Search

Background Breast-conserving surgery is a critical step in treatment with the goal of removing all cancer cells while minimizing the removal of healthy tissue. 15% to 25% of lumpectomy patients have positive margins and require a second surgery to achieve negative margins and reduce the risk of local recurrences. These positive margins are poorly predictive (35% PPV) of cancer left in the cavity, so most second surgeries find no residual cancer. Better detection tools are needed to guide in real-time the removal of cancer missed during the initial lumpectomy to reduce the number of second operations. The pegulicianine imaging agent is injected intravenously before surgery and its fluorescence signal is activated by proteases in tumor cells and cells at the tumor margin. The LUM Imaging System visualizes activated pegulicianine in the lumpectomy cavity via a hand-held detector and proprietary tumor detection software. This system was previously tested in multiple single-site studies and a prospective multi-site study that enrolled 234 patients and showed good ability to detect residual cancer in the lumpectomy cavity. Trial Design and Specific Aims: The current prospective, multi-center, randomized, blinded study was designed to show the clinical efficacy, system accuracy, and safety of the LUM Imaging System. It aims to demonstrate guided removal of residual cancer in the lumpectomy missed during the initial procedure and potentially reduce the rates of positive margins. This study is powered by an event-driven design that requires 70 truth-standard positive events. It is expected that approximately 390 women at fourteen medical centers across the US will be enrolled to achieve the number of events. Pegulicianine is injected 2-6 hours prior to the lumpectomy procedure. Surgeons perform standard of care (SOC) lumpectomy followed by blinded intraoperative imaging of the lumpectomy cavity with the LUM Imaging System in regions where SOC shaves will be taken. The patient is then randomized. If the random assignment is to the device arm, the surgeon is directed to excise margins that have positive LUM signal. Pathologists are blinded to the source of tissue removed (SOC vs. LUM) when conducting the pathology assessment. The amount of additional tissue volume resected is also evaluated. Patient reported outcome data is collected as a quality-of-life survey before and after the subject’s lumpectomy.Eligibility Criteria: This study seeks to enroll women with primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. Patients must not have a history of allergic reaction to polyethylene glycol, contrast agents, or have received neoadjuvant therapy to treat their current breast cancer. Use of blue dyes before imaging with the LUM System are not allowed. Additional detailed eligibility criteria are listed in the protocol. Accrual and Study Progress To date, 350 subjects have participated in this trial. This study is funded in part by the National Cancer Institute (5R44CA211013). This trial is registered as NCT03686215. Citation Format: Jorge Ferrer, David Carr, Peter Blumencranz, Irene Wapnir, Donna Dyess, Shelly Hwang, Nayana Dekhne, Daleela Dodge, Beth-Ann Lesnikoski, Kelly Hunt, Patricia Clark, Stephanie Valente, Marie Catherine Lee, Lynne Clark, Brian Schlossberg, Sean Madden, Alejandra Rodriguez, Kate Smith, Manna Chang, Barbara Smith. Pivotal study of the Lum imaging system for assisting intraoperative detection of residual cancer in the tumor bed of female patients with breast cancer: The INCITE trial [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 OT2-12-03.

BACKGROUND The Current National Comprehensive Cancer Network guidelines recommend modified radical mastectomy (MRM) as the surgical treatment of choice for nonmetastatic inflammatory breast cancer (IBC). Limited studies have looked into the outcomes of breast conserving surgery (BCS) vs. MRM for IBC. METHODS National Cancer Database (NCDB) data from 2004 to 2014 were retrospectively analyzed. Patients' demographics, tumor characteristics, and overall survival (OS) trends were compared for BCS and MRM cases of nonmetastatic IBC. Univariate and multivariate analyses were performed. RESULTS A total of 413 (3.89%) BCS and 10,197 (96.11%) MRM cases were identified. Median follow-up was 58.45 months. Compared to MRM, BCS patients were more likely to be older, be African American, have Medicare/Medicaid or be uninsured, live in lower education ZIP codes, and live in a metropolitan area (all p

One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria, and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy, and “antifolates” targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, its relationship with the oncogenic phenotype, and its therapeutic potential for cancer.

Background: Standard surgical techniques result in positive lumpectomy margins 20-40% of the time. These positive margins require surgical re-excision which places significant burden on the healthcare system and patients. The LUM Imaging System consists of a fluorescence-based imaging agent, a hand-held wide-field detector (LUM Imaging Device) used to image the surgical cavity walls intraoperatively in real-time after the resection of the main lumpectomy specimen, and a proprietary tumor detection algorithm that highlights regions in the tumor bed suspected to contain residual cancer. This imaging system was previously tested in a single-site clinical study. The current study is evaluating the imaging system in a multi-study, large patient cohort. Trial Design / Methods This trial (NCT03321929) is a non-randomized, open-label, multi-site trial designed to further refine the tumor detection algorithm utilized by the LUM Imaging System. This is a prospective, interventional feasibility study and is a pilot arm to a pivotal study which will evaluate the safety and efficacy of the LUM Imaging System. Up to 250 adult female breast cancer patients undergoing lumpectomies are being enrolled at sixteen medical centers across the US. LUM015, a fluorescence-based imaging agent, is injected prior to the subject’s lumpectomy procedure. Surgeons perform their standard of care lumpectomy followed by intraoperative imaging of the lumpectomy cavity with the LUM Imaging System. Specific Aims The primary objective is to assess performance characteristics of the LUM Imaging System and to refine the tumor detection algorithm. A secondary objective is to develop and refine the process of implementing the LUM Imaging System into institution-specific workflows during lumpectomies. Eligibility Criteria This study seeks to enroll women, over the age of 18 and with histologically or cytologically confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. In addition to be willing to follow study procedures, participating in an informed consent discussion, signing an informed consent form, and having baseline lab and screening values within protocol limits, enrolled subjects must meet the following key exclusion criteria: have no history of allergic reaction to polyethylene glycol, no history of allergic reaction to intravenous contrast agents, have not undergone any systemic therapies to treat their cancer, and will not be administered methylene blue or other dye for sentinel lymph node detection during their lumpectomy. Additional detailed eligibility criteria are listed in the protocol. Statistical Methods For categorical variables, summary tabulations of the number and percentage of patients within each category (with a category for missing data) of the parameter will be presented. For continuous variables, the number of patients, mean, median, standard deviation, minimum, and maximum values will be presented. The secondary objective will be met by evaluating a robust training and proficiency protocol for all enrolling institutions. Accrual To date, 208 subjects have participated in this LUM Imaging System trial. Contact Information Jorge Ferrer: jmferrer@lumicell.com Kate Smith: kate@lumicell.com Citation Format: Kate Smith, Jorge Ferrer, David Carr, Peter Blumencranz, Daleela Dodge, Nayana Dekhne, Irene Wapnir, Kelly Hunt, Linsey Gold, Stephanie Valente, Peter Beitsch, Donna Dyess, Shelly Hwang, Lynne Clark, Beth-Ann Lesnikoski, Anees Chagpar, Stephen Karp, Brian Schlossberg, Livia Gjylameti, Barbara Smith. Expansion into multiple institutions for training in the use of the LUM Imaging System for intraoperative detection of residual cancer in the tumor bed of female subjects with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-06-02.

Background: Standard surgical techniques result in positive lumpectomy margins 20-40% of the time. These positive margins require surgical re-excision which places significant burden on the healthcare system and patients. The LUM Imaging System consists of a fluorescent drug, a hand-held wide-field detector (LUM Imaging Device) used to image the surgical cavity walls intraoperatively in real-time after the resection of the main lumpectomy specimen, and a proprietary tumor detection algorithm that highlights regions in the tumor bed suspected to contain residual cancer. Methods: The Intraoperative Detection of Residual Cancer in Breast Cancer trial (NCT03321929) is a non-randomized, open-label, multi-site trial. This is a prospective, interventional feasibility study and is a pilot arm to a pivotal study which will further evaluate the safety and efficacy of the LUM Imaging System. This study enrolls women, over the age of 18 and with histologically or cytologically confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. LUM015, a fluorescent drug, is injected prior to the subject’s lumpectomy procedure. Surgeons perform their standard of care lumpectomy followed by intraoperative imaging of the lumpectomy cavity with the LUM Imaging System. In real-time, the LUM Imaging System highlights areas within the tumor bed that may contain, residual abnormal tumor tissue. Surgeons remove additional tissue based on the guidance of the LUM Imaging System. A maximum of two additional tissue shaves may be obtained. All excised tissue specimens are evaluated by routine pathology and correlated to the output of the LUM Imaging System. Results and Discussion: Sixteen medical centers across the United States enrolled 234 subjects into this study. Preliminary data on 141 subjects has been evaluated. The median age of enrolled women undergoing surgery using the Lumicell system was 61 years old. The histology of tumor type in women evaluated in this analysis is representative of the general population, with 21% diagnosed with ductal carcinoma in-situ (DCIS), 11% diagnosed with invasive lobular carcinoma, and 64% diagnosed with invasive ductal carcinoma (with or without DCIS features present). Most women (71%) presented with a palpable mass on physical examination prior to their lumpectomy surgery. Radiological imaging prior to lumpectomy showed scattered areas of fibroglandular density in 49% of the enrolled subjects and heterogeneously dense breast tissue in 42% of the enrolled subjects. The use of the LUM Imaging System positively impacted enrolled subjects; approximately 10% of subjects (N=14) had residual tumor detected and removed from the tumor bed guided by the LUM Imaging System after the standard of care surgery was completed. Without the use of this guidance technology, tumor tissue would have been left behind in this cohort of subjects, potentially requiring additional surgical intervention or other therapy, or local recurrence. The mean absolute volume of tissue removed due to guidance by the Lumicell System was 15 cc corresponding to about 15% of the total tissue removed. The LUM Imaging System correctly identified all positive margins in 28% of subjects with a positive margin after standard of care, and directed excision of additional tissue to create a wider margin. 12% of the patients with positive margins were converted to a negative margin by removing additional tissue guided by this imaging system. Future studies are planned that will measure the sensitivity and specificity of the device. Citation Format: Jorge Ferrer, David Carr, Peter Blumencranz, Daleela Dodge, Nayana Dekhne, Irene Wapnir, Kelly Hunt, Linsey Gold, Stephanie Valente, Peter Beitsch, Donna Dyess, Shelly Hwang, Lynne Clark, Beth-Ann Lesnikoski, Anees Chagpar, Stephen Karp, Brian Schlossberg, Sean Madden, Manna Chang, Kate Smith, David Strasfeld, W David Lee, Barbara Smith. Results from the expansion into multiple institutions for training in the use of the LUM imaging system for intraoperative detection of residual cancer in the tumor bed of female subjects with breast cancer clinical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-20-06.

Background Accurate identification of the tumor bed after breast-conserving surgery (BCS) ensures appropriate radiation to the tumor bed while minimizing normal tissue exposure. The BioZorb® three-dimensional (3D) bioabsorbable tissue marker provides a reliable target for radiation therapy (RT) planning and follow-up evaluation while serving as a scaffold to maintain breast contour. Methods After informed consent, 818 patients (826 breasts) implanted with the BioZorb® at 14 U.S. sites were enrolled in a national registry. All the patients were prospectively followed with the BioZorb® implant after BCS. The data collected at 3, 6, 12, and 24 months included all demographics, treatment parameters, and provider/patient-assessed cosmesis. Results The median follow-up period was 18.2 months (range, 0.2–53.4 months). The 30-day breast infection rate was 0.5 % of the patients (n = 4), and re-excision was performed for 8.1 % of the patients (n = 66), whereas 2.6 % of the patients (n = 21) underwent mastectomy. Two patients (0.2 %) had local recurrence. The patient-reported cosmetic outcomes at 6, 12, and 24 months were rated as good-to-excellent by 92.4 %, 90.6 %, and 87.3 % of the patients, respectively and similarly by the surgeons. The radiation oncologists reported planning of target volume (PTV) reduction for 46.2 % of the patients receiving radiation boost, with PTV reduction most commonly estimated at 30 %. Conclusions This report describes the first large multicenter study of 818 patients implanted with the BioZorb® tissue marker during BCS. Radiation oncologists found that the device yielded reduced PTVs and that both the patients and the surgeons reported good-to-excellent long-term cosmetic outcomes, with low adverse effects. The BioZorb® 3D tissue marker is a safe adjunct to BCS and may add benefits for both surgeons and radiation oncologists.

The appropriateness of substituting sentinel lymph node dissection (SLND) and regional nodal irradiation (RNI) for axillary lymph node dissection (ALND) in patients with residual lymph node (LN) disease following neoadjuvant chemotherapy (NAC) is unknown. We used the National Cancer Database (NCDB) to compare survival following SLND and ALND in breast cancer patients with residual LN disease. We analyzed NCDB patients, treated between 2006 and 2014, with cT1–3, cN1, cM0 breast cancer and residual disease in 1–3 axillary LNs (ypN1) following NAC. Patients were grouped into those who received SLND (defined as removal of ≤ 4 LNs) and RNI, or ALND and RNI. Patients were matched for all patient, tumor, and treatment characteristics. We identified 1313 eligible patients in the ALND group and 304 patients in the SLND group. For the matched cohorts, SLND was associated with significantly lower survival in both univariate and doubly robust multivariable analyses (MVA) (HR 1.7, 95% CI 1.3–2.2, P

Purpose/Objective(s) There is a paucity of data regarding the use of whole breast irradiation (WBI) as part of breast conserving therapy (BCT) in women diagnosed with breast cancer who had undergone prior breast implant-based augmentation. In this population, we investigated various factors that impact radiation related toxicities, including the need for additional cosmetic surgery. Materials/Methods A retrospective review was performed for a single institution, with queries done on health-system medical records, radiation therapy medical records, radiology mammography studies, and internal radiation therapy and toxicity databases. We included patients with a prior history of breast implant-based augmentation who subsequently underwent BCT and WBI. Patient and treatment characteristics were collected and reported, and univariate analyses comparing factors correlating with toxicity endpoints were performed. Long-term cosmesis was assessed by the patient's radiation oncologist during routine follow-up visits via the Harvard grading criteria. Results 35 patients treated at our institution between 2006 and 2020 were included in our analyses, with a median follow up time of 2.4 years. Of these, 28 were treated with conventionally fractionated WBI (1.8-2 Gy/Fx) and 7 were treated with hypofractionated WBI (2.66 Gy/Fx). Good/excellent long-term cosmesis was observed in 94% of all patients. 44% of patients ultimately needed additional cosmetic surgery at some point after radiation. Indications for additional surgery included capsular contracture (10), wound complications (2), implant rupture (1), volume loss (1), and asymmetry (1). Fourteen patients (50%) treated with conventionally fractionated WBI and one patient (14%) patient treated with hypofractionated WBI required additional surgery, but this difference was not significant on UVA (P = 0.12). Other factors found not to be significantly associated with rates of additional surgery included BMI, stage, the volume of the breast receiving 105% or 110% of the prescription dose, and presence or absence of bolus during treatment. No patients experienced local or regional recurrence. Conclusion Patients with a prior history of breast implantation often experience good long-term cosmesis with a moderate risk of needing additional future surgery. The use of hypofractionated WBI was not associated with worse cosmetic outcome or an increase in need for additional cosmetic surgery.

Objective: Increased use of oncoplastic procedures makes it difficult to distinguish the lumpectomy margins from seroma fluid. Many side effects of radiation therapy are due to overestimated treatment volumes due to seroma based planning. A novel 3-dimensional (3D) absorbable device sutured to the lumpectomy tumor bed allows greater precision in targeting of the margins while avoiding normal tissues. A multi-center clinical registry was established to assess use of this device over time. This initial report of the first 300 patients provides insight into patient selection, tumor characteristics, method of implantation and initial outcomes. Methods: A bioabsorbable 3D implantable marker with a fixed array of 6 titanium clips was sutured to the site of the excised breast cancer during lumpectomy with or without oncoplastic closure techniques. The framework of the implant resorbs slowly over time, while the clips remain permanently. The marker was utilized for boost or partial breast irradiation (PBI) planning or treatment targeting. To date the registry has accrued 300 patients from 12 centers. Data includes patient demographics, breast size, tumor characteristics, surgical and radiotherapy techniques, cosmesis and follow-up. Results: Data on 300 patients with median follow-up 10.4 months was analyzed. Median age was 64.4 years, 83% of women were postmenopausal and 10% had comorbidities. Breast size was evenly distributed between cup size B, C and >D. Cancer histology was in-situ (20%), invasive ductal (62%), invasive lobular (9%) and others (10%) measuring T1 (80%) and T2 (20%). Laterality and tumor location within the breast were typical. Most patients had oncoplastic rearrangement (74%) at the time of lumpectomy. Re-excision (including mastectomy for extensive disease) occurred in 14% of patients. Infections occurred in 2% of patients and in one patient the device was removed during surgical debridement of the postoperative infection. No devices have been removed during follow-up due to misplacement or patient-generated concerns. No cancer recurrences have been reported. In most cases, size of the device reflected size of the tumor with the 2X2cm (47%) and 2X3cm (32%) devices used most often. The device was utilized by radiation oncologists for boost or PBI planning and treatment. Data on ease of setup and boost planning is being collected. Early reports regarding cosmetic appearance show a trend for excellent or good cosmesis as judged separately by both physicians and patients (93% and 92%). Conclusions: A novel 3-dimensional absorbable device sutured into the tumor bed provides clarity in radiation targeting of tissues at greatest risk while sparing adjacent normal tissues mobilized during surgery. This initial report of a multi-center clinical registry on over 300 patients characterizes patient selection, treatment methods and follow-up. Ongoing collection of additional data, regarding radiation regimen, cosmesis and patient satisfaction, have been accumulated and provide insight regarding clinical attributes of this implantable marker. Citation Format: Kaufman CS, Cross MJ, Goyal S, Barone J, Devisetty K, Dekhne NS, Edmonson D, Gass JS, Graham CL, Hong RL, Patton BJ, Phillips RF, Schonholz SM, Smith LA, Tafra L, Smith AB, Dilworth J. Use of an absorbable implant to mark the lumpectomy cavity: Initial report of 300 patients in a multi-center registry database [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-13-04.

Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, β-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure–activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

Background Breast-conserving surgery is a critical first step in treatment with the goal of removing all cancer cells while minimizing the removal of healthy tissue. 15% to 25% or more of lumpectomy patients have positive margins and require a second surgery to achieve negative margins and reduce the risk of local recurrences. These positive margins are poorly predictive (35% PPV) of cancer left in the cavity, so most second surgeries find no residual cancer. Better detection tools are needed to identify residual cancer in real time during the initial lumpectomy to reduce the number of second operations. LUM015 is a protease-activated fluorescent imaging agent that accumulates in tumor cells and tumor associated macrophages. The LUM Imaging System camera visualizes the intravenously injected LUM015 in the lumpectomy cavity via a hand-held wide field detector and proprietary tumor detection software. This imaging system was previously tested in multiple single-site studies and a large, prospective multi-site study that enrolled 234 patients and showed good ability to detect residual cancer in the lumpectomy cavity. Trial Design The current prospective, multi-center, randomized, blinded study was designed to demonstrate the clinical efficacy, system accuracy, and safety of the LUM Imaging System. It aims to identify residual cancer in the lumpectomy cavity to reduce the rates of positive margins. This study seeks to enroll women with primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. Approximately 310 women at 14 US medical centers will be enrolled. This study is powered by an event-driven design that requires 70 patients to be enrolled that have a ‘truth-standard positive' event, which is the identification of cancer in a protocol defined tissue type. To be eligible, patients must not have a history of allergic reaction to polyethylene glycol, intravenous contrast agents, or systemic therapies to treat their cancer. Use of blue dyes before imaging with the LUM System are not allowed. LUM015 is injected prior to the lumpectomy procedure. Surgeons perform standard of care (SOC) lumpectomy and perform blinded intraoperative imaging of the lumpectomy cavity with the LUM Imaging System in regions where SOC shaves will be taken. The patient is then randomized. If the random assignment is to the device arm, the surgeon is directed to excise margins that the LUM System indicates are positive for cancer. Pathologists are blinded to the type of tissue removed when conducting the pathology assessment. Patient reported outcome data is collected as a quality-of-life survey before and after the subject's lumpectomy. Additional detailed eligibility criteria are in the protocol. To date, 166 patients have participated in this trial. This trial is registered as NCT03686215. Citation Format: Jorge Ferrer, David Carr, Peter Blumencranz, Irene Wapnir, Donna Dyess, Shelly Hwang, Nayana Dekhne, Daleela Dodge, Beth-Ann Lesnikoski, Kelly Hunt, Patricia Clark, Stephanie Valente, M. Catherine Lee, Lynne Clark, Brian Schlossberg, Sean Madden, Alejandra Rodriguez, Kate Smith, Manna Chang, Barbara Smith. Pivotal Study of the LUM Imaging System for assisting intraoperative detection of residual cancer in the tumor bed of female patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT259.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is nearly 100%. Thus, new tumor-selective therapies for EOC are urgently needed. One such treatment option involves targeting tumors via folate receptor α (FRα), which is overexpressed in up to 90% of EOCs and shows increasing expression with higher stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359, AGF362 and AGF363) that inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibition at cytosolic enzyme targets including those in de novo purine biosynthesis. Potent inhibition was seen with several FRα-expressing EOC tumor cells. Inhibitory potencies were in order, AGF347 > AGF359 > AGF362 > AGF363. Drug effects were substantially reduced with excess folic acid (FA), confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Targeted metabolomics, using L-[2,3,3-2H]serine as a tracer in wild-type or SHMT2 knockdown SKOV3 cells, confirmed all compounds inhibited cytosolic and mitochondrial C1-metabolism (at SHMT2). Apoptosis was detected for all compounds by Annexin V/PI, with partial rescue of apoptosis observed upon addition of glutathione (GSH). Glutathione pools (GSH and total GSH+GSSG) were significantly perturbed by drug treatment with all inhibitors in SKOV3 cells, comparable to GSH levels observed in SHMT2 KD cells. In vivo efficacy studies with SKOV3 xenografts treated with either AGF347 or cisplatin in SCID mice showed cisplatin resistance, while AGF347 demonstrated efficacy and delay in disease progression with a median tumor growth delay of 10 days, with the longest delay being 15 days. Our studies describe a series of novel inhibitors targeting mitochondrial and cytosolic C1-metabolism, selectively delivered via FRα, which show direct cytotoxic effects against cisplatin resistant EOC in vitro and in vivo, and display additional mechanisms of cytotoxicity mediated through glycine depletion. Citation Format: Adrianne C. Wallace-Povirk, Carrie O'Connor, Xun Bao, Jade Katinas, Jennifer Wong-Roushar, Aamod Dekhne, Zhanjun Hou, Md. Junayed Nayeen, Khushbu Shah, Jose Cardiel Nunez, Jing Li, Seongho Kim, Lisa Polin, Charles E. Dann, Aleem Gangjee, Larry H. Matherly. Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2348.

Background: For many women with breast cancer, breast-conserving surgery is a critical first step in treatment with the goal of removing all cancer cells while minimizing the removal of healthy tissue. 15% to 25% or more of lumpectomy patients have positive margins and require a second surgery to achieve negative margins and reduce the risk of local recurrences. These positive margins are poorly predictive (35% PPV) of cancer left in the cavity, so most second surgeries find no residual cancer. Better detection tools are needed to identify residual cancer in real time during the initial lumpectomy to reduce the number of second operations. LUM015 is a protease-activated fluorescent imaging agent that accumulates in tumor cells and tumor associated macrophages. The LUM Imaging System camera visualizes the intravenously injected LUM015 in the lumpectomy cavity via a hand-held wide field detector and proprietary tumor detection software. This imaging system was previously tested in multiple single-site studies and a large, prospective multi-site study that enrolled 234 patients and showed good ability to detect residual cancer in the lumpectomy cavity. Trial Design: The current prospective multi-center, two-arm randomized, blinded study was designed to demonstrate the clinical efficacy, system accuracy, and safety of the LUM Imaging System. It aims to identify residual cancer in the lumpectomy cavity in order to reduce the rates of positive margins. This study seeks to enroll women with confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. The planned enrollment is 170 women being treated at fifteen medical centers across the US. To be eligible, patients must not have a history of allergic reaction to polyethylene glycol, intravenous contrast agents, or systemic therapies to treat their cancer. Use of blue dyes before imaging with the LUM System are not allowed. LUM015 is injected prior to the lumpectomy procedure. Surgeons perform standard of care (SOC) lumpectomy and perform blinded intraoperative imaging of the lumpectomy cavity with the LUM Imaging System in any regions where SOC shaves will be taken. The patient is then randomized either the further use of device or not. If the random assignment is to the device arm, the surgeon is directed to excise margins that the LUM System indicates are positive for cancer. Pathologists are blinded to the type of tissue removed when conducting the pathology assessment. Patient reported outcome data is collected as a quality of life survey before and after the subject's surgery. Additional detailed eligibility criteria are listed in the protocol. To date, 22 subjects have participated in this trial. This trial is registered as NCT03686215. Citation Format: Jorge Ferrer, David Carr, Irene Wapnir, Kelly Hunt, Peter Blumencranz, Nayana Dekhne, Daleela Dodge, Donna Lynn Dyess, Shelley Hwang, Stephanie Valente, Lynne Clark, Beth-Ann Lesnikoski, Anees Chagpar, Paticia Clark, Marie Catherine Lee, Brian Schlossberg, Sean Madden, Livia Gjylameti, Manna Chang, Kate Smith, Barbara Smith. Pivotal study of the LUM Imaging System for assisting intraoperative detection of residual cancer in the tumor bed of female patients with breast cancer [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 CT281.

// Bryan J. Thibodeau 1 , Vincent Lavergne 2 , Nayana Dekhne 3 , Pamela Benitez 3 , Mitual Amin 4 , Samreen Ahmed 1 , Jean L. Nakamura 2 , Philip R. Davidson 5 , Alice O. Nakamura 5 , Inga S. Grills 6 , Peter Y. Chen 6 , Jessica Wobb 6 and George D. Wilson 1, 6 1 Beaumont BioBank, Beaumont Health, Royal Oak, MI, USA 2 Department of Radiation Oncology, University of California, San Francisco, CA, USA 3 Department of Surgery, Beaumont Health, Royal Oak, MI, USA 4 Department of Pathology, Beaumont Health, Royal Oak, MI, USA 5 Department of Finance and Statistical Analysis, University of Alberta, Edmonton, Alberta, Canada 6 Department of Radiation Oncology, Beaumont Health, Royal Oak, MI, USA Correspondence to: George D. Wilson, email: george.wilson@beaumont.edu Keywords: angiosarcoma; radiation-associated; breast; mutational signature; next generation sequencing Received: September 15, 2017 Accepted: October 13, 2017 Published: January 19, 2018 ABSTRACT Purpose: Radiation-associated breast angiosarcomas are a rare complication of radiation therapy for breast carcinoma. With relatively little is known about the genetic abnormalities present in these secondary tumors, we examined genomic variation in biospecimens from radiation-associated breast angiosarcomas. Experimental Design: Patients were identified that had a previous breast cancer diagnosis, received radiation therapy, and developed angiosarcoma in the ipsilateral breast as the earlier cancer. Tumor regions were isolated from archival blocks using subsequent laser capture microdissection. Next generation sequencing was performed using a targeted panel of 160 cancer-related genes. Genomic variants were identified for mutation and trinucleotide-based mutational signature analysis. Results: 44 variants in 34 genes were found in more than two thirds of the cases; this included 12 variants identified as potentially deleterious. Of particular note, the BRCA1 DNA damage response pathway was highly enriched with genetic variation. In a comparison to local recurrences, 14 variants in 11 genes were present in both the primary and recurrent lesions including variants in genes associated with the DNA damage response machinery. Furthermore, the mutational signature analysis shows that a previously defined IR signature is present in almost all of the current samples characterized by predominantly C→T substitutions. Conclusions: While radiation-associated breast angiosarcomas are relatively uncommon, their prognosis is very poor. These data demonstrate a mutational pattern associated with genes involved in DNA repair. While important in revealing the biology behind these tumors, it may also suggest new treatment strategies that will prove successful.

Folate-dependent one-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a number of metabolites critical to tumor propagation. Folates are taken up by the plasma membrane facilitative transporters, reduced folate carrier (RFC; major tissue transporter) and proton-coupled folate transporter (PCFT; narrow physiological niche, but commonly expressed in solid tumors), and then transported into mitochondria via the mitochondrial folate transporter (MFT; SLC25A32). Although drug-targeting of cytosolic 1CM remains a clinical mainstay for a variety of cancers, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in a host of different cancers and, overall, SHMT2 is the fifth-most differentially expressed metabolic enzyme in cancer versus normal tissues. Despite its unequivocal oncogenic importance and therapeutic potential, there are no clinically relevant inhibitors of SHMT2. In this study, we characterized cellular pharmacodynamics of novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320, and AGF347) which show in vitroantitumor efficacy toward H460 lung, HCT-116 colon, and MIA PaCa-2 pancreatic cancer cells. Inhibition of mitochondrial SHMT2 and cytosolic 1CM at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) by this series was established by in vitro targeted metabolomics in H460, HCT-116, and MIA PaCa-2 cells and in vitro cell-free assays with purified enzymes. By depleting SHMT2-derived formate, these compounds potentiate their own direct inhibition of GARFTase and AICARFTase. Depletion of adenine nucleotide pools in vitro by all compounds led to inhibition of mTOR signaling to S6K1 in HCT116 cells. Subcellular fractionation of MIA PaCa-2 and MFT-null and human MFT-transfected glyB Chinese hamster ovary cells confirmed synthesis of polyglutamyl forms of AGF347 in both cytosol and mitochondria with mitochondrial uptake of AGF347 in part mediated by MFT. Treatment by all compounds decreased the cellular GSH/GSSG ratio, indicating depleted ability to combat oxidative stress. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice (n=5) with median tumor growth delay (T-C) in 4 mice >38 days and 1 of 5 tumor-free survivors (cured). In vivo metabolomics on tumor xenografts confirmed inhibition of serine catabolism and purine biosynthesis. Collectively, our studies establish the exceptional therapeutic potential of inhibitors dual-targeting mitochondrial and cytosolic 1CM. Citation Format: Aamod Dekhne, Khushbu Shah, Gregory S. Ducker, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Arpit Doshi, Xun Bao, Hasini Kalpage, Jenney Liu, Seongho Kim, Adrianne Wallace-Povirk, Changwen Ning, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Jing Li, Maik Hüttemann, Joshua D. Rabinowitz, Charles E. Dann, Aleem Gangjee, Larry Matherly. Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2992.

Background: Obtaining tumor-free margins is critical for local control in breast conserving surgery. Currently, 20-40% of lumpectomy patients have positive margins that require surgical re-excision; this is a significant burden for patients, surgeons and the healthcare system. Therefore, it is of paramount importance that all cancer be resected from the tumor bed during lumpectomy surgery. The LUM Imaging System consists of a fluorescence-based imaging agent, a hand-held wide-field detector (LUM Imaging Device) used to image the surgical cavity walls intraoperatively in real-time after the resection of the main lumpectomy specimen, and a proprietary tumor detection algorithm that highlights regions in the tumor bed suspected to contain residual cancer. Methods: This multi-site clinical study is designed to refine the tumor detection algorithm developed in previous studies by correlating images generated by the LUM Imaging System with histopathology results. This is a prospective, interventional feasibility study and is an introductory arm to a pivotal study which will evaluate the safety and efficacy of the LUM Imaging System in reducing the positive margin rate during standard of care lumpectomies. Up to 250 adult female breast cancer patients undergoing lumpectomies are being enrolled at sixteen medical centers across the US. The novel imaging agent, LUM015, is injected into all enrolled subjects prior to their lumpectomy procedure. Subjects undergo the standard of care lumpectomy followed by intraoperative use of the LUM Imaging System which helps guide surgeons in identifying potential residual tumor tissue. The primary objective is to assess performance characteristics of the LUM Imaging System and to fine-tune the tumor detection algorithm. A secondary objective is to develop and refine the process of implementing the LUM Imaging System into institution-specific workflows during lumpectomies. A comprehensive process to evaluate the LUM Imaging System has been implemented and will be reported. This study is being conducted under an Investigational Device Exemption (IDE) approved by the FDA. To date, 72 subjects have been imaged using the LUM Imaging System. Successful implementation of standardized tissue collection, naming conventions, and LUM Imaging System training is intended to improve the quality and integrity of the data collected in the upcoming pivotal clinical trial to evaluate the safety and efficacy of the LUM Imaging System. This study is partially funded by a grant from the National Cancer Institute (award 1R44CA211013) to support biomedical technology development and Lumicell Inc. and is registered on Clinical Trials.gov as NCT03321929. Citation Format: Kate Smith, Jorge M. Ferrer, Barbara L. Smith, E. Shelley Hwang, Kelly K. Hunt, Daleela G. Dodge, Stephen E. Karp, Stephanie A. Valente, Irene L. Wapnir, Lynne P. Clark, David R. Carr, Peter D. Beitsch, Donna L. Dyess, Beth-Ann Lesnikoski, Peter W. Blumencranz, Nayana S. Dekhne, Linsey P. Gold, Anees Chagpar, Katherine Kacena, Livia Gjylameti, Felix Geissler. Expansion into multiple institutions for training in the use of the LUM Imaging System for intraoperative detection of residual cancer in the tumor bed of female subjects with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT220.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Even though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is virtually 100%. Thus, there is an urgent need for new tumor-selective therapies for EOC. One such treatment option involves targeting tumors via folate receptor alpha (FRα) which is overexpressed in up to 90% of EOCs and shows increasing expression with stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347, AGF359 and AGF362) which inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibitions at cytosolic enzyme targets including those in de novo purine biosynthesis and SHMT1. Potent inhibition was seen toward isogenic Chinese hamster ovary (CHO) cell lines individually expressing FRα, the reduced folate carrier (RFC, ubiquitously expressed tissue folate transporter) and the proton-coupled folate transporter (PCFT, expressed in a limited number of normal tissues and inactive at normal pH, and several solid tumors at acidic pH including EOC), and with FRα-expressing tumor cells, including KB and IGROV1, a human EOC cell line. Inhibitory potencies were in order, AGF347 > AGF362 >> AGF291 = AGF320 = AGF359. Drug effects were substantially reduced with excess folic acid, confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Excess folic acid abrogated drug effects to varying degrees, suggesting significant uptake by the PCFT and/or the RFC, in addition to FRα. Short-term (5 minutes) cell uptake assays with the EOC cell lines and [3H]AGF347 confirmed transport by PCFT and RFC. With sustained exposures resulting in steady state AGF347 accumulations under both physiologic (pH 7.2) and acidic (pH 6.8, approximating the tumor microenvironment) conditions, FRα uptake predominated. [3H]AGF347 treatment of IGROV1 EOC resulted in substantial drug accumulation in both cytosol and mitochondria. Inhibition of cell proliferation for all analogs was reversed by addition of both glycine and adenosine, implicating C1 metabolism in mitochondria including SHMT2 and de novo purine biosynthesis in the cytosol as the targeted pathways; protection by 5-aminoimidazole-4-carboxamide (AICA) with or without glycine was incomplete, implying direct targeting of AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme in purine biosynthesis. Our studies describe first-in-class FRα-selective compounds targeting mitochondrial and cytosolic C1 metabolism, with potent activity against EOC, including cisplatin resistant EOC. Citation Format: Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Md. Junayed Nayeen, Khushbu Shah, Aleem Gangjee, Larry Matherly. Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4800.

Targeting cellular one-carbon (1C) metabolism in cancer cells with pyrrolo[3,2-d]pyrimidine analogs provides in vivo antitumor efficacy. We discovered a first-in-class series of 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347) with inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial 1C metabolism and a reported oncodriver. In addition, AGF291, AGF320 and AGF347 inhibited de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), and serine hydroxymethyltransferase 1 (SHMT1). In vivo studies demonstrated excellent antitumor efficacy for AGF347 against MIA PaCa-2 pancreatic adenocarcinoma xenografts in SCID mice with a median tumor growth delay (T-C) of >38 days for 4 mice, and 1 of 5 mice tumor-free 122 days post-treatment. AGF359, a new analog in this series, was a potent inhibitor of KB human tumor cell proliferation in vitro. AGF359 inhibition of KB human tumor cells was reversed with glycine and adenosine, establishing mitochondrial 1C metabolism and de novo purine biosynthesis as the targeted pathways; AICA plus glycine was incompletely protective, implicating AICARFTase as a direct cellular target. AGF359, like AGF291, AGF320, and AGF347, inhibited purified human SHMT2 (Ki = 0.399 ± 0.174 µM), SHMT1 (Ki = 0.70 ± 0.088 µM) and AICARFTase (Ki = 8.86 ± 2.83 µM). There was no inhibition of 5,10-methylene tetrahydrofolate dehydrogenase 2 (MTHFD2). Further structural modifications of these multi-targeted agents afforded pyrrolo[3,2-d]pyrimidine analogs AGF307 and AGF312. Cell-based glycine/nucleotide rescue experiments in KB tumor cells established that, as with AGF359, AGF307 and AGF312 were dual inhibitors of the mitochondrial 1C metabolism (AGF307) and de novo purine biosynthesis (AGF307, AGF312). These compounds are currently in further preclinical evaluation as a prelude to possible clinical development as antitumor agents. Citation Format: Md Junayed Nayeen, Khushbu Shah, Aamod Dekhne, Changwon Ning, Carrie O'Connor, Jade M. Katinas, Jennifer Wong, Zhanjun Hou, Charles E. Dann III, Larry H. Matherly, Aleem Gangjee. Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic de novo purine synthesis enzymes as anti-tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 811.

Pancreatic cancer (PaC) represents the 4th leading cause of cancer-related deaths in the US with a mortality rate of 99%. The 5-year overall survival rate for PaC is currently 8%. One-carbon (C1) metabolism is frequently altered in cancer. For PaC, TCGA data sets show that elevated expression of key enzymes involved in cytosolic [5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and serine hydroxymethyltransferase (SHMT)1] and mitochondrial [SHMT2 and methylene tetrahydrofolate dehydrogenase 2 (MTHFD2)] C1 metabolism is associated with poor survival. Antifolate therapeutics disrupt cytosolic C1 pathways required for syntheses of thymidylate, purines, and certain amino acids, and are a mainstay for therapy of several cancers. Antifolate uptake into tumors and tissues involves the reduced folate carrier, the major tissue folate transporter, and the proton-coupled folate transporter (PCFT), which shows a more limited tissue distribution but is widely expressed in human solid tumors and is active only at acidic pHs characterizing the tumor microenvironment. We discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359) with PCFT transport that potently inhibited proliferation of PaC cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIA PaCa-2 and PANC-1), of which HPAC (KRAS mutant) and BxPC-3 (KRAS wild-type) cells were most sensitive. The PaC cell lines all expressed PCFT transcripts and proteins that were active for PCFT transport with 3H-AGF347 at acid pH. When HPAC cells were incubated with 3H-AGF347 over 48 h, drug accumulated in both cytosol and mitochondria. 3H-AGF347 was extensively metabolized to polyglutamates. Treatment of PaC cells with AGF347 and AGF359 inhibited proliferation by inducing glycine and adenosine auxotrophy that was rescued by excess glycine and adenosine. This implied that both mitochondria and cytosolic C1 metabolism was inhibited. Inhibition of mitochondrial SHMT2 and cytosolic SHMT1, glycinamide ribonucleotide formyltransferase and/or AICARFTase was confirmed by in vitro targeted metabolomics and assays with purified enzymes. Tumor cell killing was confirmed (with HPAC and BxPC-3) by colony-forming assays with AGF347 and AGF359 and drug-induced apoptosis with AGF347 was demonstrated (with HPAC) by annexin V-PI staining and flow cytometry. AGF347 and AGF359 depleted purine nucleotides and inhibited mTOR signaling via S6K1 at least in part (for BxPC-3) via activation of AMPK, likely due to elevated ZMP accompanying suppression of AICARFTase. Collectively, our studies identify first-in-class inhibitors and establish the considerable therapeutic potential of dual-targeting mitochondrial and cytosolic C1 metabolism in PaC independent of KRAS mutation status and reflecting cellular uptake by PCFT. Citation Format: Changwen Ning, Aamod Dekhne, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Josephine Frühauf, Xun Bao, Carrie O’Connor, Adrianne Wallace-Povirk, Jing Li, Charles E. Dann, Aleem Gangjee, Larry H. Matherly, Zhanjun Hou. Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4794.

One-carbon (C1) metabolism supports a number of physiological and pathophysiological processes ranging from stem cell renewal to cancer progression. Clinically used antifolates are transported into both tumor and normal cells by the ubiquitously expressed reduced folate carrier (RFC). Uptake of targeted agents via tumor-specific folate receptors (FRs) over RFC would permit tumor-selectivity, while limiting dose-limiting toxicities associated with standard chemotherapy. Serine catabolism in mitochondria is the major source of glycine and C1 units for cytosolic biosynthesis, preserves redox balance and minimizes reactive oxygen species, and is an important source of ATP. Among the mitochondrial C1 enzymes, serine hydroxymethyltransferase 2 (SHMT2) and 5,10-methylene tetrahydrofolate (me-THF) dehydrogenase 2 (MTHFD2) are highly expressed in tumors versus normal tissues. SHMT2 has been suggested to be an important oncodriver. However, there are no clinically relevant inhibitors of these enzymes. To generate potential inhibitors of these enzymes, we synthesized 5-substituted pyrrolo[3,2-d]pyrimidine analogs as structural hybrids of cytotoxic 5-substituted pyrrolo[2,3-d]pyrimidines and me-THF. The 5-substituted pyrrolo[3,2-d] pyrimidine with a four carbon bridged phenyl side chain AGF300 afforded selective uptake via FRα over RFC, with inhibition of mitochondrial C1 metabolism and de novo purine biosynthesis, resulting in inhibition of KB human tumor cell proliferation. Inhibition of KB cells by AGF300 was reversed by glycine and adenosine. As previous studies of related 5-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge plays an important role in determining tumor cell potency and transport selectivity, we replaced the carbon adjacent to the phenyl ring in AGF300 with heteroatoms, including O (AGF323), S (AGF346) or NH (AGF350). These compounds were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells singly expressing human FRα (RT16) or RFC (PC43-10). Incorporation of the O, S and NH in the pyrrolo[3,2-d]pyrimidine analogs preserved excellent inhibition of FRα-containing CHO cells (IC50s of 57 nM, 77 nM and 50 nM, respectively); there was no inhibition of cells with RFC uptake up to 1000 nM. AGF323, AGF346 and AGF350 inhibited proliferation of KB cells which was reversed by excess glycine and adenosine. This establishes that for AGF323, AGF346 and AGF350, both mitochondrial and cytosolic C1 metabolism were inhibited. The development of novel compounds targeting mitochondrial and cytosolic C1 pathways with tumor-selective uptake is highly significant in that this would overcome the drawbacks of currently used cytotoxic agents for cancer. Citation Format: Nian Tong, Khushbu Shah, Aleem Gangjee, Carrie O’Connor, Adrianne W. Porvirk, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Multi-targeted novel 5-substituted pyrrolo[3,2-d]pyrimidines with tumor-selective targeting and inhibition of cytosolic de novo purine biosynthesis and mitochondrial one-carbon metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 789.

The purpose of the study was to show that delayed axillary lymph node dissection (ALND) has higher rates of lymphedema compared with immediate ALND, using data from NSABP-B32 at Beaumont Hospital.NSABP B-32 at Beaumont had 207 patients with follow-up data on 199 patients, randomizing clinically negative axilla to sentinel lymph node biopsy (SLNB)+ALND (GrA N=98), and SLNB+cytology±ALND (GrB N=101). All patients had preoperative volumetric arm measurements and only node negatives had routine postoperative measurements assessing lymphedema for 36 months. We contacted node-positive patients for postoperative measurements for this study. Twenty-four and 15 cytology-positive patients had SLNB+ALND in GrA and GrB, respectively (SubGrA1 N=24; SubGrB1 N=15). Fourteen hematoxylin and eosin-positive patients had delayed ALND (SubGrB2a N=14).Lymphedema rate for node-positive SLNB+ALND was 10.3% [SubGrA1 (3/24)+SubGrB1 (1/15)=4/39] and node-negative SLNB+ALND was 6.8% (SubGrA2=5/74). Lymphedema was 14.3% for delayed ALND in SubGrB2a (2 of 14) and 0% for 72 SLNBs in SubGrB2b. Our study comparing immediate and delayed ALND lymphedema was not statistically significant (10.3% vs. 14.3%, P=0.65). Comparing node-negative ALND (SubGrA2= 5/74=6.8%) to node-positive ALND (A1+B1+B2a=6/53=11.3%) was not statistically significant (P=0.52). Comparing lymphedema for node-negative ALND (SubGrA2) to SLNB (SubGrB2b) only approached significance (6.8% vs. 0%, P=0.058).The rate of lymphedema was higher in delayed ALND but not statistically significant. Comparison, however, is difficult, given the limited sample size. We urge the other centers of NSABP-B32 to validate this, by contacting the node-positive patients for measurements. The lymphedema rate for SLNB alone was 0% and approached statistical significance when compared with node-negative ALND.

Background This study reports the rate of breast reconstruction failure and cosmetic outcomes after postmastectomy radiation therapy (PMRT) with temporary tissue expanders (TEs) or implants in place. Patients and Methods Ninety-four patients underwent mastectomy (93 unilateral, 1 bilateral; 95 cases total) and immediate TE reconstruction followed by PMRT. Ninety TEs and 5 permanent implants were irradiated. All patients received a dose of 5400 cGy given in 180-cGy fractions to the reconstructed breast. Twenty-one patients (22%) received tangents alone and 74 patients (78%) were treated with tangents and a supraclavicular field using a monoisocentric technique. Bolus was used in 91 patients (96%). Eighty-eight patients (93%) received chemotherapy and 78 patients (82%) received endocrine therapy. Results With a median follow-up of 24.1 months, 19 patients (20%) experienced failure of reconstruction. The 1-, 2-, and 3-year actuarial rate of reconstruction failure was 9.7%, 19.3%, and 25.5%, respectively. Infection was the most common cause of failure. Of the 19 failures, 8 patients underwent salvage procedures with flap reconstruction. Univariate analysis was performed examining age, chemotherapy use, hormone therapy use, use of a supraclavicular field, smoking status, diabetes, hypertension, and menopausal status. No risk factors were found to be associated with reconstruction failure. In patients who did not experience reconstruction failure, good/excellent cosmesis was observed in 75% of patients. Conclusion In the current series of women with a high risk of locoregional recurrence, PMRT with a TE/implant in place provides good cosmesis in the majority of women, with an acceptable risk of expander or implant loss.

Limited data are available on the implementation of a high-risk assessment program in the construct of a community clinical oncology program. The development of a high-risk breast cancer screening program at our institution identified 15%-20% of patients screened as high risk, with limited increases in structural or personnel requirements. Identification of patients as high risk could potentially improve outcomes by allowing for individualized prevention strategies and more appropriate high-risk surveillance.The implementation of a program that incorporates a risk assessment questionnaire (RAQ) to identify high-risk patients in a community-based health system was evaluated.Women with no history of breast cancer or ductal carcinoma in situ who were undergoing annual screening mammography were offered an RAQ. Cases determined to be high risk (Gail lifetime risk ≥20% or as indicated through personal and/or family history) were offered referral to our High-risk Breast Cancer Program. A retrospective data review was conducted on completed questionnaires.A total of 5878 women underwent evaluation with the RAQ between September 2009 and August 2010. The mean age of the cohort was 55 years old, with 84.9% (4990) of participants being white, and 6.8% (400) African American. In the entire population, 45.7% (2446) had at least 1 first-degree relative with breast cancer (BC), and 923 (17.2%) women were found to be high risk by the Gail model. Beyond the Gail model, 53 (0.9%) women had undergone prior chest radiation, 34 (0.6%) had a male relative with BC, 200 (3.4%), had 3 or more relatives with BC on one side of their family, 308 (5.2%) had a relative with breast and ovarian cancer on one side of the family, and 105 (1.8%) noted 2 relatives with BC with onset under age 50 years on the same side of the family.Our experience indicates that the identification of women at high risk for BC can be easily incorporated into an annual screening mammography visit. Identification of these patients as high risk can allow for individualized, more-appropriate surveillance and prevention.

To compare outcomes after accelerated partial breast irradiation (APBI) between node-negative and node-positive patients.A total of 534 patients with early-stage breast cancer received APBI including 39 node-positive (N+) cases. Clinical, pathologic, and treatment-related factors were compared between node-negative (N-) and N+ cohorts. Local recurrence (LR), regional recurrence (RR), axillary failure (AF), distant metastases (DM), disease-free survival (DFS), cause-specific survival (CSS), and overall survival (OS) were analyzed.N+ patients were younger (p = 0.04), had larger tumors (p0.001), and were more likely to receive chemotherapy (p0.001). Mean follow-up was 7.8 years for N+ patients and 6.3 years for N- patients (p = 0.06). No differences were seen in 5-year actuarial rates of LR (2.2% vs. 2.6%, p = 0.86), AF (0% vs. 0%, p = 0.69), DFS (90.0% vs. 88.0%, p = 0.79), or OS (91.0 vs. 84.0%, p = 0.65) between the two groups, whereas higher rates of RR (0% vs. 6.1%, p0.001) and DM (2.2% vs. 8.9%, p = 0.005) were noted in N+ patients. A trend for improved CSS (p = 0.06), was seen in N- patients. Age, tumor size, receptor status, T-stage, chemotherapy, APBI technique, and nodal status (p = 0.86) were not associated with LR, while a trend for an association with LR was noted with close/positive margins, (p = 0.07), and failure to receive adjuvant hormonal therapy (p = 0.06).No differences were seen in the rates of LR or AF between N- and N+ patients after APBI. These results support the continued enrollment of node-positive patients in Phase III trials evaluating the efficacy of APBI including the National Surgical Adjuvant Breast and Bowel Project-B39/Radiation Therapy Oncology Group 0413.

Purpose: To examine the rate of breast reconstruction failure and cosmetic outcomes following post-mastectomy radiation therapy (PMRT) with temporary tissue expander or implant in place. Methods and Materials: Ninety-four patients with 95 primary breast malignancies underwent mastectomy and immediate tissue expander reconstruction followed by PMRT. Ninety tissue expanders and five implants were irradiated. All patients received a dose of 5,400 cGy given in 180 cGy fractions. Twenty-one patients (22%) received tangents alone and seventy-four patients (78%) were treated with tangents and a supraclavicular field via a mono-isocentric technique. Bolus was used in 91 patients (96%). Eighty-eight patients (93%) received chemotherapy and 78 patients (82%) received endocrine therapy. Results: With a median follow up of 22.4 months, nineteen patients (20%) developed a reconstruction failure. Ten patients lost their tissue expanders with a median time to reconstruction failure of 7 months (range 3–9 months) while ten patients lost their permanent implant with a median time to loss of 19 months (range 9–31 months) following PMRT. The one and two-year actuarial rate of reconstruction failure was 11.2% and 24.9%, respectively. The most common cause of failure was infection (37%), followed by skin break down (21%), extrusion of expander or implant (16%), fibrosis/eschar (11%), trauma (11%) and pain (5%). Out of the 19 failures, eight patients were salvaged with an autologous flap reconstruction. Univariate analysis was performed to examine the association between age, chemotherapy, hormonal therapy, smoking status, hypertension, diabetes, menopause status, weight and the use of a third supraclavicular RT field on reconstruction failure. However no risk factors were found to be associated with reconstruction failure. In patients who did not have reconstruction failure, good/excellent cosmesis was observed in 79% of patients and fair/poor cosmesis was observed in 21% of patients. Conclusions: In our series of women with a high risk of recurrence, PMRT with a tissue expander in place followed by a prosthetic implant provides good cosmesis in the majority of women with an acceptable risk of expander or implant loss. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-13-04.

Tumor-specific folate receptor (FR) targeting over the ubiquitous reduced folate carrier (RFC) allows for specific tumor targeting without the attending dose-limiting toxicity of all clinically used cytotoxic agents. In an attempt to provide selectivity for FRα and/or β, folic acid has been conjugated with a variety of cytotoxic payloads to alleviate dose-limiting toxicities since folic acid is not transported via RFC (the major tissue folate transporter) to any significant extent. These conjugates have been unsuccessful in clinical trials likely in part due to the instability of the linkers used for conjugation. Premature cleavage results in the cytotoxic component being released prior to tumor penetration, thus precluding any possibility of selective tumor targeting. We designed novel pyrrolo[3,2-d]pyrimidine compounds in which both the targeting component and the cytotoxic component are in the same molecule. These compounds do not require any conjugation or linkers and hence do not suffer any premature cleavage. In this report, we synthesized and evaluated a “first-in-class” series of fluorinated analogues that target FRα/β and have limited transport via RFC, thus providing selectivity for tumors expressing FRα and/or FRβ. Two lead molecules, AGF347 and AGF355, were potent inhibitors of KB human tumor cell proliferation in vitro (IC50 = 1.05 nM and 6.25 nM respectively). Based on protection experiments with glycine, thymidine, adenosine and 5-aminoimidazole-4-carboxamide (AICA), AGF347 and AGF355 were identified as dual inhibitors of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial one-carbon metabolism and reported oncodriver, and AICA ribonucleotide formyltransferase (AICARFTase), the 2nd folate-dependent step in de novo purine biosynthesis. These compounds are currently being evaluated in preclinical studies as potential candidates for clinical trials. Citation Format: Md Junayed Nayeen, Khushbu Shah, Aleem Gangjee, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. First-in-class, fluorinated folate receptor specific agents that target tumor cells via inhibition of serine hydroxymethyltransferase 2 (SHMT2) and 5-aminoimidazole-4-carboxamide formyltransferase (AICARFTase) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1656.

Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the proton-coupled folate transporter (PCFT), a folate transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced folate carrier (the major tissue folate transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors. Citation Format: Aamod S. Dekhne, Gregory S. Ducker, Josephine Frühauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Aleem Gangjee, Joshua D. Rabinowitz, Larry H. Matherly. Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3980.

Design, modeling and synthesis of pyrrolo[3,2-d]pyrimidine analogs were carried out based on the concept of dual mechanisms-of-action for achieving maximal antitumor efficacy. Tumor-targeted delivery via agents that are selectively transported by the folate receptor (FRα/β) and/or PCFT, overexpressed in tumor cells such as epithelial ovarian cancer and non-small cell lung cancer, over the ubiquitously expressed reduced folate carrier (RFC) would afford tumor selectivity. Gangjee, Matherly and coworkers previously reported 5-substituted pyrrolo[2,3-d]pyrimidines as inhibitors of de novo purine nucleotide biosynthesis glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). To synthesize potential dual inhibitors of de novo purine biosynthesis at cytosolic AICARFTase and mitochondrial one-carbon metabolism at serine hydroxymethyl transferase (SHMT) 2, we generated structural hybrids of these inhibitors and 5,10-methylenetetrahydrofolate (the SHMT2 cofactor), 5-substituted pyrrolo[3,2-d]pyrimidine analogs AGF291, AGF299, AGF300, AGF318, AGF320, and AGF331. Molecular modeling of these compounds utilizing X-ray crystal structures of the transporters (FRα/β) and enzyme targets (AICARFTase, SHMT2) predicted excellent activities. The influence of bridge lengths and the aromatic moieties of these novel compounds were evaluated by testing their anti-proliferative activities in Chinese hamster ovary cells expressing human FRα or PCFT, and in FRα- and/or PCFT expressing nasopharyngeal carcinoma (KB) and lung cancer (H460) cells. All the pyrrolo[3,2-d]pyrimidine analogs were inhibitory toward FR- and/or PCFT-expressing cells. Compared to its pyrrolo[2,3-d]pyrimidine analog, AGF300 increased inhibition of cells via FR uptake from IC50 8.6 nM to 2.1 nM (by 4-fold) and decreased uptake through RFC from 56.5 nM to 516 nM (by 10-fold), suggesting a FR-targeted delivery of the agent. Toward H460 xenografts in SCID mice, AGF291 was efficacious. Protection studies with thymidine, glycine, adenosine and AICA and radiotracer studies with [3-14C]serine identified the likely intracellular targets of the pyrrolo[3,2-d]pyrimidine analogs as SHMT2 and AICARFTase. Mitochondrial one-carbon metabolism originating with SHMT2 is a critical source of reducing equivalents and one-carbon units for cytosolic biosynthesis and SHMT2 has been implicated as an oncodriver in several tumor types. While inhibition of SHMT2 was the subject of exploratory studies, our novel pyrrolo[3,2-d]pyrimidine series are “first-in-class” in terms of their in vitro and in vivo antitumor efficacies attributable to dual targeting SHMT2 and AICARFTase. Citation Format: Khushbu Shah, Aamod Dekhne, Zhanjun Hou, Larry Matherly, Aleem Gangjee. 5N-substituted pyrrolo[3,2-d]pyrimidines: Tumor-targeted agents with first-in-class dual inhibition of serine hydroxymethyl transferase 2 and 5-amino-4-imidazolecarboxamide ribonucleotide formyl transferase enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1658.

Background: Standard surgical techniques result in positive lumpectomy margins 20-40% of the time. These positive margins require surgical re-excision which places significant burden on the healthcare system and patients. The LUM Imaging System consists of a fluorescence-based imaging agent, a hand-held wide-field detector (LUM Imaging Device) used to image the surgical cavity walls intraoperatively in real-time after the resection of the main lumpectomy specimen, and a proprietary tumor detection algorithm that highlights regions in the tumor bed suspected to contain residual cancer. This imaging system was previously tested in a single-site clinical study. The current study is evaluating the imaging system in a multi-study, large patient cohort. Trial Design / Methods This trial (NCT03321929) is a non-randomized, open-label, multi-site trial designed to further refine the tumor detection algorithm utilized by the LUM Imaging System. This is a prospective, interventional feasibility study and is a pilot arm to a pivotal study which will evaluate the safety and efficacy of the LUM Imaging System. Up to 250 adult female breast cancer patients undergoing lumpectomies are being enrolled at sixteen medical centers across the US. LUM015, a fluorescence-based imaging agent, is injected prior to the subject’s lumpectomy procedure. Surgeons perform their standard of care lumpectomy followed by intraoperative imaging of the lumpectomy cavity with the LUM Imaging System. Specific Aims The primary objective is to assess performance characteristics of the LUM Imaging System and to refine the tumor detection algorithm. A secondary objective is to develop and refine the process of implementing the LUM Imaging System into institution-specific workflows during lumpectomies. Eligibility Criteria This study seeks to enroll women, over the age of 18 and with histologically or cytologically confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy. In addition to be willing to follow study procedures, participating in an informed consent discussion, signing an informed consent form, and having baseline lab and screening values within protocol limits, enrolled subjects must meet the following key exclusion criteria: have no history of allergic reaction to polyethylene glycol, no history of allergic reaction to intravenous contrast agents, have not undergone any systemic therapies to treat their cancer, and will not be administered methylene blue or other dye for sentinel lymph node detection during their lumpectomy. Additional detailed eligibility criteria are listed in the protocol. Statistical Methods For categorical variables, summary tabulations of the number and percentage of patients within each category (with a category for missing data) of the parameter will be presented. For continuous variables, the number of patients, mean, median, standard deviation, minimum, and maximum values will be presented. The secondary objective will be met by evaluating a robust training and proficiency protocol for all enrolling institutions. Accrual To date, 208 subjects have participated in this LUM Imaging System trial. Contact Information Jorge Ferrer: jmferrer@lumicell.com Kate Smith: kate@lumicell.com Citation Format: Kate Smith, Jorge Ferrer, David Carr, Peter Blumencranz, Daleela Dodge, Nayana Dekhne, Irene Wapnir, Kelly Hunt, Linsey Gold, Stephanie Valente, Peter Beitsch, Donna Dyess, Shelly Hwang, Lynne Clark, Beth-Ann Lesnikoski, Anees Chagpar, Stephen Karp, Brian Schlossberg, Livia Gjylameti, Barbara Smith. Expansion into multiple institutions for training in the use of the LUM Imaging System for intraoperative detection of residual cancer in the tumor bed of female subjects with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-06-02.

BACKGROUND: Data on patients who received breast-conserving therapy (BCT) for early stage breast cancer were examined to detect differences in disease presentation, management techniques, long-term treatment outcomes, and toxicities based on race. METHODS: Six hundred ninety-nine women with breast cancer (39 African-American [AA] women and 660 Caucasian [C] women) who received BCT were analyzed on race, clinical and pathologic characteristics at presentation, management techniques, treatment-related toxicities, recurrence, and survival. The median follow-up was 12.2 years. RESULTS: At diagnosis, AA women were younger (aged

The goal of the current study is to help refine guidelines for the need for re-excision and the appropriate amount of breast tissue to re-excise in patients with early breast cancer following excisional breast biopsy when treated with breast-conserving therapy (BCT).The study population consisted of 441 patients derived from a dataset of 607 consecutive cases of stage I and II breast cancer treated with BCT, in which patients underwent primary excisional diagnostic biopsy and subsequent re-excision prior to the initiation of radiation therapy (RT). A single pathologist reviewed all specimens. Re-excision was indicated because tumor was found close to or involving the resection margin. In 333 of the 441 cases, it was possible to measure the extension of carcinoma into the re-excision specimen. Margins were classified as negative (carcinoma4.2 mm from the margin), near (4.2 mm from the margin) or positive. Any carcinoma identified near the final margin was quantified by width of invasive carcinoma and number of ductal carcinoma in situ (DCIS) ducts near the margin and subdivided into three distinct groups: least, intermediate, and greatest amount. These factors were then analyzed to determine the likelihood and extent of residual carcinoma in re-excision specimens. Statistical analysis was performed using Systat version 10 (SPSS Inc., Chicago, IL).The quantity of carcinoma near the initial biopsy margin and the invasive carcinoma-to-specimen dimension ratio demonstrated a significant association with increasing amounts of residual carcinoma at re-excision. Combination of these two variables allowed for a statistically significant (P0.001) calculation of risk index for identifying significant residual invasive carcinoma or DCIS in the adjacent breast parenchyma at re-excision, and yielded stratification into low- (6%), intermediate- (27%), and high-risk (44%) groups. In re-excision specimens, the observed distance of carcinoma extension into adjacent breast tissue was associated with a statistically significant decrease in the ratio of the initial excisional biopsy specimen dimensions and invasive carcinoma dimensions. Combining the initial margin status with the specimen-to-invasive carcinoma maximum dimension ratio yielded an accurate predictor of the maximum distance of tumor extension.Evaluation of the initial excisional biopsy margin status in correlation with the invasive carcinoma-to-specimen maximum dimension ratio may be helpful for (1) identifying patients who require re-excision prior to RT and (2) predicting the quantity of additional breast tissue to excise to ensure adequate surgical margins with BCT.

The authors reviewed changes in the initial clinical presentation, management techniques, and patterns of disease recurrence over time (1981-1996) in patients with breast carcinoma treated with breast-conserving therapy (BCT) at a single institution. The goals of the current study were to determine the frequency and use of optimal local and systemic therapy techniques and to evaluate the impact of these changes on treatment efficacy.Six hundred seven patients with American Joint Committee on Cancer Stage I or II invasive breast carcinomas treated with BCT at William Beaumont Hospital (Royal Oak, MI) constituted the study population. All patients received at least an excisional biopsy of the primary tumor, an axillary lymph node staging procedure, and postoperative radiotherapy (RT) (a median tumor bed dose of 61 Gray [Gy] was administered). All sides were reviewed by one pathologist. Numerous clinicopathologic and treatment-related factors were analyzed to monitor changes that occurred over time. Changes in patterns of disease recurrence and treatment efficacy over time also were analyzed.Over the time period analyzed, changes at initial presentation included an increase in the mean age at diagnosis (age 56.1 years vs. 61.4 years; P0.001), a decrease in the number of patients with clinically palpable tumors (78% vs. 36%; P0.001), a decrease in the mean tumor size (2.2 cm vs. 1.6 cm; P0.001), but no change in the percentage of patients with negative lymph nodes (79% vs. 78%; P = 0.83). No differences over time were observed in mean tumor grade (2.0 vs. 1.9; P = 0.2) or the presence of angiolymphatic invasion (27% vs. 26%; P = 0.25). Changes in surgical management and pathologic assessment included the more frequent use of reexcision (46% vs. 81%; P0.001), larger mean total volumes of breast tissue specimens excised (115 cm3 vs. 189 cm3; P = 0.001), a larger percentage of patients with final negative surgical margins (74% vs. 97%; P0.001), and a small increase in the mean number of lymph nodes excised (13.8 lymph nodes vs. 14.1 lymph nodes; P = 0.01). The only other significant change in the pathologic management of patients over time included a doubling in the mean number of slides examined (10.6 slides vs. 21.1 slides; P0.001). Changes in adjuvant local and systemic therapy included an increase in the percentage of patients treated with60 Gy to the tumor bed (66% vs. 95%; P0.001), a doubling in the mean number of days from the last surgery to the start of RT (24 days vs. 50 days; P0.001), and a decrease in the use of regional lymph node RT (24% vs. 8%; P0.001). The use of adjuvant tamoxifen increased from 10% to 61% (P0.001). Finally, improvements were observed in the 5-year and 12-year actuarial rates of local disease recurrence (8% vs. 1% and 21% vs. 9%, respectively; P = 0.001) and distant metastases (12% vs. 4% and 22% vs. 9%, respectively; P = 0.006). No changes in the mean number of years to ipsilateral (6.5 years vs. 6.4 years; P = 0.59) or distant disease recurrence (4.6 years vs. 3.8 years; P = 0.73) were observed.The impact of screening mammography and substantial changes in surgical, pathologic, RT, and systemic therapy recommendations were observed over time in the study population. These changes were associated with improvements in 5-year and 12-year local and distant control rates and suggested that improvements in outcome can be realized through adherence to best practice guidelines and continuous monitoring of treatment outcome data.

Three specialized systems exist for membrane transport of folates and antifolates across biological membranes. The reduced folate carrier (RFC) is ubiquitously expressed in tumors and normal tissues. Folate receptors (FR) α and β, and the proton-coupled folate receptor (PCFT) exhibit narrow patterns of tissue expression and serve specialized physiologic roles. FRs are expressed in a number of cancer cells (e.g., FRα in ovarian cancer and non-small cell lung cancer; FRβ in acute myelogenous leukemia), whereas PCFT is expressed in a large number of human solid tumors but not leukemias. FRs are either inaccessible to the circulation (FRα) or are nonfunctional (FRβ) in normal tissues. PCFT transport is limited in most normal tissues, given the requirement of acidic pH for optimal activity. These properties facilitate tumor targeting of cytotoxic compounds with specificities for PCFT or/and FR transport. Clinically used antifolates such as methotrexate and pemetrexed are substrates for RFC and their uptake by both normal tissues and tumors confers dose-limiting toxicity due to limited tumor selectivity. We previously reported a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate with a 4-carbon bridge and a phenyl glutamate side chain (AGF127). Previous studies of related 6-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge region plays an important role in determining tumor cell potency and transport selectivity. Thus, it was of interest to vary the length and insert heteroatoms into the side chain -(CH2)4- of AGF127, including -(CH2)n-S- (compound 1, n= 3 and compound 2, n = 4) and -(CH2)n-O- (compound 3, n= 3 and compound 4, n = 4), to determine the impact on drug potency and transport selectivity. The novel analogs were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells expressing human FRα (RT16), RFC (PC43-10), and PCFT (R2/PCFT4). AGF127 showed potent inhibition of CHO cells expressing FRα (IC50 = 8.6 nM), and reduced activity toward CHO cells expressing RFC (57 nM) or PCFT (840 nM). Compounds 1, 2, and 3, showed reduced inhibitions toward FRα-expressing RT16 cells (4-35-fold) compared to AGF127; however, compound 4 was equipotent to AGF127. Like AGF127, none of the series were effective PCFT substrates. Compound 3, like AGF127, inhibited RFC-expressing PC43-10 cells (IC50 = 37 nM); RFC activity was reduced 6- to >20-fold for compounds 1, 2, and 4. Toward FRα-, RFC-, and PCFT-expressing KB human tumor cells, the order of potency was 4 = AGF127 > 2 = 3 = 1. Compound 4 was also more active (15-fold) than pemetrexed with KB cells. Collectively, the potent and selective activity of compound 4 toward FRα-expressing tumor cells and limited activity toward RFC-expressing cells offers significant advantages over AGF127 and clinically used non-targeted antifolates, suggesting that further preclinical evaluation is warranted. Citation Format: Aleem Gangjee, Rishabh Mohan, Manasa Ravindra, Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2017-3239

52 Background: Our tertiary care hospital addressed recent survivorship care initiatives by implementing a Nurse Practitioner (NP)-led Breast Care Survivorship Clinic (BCSC). The monthly, NP-led multidisciplinary clinic (MDC) integrated the unique needs of Breast Cancer Survivors (BCS). After 14 months, an internal evaluation prompted three significant changes to the NP-led BCSC. First, the new BCBS would become a Nurse Navigator (NN)-led risk-based model that would allow survivorship care to be individually tailored to each BCS’s needs. Secondly, patients would not meet with rest of MDC team (i.e. social work, nutrition, physical therapy) during this visit. Patients were instead referred to the Survivorship Care Workshop Series (SCWS), a bi-monthly educational program developed to complement the NN-led BCSC and address common BCS needs; needs falling outside of the SCWS content were handled individually by MDC referrals. Lastly, the NN-led BCSC was conducted at follow-up visit with surgeon to avoid an additional appointment. The NN administers a questionnaire that addresses medical and psychosocial concerns, refers BCS to relevant SCWS lecture and/or MDC referrals based on questionnaire results, and finally, delivers the SCP. Methods: A comparison of the NP-led versus the NN-led BCSC was performed on the following factors: average total time and effort (T&E) for preparation and delivery of SCPs; number of actual SCPs delivered to BCS; and cost analysis of each model. Results: There were 58 SCPs completed, and 37/58 were successfully delivered to patients during the NP-led BCSC (9/2014-10/2015). There were 95 SCPs completed, and 91/95 were successfully delivered during the NN-led BCSC (2/2016-7/2016). The average total time spent by NP was two hours (n = 37) per patient compared to the average total time of 45 minutes spent by NN (n = 91) for preparation and delivery of SCPs. The estimated annual cost of the NP-led model includes T&E for NP and MDC team, is $273,946 compared to cost of the NN-model, which includes NN T&E as well a full-time SCWS program coordinator, is $86,651. Conclusions: The NN-led risked-based BCSC model is less resource-intensive, less expensive and allows for increased delivery of SCPs to BCS.

Objective Use of routine sentinel lymph node biopsy (SLNB) in contralateral prophylactic mastectomy (CPM) is controversial. This retrospective study was undertaken to determine the frequency of SLNB in CPM at a community hospital and its utility as a guide to patient decision making. Methods Between 2007 and 2009, 170 patients underwent CPM at a suburban, tertiary care facility. The CPM was either immediate or delayed, or was for ipsilateral recurrent breast cancer. Thirty-seven (21.8%) of 170 patients had SLNB performed with CPM. The mastectomy specimens underwent standard pathologic evaluation by using intraoperative touch preparation cytology and postoperative hematoxylin and eosin staining and immunohistochemistry. Results No patients who underwent SLNB had positive nodes on touch preparation or final hematoxylin and eosin staining (0/37 [0%]). Fourteen (8.2%) of 37 patients had additional nodes identified in the specimens. These were either axillary tail or intramammary nodes. The median number of SLNs removed was 2 (range, 1-5), none of these were positive. There were 3 incidental cancers diagnosed on final pathology. Two invasive cancers (T1a and grade I) and 1 ductal carcinoma in situ were identified. SLNB was only performed on the patient with DCIS. Neither SLNB nor subsequent axillary lymph node dissection was performed in the invasive cancers. Conclusions SLNB was performed in 37 (21.8%) of patients who underwent CPM in a community hospital. Only 3 (1.76%) of 170 patients who underwent CPM had findings on final pathology that would have justified axillary staging. This correlates with other published data regarding SLNB in CPM. Because SLNB is associated with significant morbidity, guidelines for SLNB in prophylactic mastectomy need to be established so to avoid overtreatment.

Purpose To determine the rates of breast cancer–related lymphedema (BCRL) in patients undergoing whole-breast irradiation as part of breast-conserving therapy (BCT) and to identify clinical, pathologic, and treatment factors associated with its development. Methods and Materials A total of 1,861 patients with breast cancer were treated at William Beaumont Hospital with whole-breast irradiation as part of their BCT from January 1980 to February 2006, with 1,497 patients available for analysis. Determination of BCRL was based on clinical assessment. Differences in clinical, pathologic, and treatment characteristics between patients with BCRL and those without BCRL were evaluated, and the actuarial rates of BCRL by regional irradiation technique were determined. Results The actuarial rate of any BCRL was 7.4% for the entire cohort and 9.9%, 14.7%, and 8.3% for patients receiving a supraclavicular field, posterior axillary boost, and internal mammary irradiation, respectively. BCRL was more likely to develop in patients with advanced nodal status (11.4% vs. 6.3%, p = 0.001), those who had a greater number of lymph nodes removed (14 nodes) (9.5% vs. 6.0%, p = 0.01), those who had extracapsular extension (13.4% vs. 6.9%, p = 0.009), those with Grade II/III disease (10.8% vs. 2.9%, p p = 0.02). Regional irradiation showed small increases in the rates of BCRL ( p = not significant). Conclusions These results suggest that clinically detectable BCRL will develop after traditional BCT in up to 10% of patients. High-risk subgroups include patients with advanced nodal status, those with more nodes removed, and those who receive chemotherapy, with patients receiving regional irradiation showing a trend toward increased rates.

Purpose To examine 10-year results of a single institution's experience with radiotherapy limited to the region of the tumor bed (i.e., accelerated partial breast irradiation, [APBI]) in selected patients treated with breast-conserving therapy (BCT) and compare them with results of matched BCT patients who underwent whole-breast irradiation (WBI). Patients and Methods A total of 199 patients with early-stage breast cancer were treated prospectively with BCT and APBI using interstitial brachytherapy. To compare potential differences in local recurrence rates on the basis of the volume of breast tissue irradiated, patients in the APBI group were matched with 199 patients treated with WBI. Match criteria included tumor size, nodal status, age at diagnosis, margins of excision, estrogen receptor status, and use of adjuvant tamoxifen therapy. Local–regional control, disease-free survival, and overall survival were analyzed between treatment groups. Results Median follow-up for surviving patients was 9.6 years (range, 0.3–13.6 years). Eight ipsilateral breast tumor recurrences (IBTRs) were observed in patients treated with APBI. The cumulative incidence of IBTR at 10 years was 5%. On matched-pair analysis, the rate of IBTR was not statistically significantly different between the patient groups (4%, 95% confidence interval [CI] 1.3–6.7% for WBI therapy patients vs. 5%, 95% CI 1.5–8.5% for APBI patients; p = 0.48). Conclusions Radiation therapy limited to the region of the tumor bed (APBI) produced 10-year local control rates comparable to those from WBI in selected low-risk patients.

BACKGROUND. The molecular classification system categorizes invasive breast carcinomas according to their key driving biomarkers. In the current study, the authors evaluated whether response to neoadjuvant chemotherapy was correlated with the molecular classification groups. METHODS. Using immunohistochemistry, the molecular classification group (luminal-A, luminal-B, HER-2–variant, HER-2–classic, and basal phenotype) was retrospectively determined in 68 breast cancer patients who received neoadjuvant treatment. RESULTS. A total of 28 carcinoma patients (41.2%) achieved a compete pathologic response (CPR), including 2 of 15 patients classified as having luminal-A (13.3%), 4 of 16 patients classified as having luminal-B (25.0%), 10 of 12 patients classified as having HER-2–classic (83.3%), none of the 4 patients classified as having HER-2–variant, and 12 of 21 patients classified as having basal phenotype (57.1%) neoplasms. The CPR rate among patients with the HER-2–classic and basal neoplasms was 67% (22 of 33 neoplasms), compared with 17.1% (6 of 35 neoplasms) in the non-HER-2–classic/basal combined group (P < .001). Eleven carcinomas were initially diagnosed as invasive lobular carcinomas (pleomorphic and classic), 4 of which were luminal-A, 4 of which were luminal-B, 2 of which were HER-2–classic, and 1 of which was basal. On review, only 3 of these 11 cases remained classified as classic lobular carcinoma, all of which were classified as luminal-A, and none of these patients achieved a CPR. Four of the other 8 patients achieved a CPR. CONCLUSIONS. The molecular classification system is useful for identifying carcinoma patients who are most likely and those who are least likely to achieve a CPR. In the current study, all the morphologically classic lobular carcinomas were classified as luminal-A neoplasms, which may explain the low rate of CPR reported. Cancer 2007. © 2007 American Cancer Society.

45 Background: To assess if partial vs whole breast irradiation (RT) in treatment of the various biological (bio-) subtypes of breast cancer would lead to differing clinical results, a match pair analysis of APBI vs WBI was undertaken. Methods: Between 3/1993 and 9/2013 all breast CA patients (pts) treated at one institute with either APBI or WBI were matched 1:1 by follow-up (FU) +/- 15 yrs, stage and bio-subtype. This yielded 772 pts of whom 640 were luminal A, 42 luminal B, 58 triple neg (TNBC), 6 HER2+, and 26 triple +. Outcomes were analyzed across the various bio-subtypes for all the pts (APBI + WBI), APBI alone and WBI alone. The endpoints assessed were local recurrence (LR), true recurrence/marginal miss (TR/MM), elsewhere failure (EF), regional recurrence (RR), distant metastasis (DM), disease-free survival (DFS), and contralateral breast failure (CLBF). Results: Mean age was 66 (32-94) with a mean FU of 5.2 years (0.1-18.3). Regarding systemic therapy, 75% received endocrine Rx, while chemotherapy was documented in 21%. For all pts (APBI+WBI) there was no significant difference across the various bio-subtypes with respect to 5, 10 and 15 yr actuarial LR, TR/MM, EF, RR, DM, DFS and CLBF. Likewise, for the 386 APBI-alone treated pts, no significant difference was found between the various bio-subtypes for all the same endpoints reported. However, for the 386 WBI pts, a significant difference was seen in the 15-year actuarial LR between luminal A and TNBC (1.5% vs 7.4%, p = 0.007). Significance was also found in the 15-year actuarial DFS between luminal A/B and TNBC (98.0%, 95.2%; 86.0%, respectively, p = 0.009). Conclusions: In comparing partial to whole breast RT across the various biological subtypes of breast CA, APBI is at least as effective as WBI. However, the retrospective nature of this study along with the limited numbers of pts in the HER2+ and triple + subsets are weaknesses which are acknowledged to exist in this dataset. The addition of further pts in all the biological subtypes, particularly the HER2+ and triple + subsets, along with results from randomized trials incorporating biomarker data will be needed to substantiate these findings.

40 Background: In response to many recent publications and mandates to assure referrals to genetic counseling for oncology patients, the Royal Oak (RO) Breast Care Center (BCC) at Beaumont Health System (BHS) evaluated the surgical outcomes of genetic referral(GR) in breast cancer (BC) patients. The goal of this study was to determine the impact of GR on surgical decision making and evaluate outcomes in this population. Methods: A retrospective chart review was performed, to identify patients who had a BC diagnosis and met criteria for GR from July 2012- July 2014. Age, histology, laterality of cancer, prior history of cancer, neoadjuvant chemotherapy, plastic surgery consultation, MRI, reason for MRI, additional testing, type of surgery, laterality of surgery, reconstruction, lymph node surgery, and time from diagnosis to surgery were evaluated using Chi Square analysis. Results: A total of 506 patients with a new BC diagnosis seen at the RO BCC at BHS within the inclusion dates were analyzed. There were 191 patients referred to the GP for counseling and possible genetic testing. Eighty percent of the referred patients underwent genetic testing. Twelve patients tested positive for deleterious mutations in BRCA 1 or 2. A statistically significant difference was found in the BC patients referred to the GP with respect to age, MRI usage, neoadjuvant chemotherapy, type and laterality of surgery, ALND, plastic surgery consult & reconstruction, and time from diagnosis to surgery. MRI usage in patients referred to GP was 43.5% vs. 12.7% in those not referred. Mastectomy rate in patients referred to GP was 51.3% vs. 25.5% (p