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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: 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.

Purpose. Hormonal status significantly affects background normal parenchymal enhancement (BPE) degree on breast MRI. A previous study reported the association between lack of BPE suppression and non pathologic complete response (pCR) after neoadjuvant chemotherapy for HR+ invasive cancer1. For DCIS, local control is the treatment goal2,3. Surgical outcomes which might be related to local control include presence of invasive components at surgery, re-excision after breast conserving surgery (BCS), and residual DCIS extent. We hypothesized that imaging outcomes (pre-surgery MRI volume and volume reduction from baseline) might also be related to local control. We investigated the association between BPE degree/suppression and 5 surgical/imaging outcomes after neoadjuvant endocrine therapy (NET) for DCIS. Methods. CALGB 40903 was a single arm trial of NET for ER+ DCIS without invasion4. Clinical and image data from a subset of 59 post-menopausal patients from this trial was available for analysis. Patients took letrozole 2.5 mg/day for 6 months before surgery (BCS, 53; mastectomy, 6). Dynamic contrast-enhanced MRIs were obtained at baseline, 3 months, and 6 months (pre-surgery). MRI volume was measured by summing all voxels with pre-defined level of enhancement within a 3D bounding box. MRI volume reduction was measured by percent change from baseline. BPE degree was calculated as the mean early percent enhancement of the central 50% axial slices on a contralateral breast. BPE suppression was dichotomized as suppressed or non-suppressed relative to baseline. For analyses on re-excision, only BCS patients were included. For analyses on residual DCIS extent, patients who achieved pCR or had invasive components were excluded. Mann-Whitney, Spearman’s rho, and Fisher’s exact tests were used for analyses, with P Table 1.Association of BPE Degree with Surgical and Imaging OutcomesOutcomeTime pointOutcome statusNo. of patientsMedian [1st, 3rd quartiles]P-valuePresence of invasive componentBaselineAbsent47 (92)25.0 [19.6, 30.0]0.282Present4 (8)17.2 [15.3, 25.5]3 monthsAbsent48 (91)23.5 [17.8, 31.0]0.668Present5 (9)16.6 [16.3, 34.7]6 monthsAbsent45 (90)24.6 [18.1, 33.0]0.343Present5 (10)17.2 [12.8, 31.2]OutcomeTime pointOutcome statusNo. of patientsMedian [1st, 3rd quartiles]P-valueRe-excision after BCSBaselineNot performed40 (87)22.6 [19.0, 27.1]0.019*Performed6 (13)43.0 [29.3, 53.9]3 monthsNot performed40 (85)22.1 [16.5, 29.5]0.018*Performed7 (15)36.7 [29.2, 40.3]6 monthsNot performed37 (84)19.8 [16.5, 31.4]0.009*Performed7 (16)38.3 [31.4, 41.0]OutcomeTime pointOutcome statusNo. of patientsSpearman’s rhoP-valueResidual DCIS extentBaselineNA3800.9973 monthsNA38-0.270.1016 monthsNA37-0.230.164MRI volumeBaselineNA510.130.3733 monthsNA530.170.2326 monthsNA500.420.003*MRI volume reductionBaselineNA510.070.6093 monthsNA530.290.0336 monthsNA500.50< 0.001** P Table 2.Association of BPE suppression with Surgical and Imaging OutcomesOutcomeTime pointBPE suppression groupNo. of patientsNo. of patients w/o invasive componentNo. of patients w/invasive componentP-valuePresence of invasive component3 monthssuppressed26 (53)24 (53)2 (50)1.000non-suppressed23 (47)21 (47)2 (50)6 monthssuppressed25 (53)22 (51)3 (75)0.611non-suppressed22 (47)21 (49)1 (25)OutcomeTime pointBPE suppression groupNo. of patientsNo. of patients w/o re-excisionNo. of patients w/re-excisionP-valueRe-excision after BCS3 monthssuppressed26 (53)22 (51)4 (67)0.671non-suppressed23 (47)21 (49)2 (33)6 monthssuppressed25 (53)21 (51)4 (67)0.670non-suppressed22 (47)20 (49)2 (33)OutcomeTime pointBPE suppression groupNo. of patientsMedian[1st, 3rd quartiles]P-valueResidual DCIS extent3 monthssuppressed19 (53)23mm[7, 36]0.172non-suppressed17 (47)11mm[4, 22]6 monthssuppressed18 (51)20mm[6.5, 34]0.487non-suppressed17 (49)18mm[4, 24]MRI volume3 monthssuppressed26 (53)0.4cc[0.2, 1.9]0.613non-suppressed23 (47)0.6cc[0.1, 1.6]6 monthssuppressed25 (53)0.3cc[0.1, 0.8]0.015*non-suppressed22 (47)1.1cc[0.4, 2.8]MRI volume reduction3 monthssuppressed26 (53)-73.0%[-91.4, -45.0]0.684non-suppressed23 (47)-71.1%[-92.1, -46.7]6 monthssuppressed25 (53)-84.8%[-96.2, -66.5]0.001*non-suppressed22 (47)-48.6%[-75.0, 57.3]* P Citation Format: Natsuko Onishi, David C Newitt, Jessica E Gibbs, Alex A Nguyen, Rita Freimanis, Elissa R Price, Shelly Hwang, Nola M Hylton. Background parenchymal enhancement as an imaging biomarker in neoadjuvant endocrine therapy for DCIS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-03-08.

Background Risk factors for psychosocial distress following a breast cancer diagnosis include younger age, history of depression, inadequate social support, and serious comorbid conditions. Although these quality of life (QOL) concerns have been studied in women with ductal carcinoma in situ (DCIS), Latina women have been understudied. Methods Data were from a cross-sectional telephone survey of Latina and Euro-American women with DCIS recruited through a population-based cancer registry. The sample included 396 Euro-American women and 349 Latina women; 156 were interviewed in English and 193 in Spanish, with a median of 2 years after diagnosis. Regression models were created for measures in each of the following four QOL domains: physical, psychological, social, and spiritual. Results Younger age, no partner, and lower income were related to lower QOL in various domains. Physical comorbidities were associated with lower physical, psychological, and social QOL; lingering effects of surgery and prior depression were associated with lower QOL in all domains. English-speaking and Spanish-speaking Latinas (SSLs) reported higher spiritual QOL, and SSLs reported lower social QOL than Euro-American women. Conclusions Despite having lower mortality, women with DCIS are treated with surgery and radiation therapy as if they have invasive cancer, and the aftereffects of treatment can impact their QOL. SSLs are at risk for lower QOL partly because of poverty. However, Latinas' greater spiritual QOL may mitigate some of the psychological and social effects of treatment. Implications It is important to incorporate these findings into treatment decision making (choice of surgical treatment) and survivorship care (monitoring women with a history of depression or physical comorbidity). Copyright © 2012 John Wiley & Sons, Ltd.

Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin- and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches.

Purpose Optic nerve (ON) ischemia associated with nonarteric anterior ischemic optic neuropathy (NAION) results in axon and myelin damage. Myelin damage activates the intraneural Ras homolog A (RhoA), contributing to axonal regeneration failure. We hypothesized that increasing extrinsic macrophage activity after ON infarct would scavenge degenerate myelin and improve postischemic ON recovery. We used the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to upregulate ON macrophage activity, and evaluated GM-CSF's effects after ON ischemia in the NAION rodent model (rAION). Methods Following rAION induction, GM-CSF was administered via intraventricular injection. Retinal ganglion cell (RGC) stereologic analysis was performed 1 month postinduction. The retinae and optic nerve laminae of vehicle- and GM-CSF-treated animals were examined immunohistochemically and ultrastructurally using transmission electron microscopy (TEM). RhoA activity was analyzed using a rhotekin affinity immunoanalysis and densitometry. Isolated ONs were analyzed functionally ex vivo by compound action potential (CAP) analysis. Results Rodent NAION produces ON postinfarct demyelination and myelin damage, functionally demonstrable by CAP analysis and ultrastructurally by TEM. Granulocyte-macrophage colony-stimulating factor increased intraneural inflammation, activating and recruiting endogenous microglia, with only a moderate amount of exogenous macrophage recruitment. Treatment with GM-CSF reduced postinfarct intraneural RhoA activity, but did not neuroprotect RGCs after rAION. Conclusions Sudden ON ischemia results in previously unrecognized axonal demyelination, which may have a clinically important role in NAION-related functional defects and recovery. Granulocyte-macrophage colony-stimulating factor is not neuroprotective when administered directly to the optic nerve following ON ischemia, and does not improve axonal regeneration. It dramatically increases ON-microglial activation and recruitment.

Risk factors for psychosocial distress following a breast cancer diagnosis include younger age, history of depression, inadequate social support, and serious comorbid conditions. Although these quality of life (QOL) concerns have been studied in women with ductal carcinoma in situ (DCIS), Latina women have been understudied.Data were from a cross-sectional telephone survey of Latina and Euro-American women with DCIS recruited through a population-based cancer registry. The sample included 396 Euro-American women and 349 Latina women; 156 were interviewed in English and 193 in Spanish, with a median of 2 years after diagnosis. Regression models were created for measures in each of the following four QOL domains: physical, psychological, social, and spiritual.Younger age, no partner, and lower income were related to lower QOL in various domains. Physical comorbidities were associated with lower physical, psychological, and social QOL; lingering effects of surgery and prior depression were associated with lower QOL in all domains. English-speaking and Spanish-speaking Latinas (SSLs) reported higher spiritual QOL, and SSLs reported lower social QOL than Euro-American women.Despite having lower mortality, women with DCIS are treated with surgery and radiation therapy as if they have invasive cancer, and the aftereffects of treatment can impact their QOL. SSLs are at risk for lower QOL partly because of poverty. However, Latinas' greater spiritual QOL may mitigate some of the psychological and social effects of treatment.It is important to incorporate these findings into treatment decision making (choice of surgical treatment) and survivorship care (monitoring women with a history of depression or physical comorbidity).

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway.

Vasopressin-regulated urea transport in the renal inner medullary collecting duct (IMCD) is mediated by two urea channel proteins, UT-A1 and UT-A3, derived from the same gene ( Slc14a2) by alternative splicing. The NH2-terminal 459 amino acids are the same in both proteins. To study UT-A1/3 phosphorylation, we made phospho-specific antibodies to UT-A sequences targeting phospho-serines at positions 84 and 486, sites identified previously by protein mass spectrometry. Both antibodies proved specific, recognizing only the phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat IMCD suspensions or whole inner medullas showed that the V2R-selective vasopressin analog 1-deamino-8-d-arginine vasopressin (dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3) and Ser486 (in UT-A1) by about eightfold. Time course studies in rat IMCD suspensions showed maximum phosphorylation within 1 min of dDAVP exposure, consistent with the time course of vasopressin-stimulated phosphorylation of the vasopressin-sensitive water channel aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. Immuno-electron microscopy studies showed that both phosphorylated forms were present mainly in intracellular compartments in the presence of vasopressin. These studies demonstrate regulated phosphorylation of both UT-A1 and UT-A3 in response to vasopressin in a manner consistent with coordinate regulation of UT-A and aquaporin-2 in the renal IMCD. The findings add to prior evidence for vasopressin-induced phosphorylation of UT-A1, providing evidence that UT-A3 may be regulated by phosphorylation as well.