'Apuzzo M, Tegtmeier B, Dadwal S. Skin, and nail, infections associated with the addition of pertuzumab to trastuzumab-based chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-10-19.

6060 Background: Advanced stage ovarian cancer patients benefit from hyperthermic intraperitoneal chemotherapy (HIPEC), prolonging overall survival by nearly 12 months. However, molecular changes triggered by HIPEC are not well characterized, and no molecular signatures of response are known. We analyzed early gene expression changes after HIPEC treatment in ovarian tumors. Methods: This is an interval subgroup analysis of a single institution Phase I trial using HIPEC with cisplatin 75 mg/m2 at time of optimal cytoreduction. Snap-frozen biopsies from tumor and normal peritoneum from 20 patients with ovarian cancer before and after HIPEC underwent whole-transcriptome sequencing using Illumina’s NovaSeq 6000 for paired 100 base-pair reads. Differential expression analysis comparing post and pre-samples was done to identify significantly changed genes, and pathway analysis was conducted using GSEA. Results: Sixty-three genes were differentially expressed (P < 0.05, fold change ≥2) between pre- and post-HIPEC tumors. Hierarchical clustering analysis of these genes confirmed that all tumors and normal tissues clustered based on pre-HIPEC versus post-HIPEC status, and not based on their patient source. Gene set enrichment analysis using a collection of 50 “hallmark” gene sets revealed that post-HIPEC tumors demonstrate significant upregulation in immune pathways (TNFA signaling via NFKB, coagulation, complement), followed by epithelial-mesenchymal transition, inflammation, apoptosis, hypoxia, angiogenesis, KRAS signaling and JAK/STAT3 signaling. In contrast, post-HIPEC normal tissues exhibited upregulation in cell cycle pathways (Myc targets V2, G2M checkpoint). As expected, both post-HIPEC tumor and normal samples shared upregulation of genes related to inflammatory response. Lastly, post-HIPEC normal samples revealed downregulation of growth and metabolism pathways; in contrast, cell cycle or DNA repair pathways were downregulated in post-HIPEC tumors. Two exceptional-responders with recurrent platinum-sensitive disease (ongoing PFS 47 and 12+ months) demonstrated the most substantial changes in gene expression. Conclusions: Exceptional ovarian cancer responders to HIPEC are characterized by extensive gene expression changes; specifically, early HIPEC-induced molecular changes are strongly associated with immune pathways changes, implicating a role for immunotherapy after HIPEC in ovarian cancer. Clinical trial information: NCT01970722.

Objectives: The addition of pertuzumab, to trastuzumab-based chemotherapy is currently considered first-line therapy for locally advanced and metastatic disease HER2 positive breast cancer and has also been suggested for use in the adjuvant setting. Over the past 12 months, we have observed an increase in the incidence of severe skin infections in patients receiving chemotherapy with pertuzumab and trastuzumab. We report the natural history of what we believe is a previously unrecognized toxicity of these regimens. Methods: Shortly after the FDA approval of pertuzumab, our clinical team appreciated an increase in invasive skin infections. We discussed this concern and identified new cases at our weekly research meeting, keeping a log of cases as they were identified. Infection control reviewed the individual patient and hospital data during this time period. Results: Eleven women were identified to have severe skin and/or nail infections; 6 after cycle 1; 2 after cycle 2, 1 after cycle 3 and 2 after cycle 6. The median age was 51 (Range 46-64); 9 received pertuzumab, trastuzumab, carboplatin, and docetaxel (PTCH) and 2 pertuzumab, trastuzumab, and docetaxel. Folliculitis of the scalp, abdomen, and/or buttocks were observed in 4 patients. Abscesses were observed in 5 patients, 4 of whom required incision and drainage. Severe paronychial infections involving one to 16 digits were observed in 3 (including one who also had folliculitis). 1 pt required surgical removal of 2 nails. Quantitative immunoglobulins were found to be low in 2 of 8 women tested; 1 patient had a total protein of 4.7 but did not have an assessment of quantitative immunoglobulins. All patients were initially treated with oral antibiotics, but 3 required hospitalization. Cultures were obtained on 6 patients, Staph aureus was identified in 2 and methicillin resistant Staph (MRSA) in 4. All patients resolved their infections and 9 of 10 were able to complete six cycles of chemotherapy. Infection control could identify no increase in Staph infections at our institution. Patients were treated at different locations and received different lot numbers of drug. Conclusions: We believe this is the first report of a substantial incidence of invasive skin and nail infections with the addition of pertuzumab to trastuzumab-based regimens not reported in the product label. Low levels of quantitative immunoglobulin in select patients suggest a possible mechanism. Citation Format: Joanne E Mortimer, Yuan Yuan, Daphne Stewart, Samuel Chung, Laura Kruper, Louise C Wong, Mary Mendelsohn, Carolyn Behrendt, Sanjeet Dadwal, Bernard Tegtmeier. Skin infections associated with the addition of pertuzumab to trastuzumab-containing chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-21.

We report a series of breast cancer patients with invasive skin and nail infections with Staphylococcus species that we attribute to the addition of pertuzumab to trastuzumab-based therapy. With the suspicion of an increased incidence of cutaneous infection in patients treated with pertuzumab and trastuzumab-based chemotherapy, treating medical oncologists identified patients receiving therapy who experienced infection. Between March and October 2014, 18 patients treated with pertuzumab and trastuzumab-based chemotherapy were found to have 21 separate skin/nail infections. Treatment was administered as neoadjuvant therapy in 12 (67 %) patients, adjuvant therapy in four (22 %) patients, and for metastatic disease in two (11 %) patients. Granulocyte growth factors were administered in 11 (61 %) patients and no patients were documented to be neutropenic. New skin and nail lesions developed as early as cycle 1 and as late as 8 months from initial therapy. The 21 separate infections documented were folliculitis and “bite-like” lesions (10), abscess (6), paronychia (3), and cellulitis (2). The appearance of these lesions was distinct from typical EGFR-associated skin changes. When cultures were obtained, Staphylococcus species were isolated. Quantitative immunoglobulins were assessed in 14 (78 %) patients and were abnormally low in six (43 %) of these patients. The skin infections resulted in treatment delay in two (11 %) patients and premature discontinuation of therapy in one patient. We believe that the skin/nail infections reported here in patients treated with the combination of pertuzumab and trastuzumab represent a previously unrecognized toxicity of adding pertuzumab to trastuzumab-based therapies.

3009 Background: IMGN853 is an antibody-drug conjugate targeting the folate receptor alpha (FOLR1), linked to maytansinoid, DM4. IMGN853 has promising single agent activity in FOLR1+ EOC. The recommended phase 2 dose (RP2D) is 6 mg/kg, based on adjusted ideal body weight (AIBW) IV every 3 wks. This study evaluates IMGN853 and G. Methods: Patients (pts) with FOLR1+ tumors including: platinum resistant EOC with ≤4 prior chemotherapy (CT) regimens, EC with ≤2 CT and TNBC ≤4 CT are eligible. FOLR1 + is defined as ≥25% of tumor staining (all tumors) ≥2+ intensity (EOC, EC) and ≥1+ (TNBC). A standard 3+3 design combines IMGN853 and G. EOC pts undergo one research biopsy to assess intratumoral vs. circulating DM4 level and biopsy vs. archival tissue FOLR1 expression. Dose-limiting toxicity (DLT) is assessed during cycle 1. Responses as per RECIST 1.1 are assessed at 12 wks. and adverse events (AEs) are evaluated by CTCAE v4.0. Results: From 10/2017 to 1/2019 a total of 15 pts. were treated (3 additional pts have consented on dose level [DL] 4):10 EOC, 3 EC and 2 TNBC. One pt. on DL1 had grade (G) 4 thrombocytopenia (PLT) DLT. Three pts were inevaluable for DLT and were replaced: 1 pt. at DL1 with G4 neutropenia without fever of unknown duration due to delayed follow up blood work, 1 pt. at DL2 and 1 pt. at DL3 due to incomplete cycle 1. No DLTs were observed on DL2-3. Day 8 cycle 1 dose modifications were required in 3 of 4 patients on DL3 (for mucositis [1 pt.], and PLT [2 pts]). We are now enrolling at the RP2D for both agents, and MTD will be determined prior to May 2019. Conclusions: IMGN853 in combination with G is achievable at clinically relevant doses and the recommended Phase 2 dose and MTD will be reported. Support: NCCN grant; with support from ImmunoGen Corp and Cancer Center Support Grant P30CA033572. Clinical trial information: NCT02996825. [Table: see text]

A characteristic feature of leukemia cells is a blockade of differentiation at a distinct stage in cellular maturation. In the 1970s and 1980s, studies demonstrating the capabilities of certain chemicals to induce differentiation of hematopoietic cell lines fostered the concept of treating leukemia by forcing malignant cells to undergo terminal differentiation instead of killing them through cytotoxicity. The first promising reports on this notion prompted a review article on this subject by us 25 years ago. In this review, we revisit this interesting field of study and report the progress achieved in the course of nearly 3 decades. The best proof of principle for differentiation therapy has been the treatment of acute promyelocytic leukemia with all-trans retinoic acid. Attempts to emulate this success with other nuclear hormone ligands such as vitamin D compounds and PPARγ agonists or different classes of substances such as hematopoietic cytokines or compounds affecting the epigenetic landscape have not been successful on a broad scale. However, a multitude of studies demonstrating partial progress and improvements and, finally, the new powerful possibilities of forward and reverse engineering of differentiation pathways by manipulation of transcription factors support the continued enthusiasm for differentiation therapy of leukemia in the future.

e12524 Background: We observed an increased incidence of severe skin and nail infections in HER2 positive patients whose treatment regimens included pertuzumab to trastuzumab-based chemotherapy; a previously unrecognized toxicity. Methods: We developed a registry for patients (pts.) who were identified by their treating physician to have severe skin/nail infections attributed to this dual HER2-directed treatment. A single infectious disease specialist (SD) oversaw the attribution of infection to drug or non-drug. Physicians were encouraged to assess quantitative immunoglobulins. Results: To date, 44 women were identified to have 62 individual episodes of infection, often at more than one site. Treatment was given as neoadjuvant therapy -24, as adjuvant therapy - 6, and for metastatic disease -14. The median age was 57 (Range 25-73); 24 received pertuzumab, trastuzumab, carboplatin, and docetaxel (PTCH); 22 pertuzumab, trastuzumab, and a taxane; 2 were on pertuzumab and trastuzumab alone. Folliculitis of the scalp, abdomen, chest wall and/or buttocks was observed in 31 patients; abscesses developed in 15;6 required surgical interventions. Paronychial infections involving one to 16 digits were observed in 6, 3 of whom required surgical intervention. With the exception of 2 pts, all had adequate WBCs. Cultures were obtained in 20 pts., and identified MSSA in 8, MRSA in 5, and Streptococcal species in 3. Two pts. with cellulitis had negative cultures; 1 pt. was cultured after initiation of antibiotics, and 1 pt. died of gram positive cocci sepsis on cycle 2 day 7 of PTCH. Her WBC on day 1 was 13.0, and she received pegfilgrastim on day 2. Immunoglobulins were found to be low in 16 of 29 (41%) women tested. In the absence of culture data, infections were managed with cephalexin, doxycycline, or topical antibiotics. The majority of patients continued on chemotherapy, but pertuzumab was deleted from the regimen in 2 pts. Conclusions: We continue to see a significant increase in gram positive invasive skin and nail infections in women with HER2 positive breast cancer treated with pertuzumab and trastuzumab-based regimens. Low levels of quantitative immunoglobulin in select patients suggest a possible mechanism.

Background and Purpose— An animal model of selective middle cerebral artery (MCA) occlusion is needed for evaluation of intra-arterial (IA) delivery of thrombolytic agents. We describe a technique for MCA thrombo-occlusion in the rabbit with real-time angiographic documentation of occlusion and thrombolytic recanalization. Methods— After femoral artery cutdown, a microcatheter was advanced from the internal carotid artery to the MCA. MCA occlusion was achieved by IA thrombin and reperfusion by IA plasmin. Results— The terminal internal carotid artery was successfully catheterized in 12 of 13 animals. Stable (2-hour) MCA occlusion was induced and verified angiographically in all 12 animals; 2 animals also had distal internal carotid artery thrombus. Recanalization was achieved rapidly after IA plasmin in 3 of 3 animals. Conclusions— We describe a new animal model of selective MCA occlusion documented by real-time angiography and used to demonstrate recanalization with IA plasmin.

e11610 Background: The addition of pertuzumab, to trastuzumab-based chemotherapy is currently considered first-line therapy for locally advanced and metastatic disease HER2 positive breast cancer a...

Summary. Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable benefit to risk profile in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg−1) designed to induce significant decreases in antiplasmin, fibrinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not significantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a direct-acting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages.

Plasminogen activators (PA) are unique agents that are currently applied as thrombolytic therapy to achieve rapid vascular reperfusion. Regimens of PA plus anticoagulants and antiplatelet drugs have attained a high degree of sophistication and predictable rates of positive clinical outcomes for acute myocardial infarction (MI), ischemic stroke, pulmonary embolism (PE), deep vein thrombosis (DVT), and thrombosed catheters. Included in the repertoire are newly approved mutants of tissue plasminogen activator (TPA), which have biochemical advantages that allow for bolus administration. Yet, despite tremendous effort devoted to enormous trials to establish the clinical efficacy of these agents in acute MI, mortality results are not superior to those with native TPA or streptokinase (SK). Furthermore, all PAs have the potential for hemorrhagic complication, most critically intracranial hemorrhage (ICH), occurring in 0.9% of patients treated with native or mutant TPA. It is possible that a limit of clinical effectiveness has been reached, beyond which more potent PAs do not achieve greater benefit without a serious increase in risk of bleeding. A breakthrough is possible, however, if the risk of ICH could be avoided. One solution is the application of the direct-acting thrombolytic enzyme, plasmin. While intravenous plasmin is not effective when administered systemically, regional infusion to a thrombus induces local thrombolysis. Unlike the PAs, plasmin treatment should not cause hemorrhage from vascular trauma sites, as it is neutralized by antiplasmin in the blood. Animal studies are fully consistent with this approach, which offers potential for achieving a truly regional thrombolytic treatment.

Plasmin is a direct-acting thrombolytic agent with an impressive safety margin in experimental models, as it is tolerated without bleeding at a 6-fold greater dose than needed for thrombolysis. At higher plasmin dosages, above a reproducible dose threshold, alpha-2-antiplasmin (AP) is consumed, accompanied by complete depletion of fibrinogen and factor VIII. As a result of clotting factor exhaustion, the primary bleeding time is prolonged. We assessed whether supplemental AP could prevent bleeding induced by a purposefully toxic dosage of plasmin, and whether AP could arrest such bleeding once it occurs. Using the rabbit ear puncture model, plasmin (8 mg/kg) alone was compared with plasmin plus supplemental AP (5 mg/kg), injected either prior to or after plasmin infusion. We dosed plasmin 8-fold higher than that required for thrombolytic efficacy, based upon our previous observations of its capacity to prolong primary bleeding in greater than 80% of treated animals. Parameters for comparison between treatment groups were: primary bleeding time at timepoints prior to, during, and after experimental infusion, incidence of rebleeding at previously stable hemostatic sites, and quantification of AP, fibrinogen, and factor VIII. Pre-treatment AP doubled the plasma concentration, preserved circulating fibrinogen and factor VIII, and prevented bleeding. Post-plasmin AP corrected the plasmin-associated depletion of AP, but fibrinogen and factor VIII concentrations remained very low, and prolonged bleeding was uncorrected. To test whether AP could be supplemented with coagulation factors in order to arrest bleeding following high dose plasmin, human cryoprecipitate was co-administered. While the fibrinogen and factor VIII levels increased to 200% and 50% of baseline values, respectively, prolonged bleeding persisted. Unexpectedly, administration of human cryoprecipitate led to excess bleeding in association with acute thrombocytopenia and gross hemolysis, presumably mediated by xenographic antibody present in the product. Thus, we find that supplemental AP administered prior to a toxic dose of plasmin prevents coagulation factor consumption and preserves normal hemostasis. Once coagulation factor depletion occurs, AP alone is insufficient to correct the hemorrhagic state that ensues. Clinically, plasmin may be useful as a regional thombolytic agent, and AP supplementation may permit higher dosing as well as protect against accidental overdose. Further studies are needed to determine whether combined AP and coagulation factor repletion may correct the prolonged bleeding induced by massive plasmin infusion.