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Obtaining tumor-free surgical margins is critical to prevent recurrence in breast-conserving surgery but it remains challenging. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor. Lumpectomy cavity walls and excised specimens of breast cancer lumpectomy patients were assessed with the LUM Imaging System (Lumicell, Inc., Wellesley MA) with and without intravenous LUM015, a cathepsin-activatable fluorescent agent. Fluorescence at potential sites of residual tumor was evaluated with a sterile hand-held probe, displayed on a monitor and correlated with histopathology. Background autofluorescence was assessed in excised specimens from 9 patients who did not receive LUM015. In vivo lumpectomy cavities and excised specimens were then imaged in 15 women undergoing breast cancer surgery who received no LUM015, 0.5, or 1 mg/kg LUM015 (5 women per dose). Among these, 11 patients had invasive carcinoma with ductal carcinoma in situ (DCIS) and 4 had only DCIS. Image acquisition took 1 s for each 2.6-cm-diameter surface. No significant background normal breast fluorescence was identified. Elevated fluorescent signal was seen from invasive cancers and DCIS. Mean tumor-to-normal signal ratios were 4.70 ± 1.23 at 0.5 mg/kg and 4.22 ± 0.9 at 1.0 mg/kg (p = 0.54). Tumor was distinguished from normal tissue in pre-and postmenopausal women and readings were not affected by breast density. Some benign tissues produced fluorescent signal with LUM015. The LUM Imaging System allows rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.

Background In breast conserving surgery, the concordance between lumpectomy margin (LM) status and the status of the corresponding lumpectomy cavity remains uncertain. Methods We analyzed pathology reports of lumpectomies from 2004 to 2006. We included those which contained both ink-directed LM and complete (≥4) separate corresponding shaved cavity margins (SCMs). SCM pathology was used as a surrogate for lumpectomy cavity status, to determine the predictive value of LM for residual disease. Results Pathology from 1,201 pairs of LM and SCM from 242 patients was compared. LM status predicted corresponding lumpectomy cavity status with 50.9% sensitivity, 69.5% specificity, 35% positive predictive value, and 81.4% negative predictive value, giving an overall accuracy of 64.9%. Conclusions Oriented LMs are not reliable for predicting lumpectomy cavity status, and therefore not reliable for directing re-excision. Taking complete, oriented SCMs at the time of lumpectomy may improve accuracy compared with traditional LM assessment.

Assessment of a pediatric breast lesion always starts with clinical evaluation. When imaging of a pediatric breast is indicated, ultrasound is the mainstay. The vast majority of pediatric breast complaints are of benign etiology, therefore the diagnostic/management approach emphasizes “first do no harm”. Correlation with age and clinical history helps to direct diagnosis. It is essential to be familiar with the imaging appearance of the normal developing breast at various Tanner stages, in order to diagnose physiologic breast findings and to minimize unnecessary biopsies in young breasts vulnerable to injury. Normal anatomic structures, developmental conditions, benign neoplastic and non-neoplastic lesions are common causes of breast complaints in children. Uncommon benign masses and rarely, secondary more than primary malignancies may present in a pediatric breast. Chest wall masses such as Ewing's sarcoma or rhabdomyosarcoma occur in children and may involve the breast via contiguous growth or locoregional metastasis. In addition, special attention should be given to any breast lesion in a child with risk factors predisposing to breast cancer, such as known extramammary malignancy, genetic mutations, prior mantle irradiation, or strong family history of breast cancer, which usually requires biopsy to exclude the possibility of malignancy. Conclusion The developing breast is vulnerable to injury, and because breast malignancy is uncommon in children, diagnostic and management approach emphasizes “first do no harm”. Understanding normal breast development and the spectrum of common and uncommon pediatric breast lesions are key to the correct diagnosis.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these micro-environments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Staying one step ahead of tumors Cancer treatments require continual adjustment. A drug that works initially will lose its potency as the tumor acquires new mutations that allow it to bypass the drug's lethal effects. To stay ahead of the tumor, oncologists need a noninvasive way to collect tumor cells from patients over the course of their treatment. Analyzing the mutations in these samples may help them choose the right drugs as the tumors change. In a small study of breast cancer patients, Yu et al. show that rare tumor cells circulating in the blood can be captured in viable form and used for this purpose. Science , this issue p. 216

A diagnosis of neuroendocrine carcinoma is often morphologically straight-forward; however, the tumor site of origin may remain elusive in a metastatic presentation. Neuroendocrine tumor subtyping has important implications for staging and patient management. In this study, the novel use and performance of a 92-gene molecular cancer classifier for determination of the site of tumor origin are described in a series of 75 neuroendocrine tumors (44 metastatic, 31 primary; gastrointestinal (n=12), pulmonary (n=22), Merkel cell (n=10), pancreatic (n=10), pheochromocytoma (n=10), and medullary thyroid carcinoma (n=11)). Formalin-fixed, paraffin-embedded samples passing multicenter pathologist adjudication were blinded and tested by a 92-gene molecular assay that predicts tumor type/subtype based upon relative quantitative PCR expression measurements for 87 tumor-related and 5 reference genes. The 92-gene assay demonstrated 99% (74/75; 95% confidence interval (CI) 0.93-0.99) accuracy for classification of neuroendocrine carcinomas and correctly subtyped the tumor site of origin in 95% (71/75; 95% CI 0.87-0.98) of cases. Analysis of gene expression subsignatures within the 92-gene assay panel showed 4 genes with promising discriminatory value for tumor typing and 15 genes for tumor subtyping. The 92-gene classifier demonstrated excellent accuracy for classifying and determining the site of origin in tumors with neuroendocrine differentiation. These results show promise for use of this test to aid in classifying neuroendocrine tumors of indeterminate primary site, particularly in the metastatic setting.

PurposeGenome-wide-association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) that are associated with an increased risk of breast cancer. Most of these studies were conducted primarily in postmenopausal breast cancer patients. Therefore, we set out to assess whether or not these breast cancer variants are also associated with an elevated risk of breast cancer in young premenopausal patients.MethodsIn 451 women of European ancestry who had prospectively enrolled in a longitudinal cohort study for women diagnosed with breast cancer at or under age 40, we genotyped 44 SNPs that were previously associated with breast cancer risk. A control group was comprised of 1142 postmenopausal healthy women from the Nurses' Health Study (NHS). We assessed if the frequencies of the adequately genotyped SNPs differed significantly (p≤0.05) between the cohort of young breast cancer patients and postmenopausal controls, and then we corrected for multiple testing.ResultsGenotyping of the controls or cases was inadequate for comparisons between the groups for seven of the 44 SNPs. 9 of the remaining 37 were associated with breast cancer risk in young women with a p-value ConclusionAfter correction for multiple testing, none of the alleles for postmenopausal breast cancer were clearly associated with risk of premenopausal breast cancer in this relatively small study.

505 Background: The aTTom study is a prospective phase III trial that randomized 6953 HR+ women to stop or continue tamoxifen (TAM) for 5 more years after completing at least 4 years of prior TAM. Results at 9 years of median follow-up demonstrated fewer breast cancer recurrences (21% vs 25%; RR= 0.86 [95% CI 0.77-0.96]; P = 0.006) and reduced breast cancer mortality (13% vs 15%; HR= 0.91 [95% CI 0.80-1.04]; P = 0.18) but increased incidence of endometrial cancer with longer TAM use (P < 0.0001). The Breast Cancer Index (BCI) is a gene expression–based signature that stratifies patients based on the risk of overall (0-10y) and late (post-5y) DR and predicted the likelihood of benefit from extended endocrine therapy in MA.17. This Translational aTTom (Trans-aTTom) study is a large-scale validation of the predictive ability of BCI for extended endocrine therapy (EET) benefit. Methods: Patients treated in the aTTom trial with available primary tumor tissue were eligible for this multi-institutional prospective-retrospective study. Primary and secondary endpoints were recurrence-free interval (RFI) and disease-free interval (DFI), respectively. Statistical significance level for RFI was set at 0.0336 as per statistical plan. Kaplan-Meier and Cox proportional hazards regression analysis with time-varying coefficients were used to test the predictive activity of BCI by HoxB13/IL17BR (H/I) status (High vs Low). Likelihood ratio test based on Cox regression was used to evaluate treatment by biomarker interaction. Results: 2637 tumors were centrally assessed for ER, PR and HER2 status leading to 1822 HR+ patients analyzed (1018 N0, 583 N+). Initial results from patients with N+ disease at 12 years of median follow-up showed 287 (49%) were classified as H/I-High and 296 (51%) were classified as H/I-Low. H/I High patients showed a statistically significant benefit of 9.8% in RFI with 10y vs 5y of TAM (HR=0.35 [95% CI 0.15-0.85]; P=0.027), whereas H/I Low patients showed no benefit (-0.2% RFI; HR=1.07 [95% CI 0.69-1.65]; P=0.77). A statistically significant interaction between continuous BCI and treatment was demonstrated (P = 0.02). Conclusions: These data provide further validation and establish level 1B evidence for BCI as a predictive biomarker for preferential benefit from EET in HR+ breast cancer.

Gaze-evoked amaurosis (GEA) is a transient monocular vision loss provoked by eccentric gaze. Gaze-evoked amaurosis has been associated with a variety of orbital lesions, most commonly optic nerve sheath meningiomas and cavernous hemangiomas. The authors describe the first report in the literature of GEA as the presenting symptom of an orbital metastasis. The patient was a 47-year-old woman with a history of breast cancer with no known history of metastasis or active disease who presented with several weeks of vision loss in the OD upon rightward gaze. She was found to have enophthalmos and optic disc edema of the OD. Imaging revealed an intraorbital lesion, and a biopsy was consistent with a scirrhous metastasis of her breast carcinoma. This case highlights the importance of considering orbital metastases among the differential for gaze-evoked amaurosis.

Context.—Most of the population in Ethiopia lives below the poverty line with severely limited access to health care. The burden of infectious diseases is high, but benign and malignant neoplasms are also encountered frequently. For diagnosis of palpable lesions in this setting, fine-needle aspiration biopsy is the method of choice.Objective.—To present findings from several patients from 3 major hospitals in Ethiopia who underwent fine-needle aspiration biopsy.Data Sources.—Representative cytopathology cases of routinely encountered problems are shown. Often patients present with clinically advanced lesions. Staffing, technique, and equipment used for fine-needle aspiration biopsy are described at Black Lion Hospital (Addis Ababa), the University of Gonder Hospital (Gonder), and Ayder Referral Hospital of Mekelle University in the Tigray region of northern Ethiopia.Conclusions.—Fine-needle aspiration biopsy is a highly effective method for diagnosis of mass lesions, especially in an environment with sparse health care resources, such as Ethiopia. This article illustrates the work of Ethiopian cytopathologists and emphasizes the constraints under which they perform their work.

Information focuses on molecular pathology of breast cancer. Presented are clinical features of breast cancer, detailed discussion of histology and molecular pathology for invasive ductal carcinoma, invasive lobular carcinoma, other subtypes of invasive breast cancer, and breast cancer progression and precursors. Phenotypes and genotypes of breast cancer are presented, along with the role of biomarkers, gene profiling, and hormone receptors. Targeted therapies and prognostic indicators are presented with practical applications of molecular pathology for the surgical pathologist.

External beam accelerated partial breast irradiation (APBI) is an increasingly popular technique for treatment of patients with early stage breast cancer following breast-conserving surgery. Here we present 5-year results of a prospective trial.From October 2003 through November 2005, 98 evaluable patients with stage I breast cancer were enrolled in the first dose step (32 Gy delivered in 8 twice-daily fractions) of a prospective, multi-institutional, dose escalation clinical trial of 3-dimensional conformal external beam APBI (3D-APBI). Median age was 61 years; median tumor size was 0.8 cm; 89% of tumors were estrogen receptor positive; 10% had a triple-negative phenotype; and 1% had a HER-2-positive subtype. Median follow-up was 71 months (range, 2-88 months; interquartile range, 64-75 months).Five patients developed ipsilateral breast tumor recurrence (IBTR), for a 5-year actuarial IBTR rate of 5% (95% confidence interval [CI], 1%-10%). Three of these cases occurred in patients with triple-negative disease and 2 in non-triple-negative patients, for 5-year actuarial IBTR rates of 33% (95% CI, 0%-57%) and 2% (95% CI, 0%-6%; P.0001), respectively. On multivariable analysis, triple-negative phenotype was the only predictor of IBTR, with borderline statistical significance after adjusting for tumor grade (P=.0537).Overall outcomes were excellent, particularly for patients with estrogen receptor-positive disease. Patients in this study with triple-negative breast cancer had a significantly higher IBTR rate than patients with other receptor phenotypes when treated with 3D-APBI. Larger, prospective 3D-APBI clinical trials should continue to evaluate the effect of hormone receptor phenotype on IBTR rates.

Unacceptably large percentage (20-40%) of breast cancer lumpectomy patients are required to undergo multiple surgeries when positive margins are found upon post-operative histologic assessment. If the margin status can be determined during surgery, surgeon can resect additional tissues to achieve tumor-free margin, which will reduce the need for additional surgeries. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to image the entire surgical margin within a short procedural time. Previously, SECM was shown to rapidly image a large area (10 mm by 10 mm) of human esophageal tissue within a short procedural time (15 seconds). When used in lumpectomy, SECM will be able to image the entire margin surface of ~30 cm2 in around 7.5 minutes. SECM images will then be used to determine margin status intra-operatively. In this paper, we present results from a study of testing accuracy of SECM for diagnosing malignant breast tissues. We have imaged freshly-excised breast specimens (N=46) with SECM. SECM images clearly visualized histomorphologic features associated with normal/benign and malignant breast tissues in a similar manner to histologic images. Diagnostic accuracy was tested by comparing SECM diagnoses made by three junior pathologists with corresponding histologic diagnoses made by a senior pathologist. SECM sensitivity and specificity were high, 0.91 and 0.93, respectively. Intra-observer agreement and inter-observer agreement were also high, 0.87 and 0.84, respectively. Results from this study showed that SECM has a potential to accurately determine margin status during breast cancer lumpectomy.

Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3′ untranslated region (3′ UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3′ UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype–specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. Cancer Res; 76(24); 7231–41. ©2016 AACR.

A large percentage of breast cancer patients treated with breast conserving surgery need to undergo multiple surgeries due to positive margins found during post-operative margin assessment. Carcinomas could be removed completely during the initial surgery and additional surgery avoided if positive margins can be determined intraoperatively. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to rapidly image the entire surgical margin at subcellular resolution and accurately determine margin status intraoperatively. In this study, in order to test the feasibility of using SECM for intraoperative margin assessment, we have evaluated the diagnostic accuracy of SECM for detecting various types of breast cancers. Forty-six surgically removed breast specimens were imaged with an SECM system. Side-by-side comparison between SECM and histologic images showed that SECM images can visualize key histomorphologic patterns of normal/benign and malignant breast tissues. Small (500 μm × 500 μm) spatially registered SECM and histologic images (n=124 for each) were diagnosed independently by three pathologists with expertise in breast pathology. Diagnostic accuracy of SECM for determining malignant tissues was high, average sensitivity of 0.91, specificity of 0.93, positive predictive value of 0.95, and negative predictive value of 0.87. Intra-observer agreement and inter-observer agreement for SECM were also high, 0.87 and 0.84, respectively. Results from this study suggest that SECM may be developed into an intraoperative margin assessment tool for guiding breast cancer excisions.

Micro-CT is a promising modality to determine breast tumour size in three dimensions in intact lumpectomy specimens. We compared the accuracy of tumour size measurements using specimen micro-CT with measurements using multimodality pre-operative imaging.A tabletop micro-CT was used to image breast lumpectomy specimens. The largest tumour dimension on three-dimensional reconstructed micro-CT images of the specimen was compared with the measurements determined by pre-operative mammography, ultrasound and MRI. The largest dimension of pathologic invasive cancer size was used as the gold standard reference to assess the accuracy of imaging assessments.50 invasive breast cancer specimens in 50 patients had micro-CT imaging. 42 were invasive ductal carcinoma, 6 were invasive lobular carcinoma and 2 were other invasive cancer. Median patient age was 63 years (range 33-82 years). When compared with the largest pathologic tumour dimension, micro-CT measurements had the best correlation coefficient (r = 0.82, p 0.001) followed by MRI (r = 0.78, p 0.001), ultrasound (r = 0.61, p 0.001) and mammography (r = 0.40, p 0.01). When compared with pre-operative modalities, micro-CT had the best correlation coefficient (r = 0.86, p 0.001) with MRI, followed by ultrasound (r = 0.60, p 0.001) and mammography (r = 0.54, p 0.001). Overall, mammography and ultrasound tended to underestimate the largest tumour dimension, while MRI and micro-CT overestimated the largest tumour dimension more frequently.Micro-CT is a potentially useful tool for accurate assessment of tumour dimensions within a lumpectomy specimen. Future studies need to be carried out to see if this technology could have a role in margin assessment.Micro-CT is a promising new technique which could potentially be used for rapid assessment of breast cancer dimensions in an intact lumpectomy specimen in order to guide surgical excision.

BACKGROUND Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

Background: The increase in breast cancer risk during pregnancy and post partum is well recognized. The cross-over to protective effect does not occur until many years later and varies with age at first birth. Recently, a genomic signature specific to the pregnant compared with the non-pregnant breast has been identified; this signature remains present in the postmenopausal parous breast. Given this, we investigated whether time interval since pregnancy affects the phenotype of breast cancers arising in young women compared with nulliparous women. Methods: We examined molecular phenotype, according to histologic grade and biomarker status, in relation to time since pregnancy in an ongoing prospective cohort study (n=355) of young women (≤40yrs) with breast cancer. Medical records were reviewed for tumor stage and receptor status. Parity was ascertained from questionnaires completed within 9 months of diagnosis. Tumor grade was determined by central pathology review. Using tumor grade and biomarker expression, cancers were categorized as luminal A (ER+ and/or PR+, HER2−, histologic grade 1 or 2); luminal B ( ER+ and/or PR+, HER2+, or ER and/or PR+, HER2− and grade 3); HER2 type (ER-, PR-, HER2+); and triple negative (ER-, PR-, HER2−). Results: The median age of the study population is 37 years (range 17–40). Overall, 80% of women had stage 1 or 2 disease; 67% of cancers were ER positive and 32% showed HER2 overexpression. The distribution of breast cancer molecular phenotypes by time interval since last pregnancy is shown in the table. Distribution of molecular phenotype by interval between last pregnancy and diagnosis In our large cohort of parous young women, we found no differences in the distribution of molecular phenotype according to time interval since pregnancy. However, nulliparous young women were more likely to develop luminal A cancers compared to parous women (40% vs. 29%; unadjusted chi square p-value=0.03) and appeared less likely to develop HER2−type and triple negative cancers (7% vs. 13%, p-value=0.09 and 17% vs. 23%, p-value=0.22 respectively). There were no differences in the distribution of luminal B cancers. Conclusions: The distribution of molecular phenotypes is similar among parous young women regardless of the time interval since parturition. Nulliparous young women appear more likely to develop luminal A cancers compared to parous women. Whether the difference in molecular phenotypes of pregnancy-associated breast cancers vs. cancers arising in nulliparous women is due to the effects of genomic alteration remains to be investigated. Effects of a prior pregnancy appear consistent across a 5-year period, in keeping with the concept of genomic alterations identified in the normal pregnant breast and thereafter. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-11-12.

Prior studies have suggested a higher prevalence of high grade, ER-negative, HER2-positive, and basal-like carcinomas in young women with breast cancer. However, the precise distribution of poor prognostic features in this population remains unclear. We examined the pathologic features and distribution of molecular phenotype in relation to patient age in a large group of young women (≤40 years) with invasive breast cancer. Medical records were reviewed for clinical characteristics, tumor stage, and receptor status. Pathologic features, including those features associated with basal-like carcinomas, were examined by central review. Using tumor grade and biomarker expression, cancers were categorized as luminal A (ER+ and/or PR+ and HER2-, histologic grade 1 or 2); luminal B (ER+ and/or PR+ and HER2+, or ER and/or PR+, HER2- and grade 3); HER2 (ER and PR- and HER2+); and triple negative (ER-, PR-, and HER2-). Among 399 women of ≤40 years, 33% had luminal A tumors, 35% luminal B, 11% HER2 (ER-negative), and 21% triple negative. Compared to published results for all breast cancers, a greater proportion of young women had luminal B tumors, and a lesser proportion had luminal A. There were no significant differences in molecular phenotype, tumor stage or grade among the different age groups of young women. However, this population of young women presented with a different distribution of molecular phenotypes compared to the general population of women with breast cancer. These findings may have implications with regard to the etiology and prognosis of breast cancer in young women.

Homeobox 9 (HOXB9), a nontransforming transcription factor overexpressed in breast cancer, alters tumor cell fate and promotes tumor progression and metastasis. Here we show that HOXB9 confers resistance to ionizing radiation by promoting DNA damage response. In nonirradiated cells, HOXB9 induces spontaneous DNA damage, phosphorylated histone 2AX and p53 binding protein 1 foci, and increases baseline ataxia telangiectasia mutated (ATM) phosphorylation. Upon ionizing radiation, ATM is hyperactivated in HOXB9-expressing cells during the early stages of the double-stranded DNA break (DSB) response, accelerating accumulation of phosphorylated histone 2AX, mediator of DNA-damage checkpoint 1, and p53 binding protein 1, at DSBs and enhances DSB repair. The effect of HOXB9 on the response to ionizing radiation requires the baseline ATM activity before irradiation and epithelial-to-mesenchymal transition induced by TGF-β, a HOXB9 transcriptional target. Our results reveal the impact of a HOXB9–TGF-β–ATM axis on checkpoint activation and DNA repair, suggesting that TGF-β may be a key factor that links tumor microenvironment, tumor cell fate, DNA damage response, and radioresistance in a subset of HOXB9-overexpressing breast tumors.

The B-cell translocation gene-2 (BTG2), a p53-inducible gene, is suppressed in mammary epithelial cells during gestation and lactation. In human breast cancer, decreased BTG2 expression correlates with high tumor grade and size, p53 status, blood and lymph vessel invasion, local and metastatic recurrence and decrease in overall survival, suggesting that suppression of BTG2 has a critical role in disease progression. To analyze the role of BTG2 in breast cancer progression, BTG2 expression was knocked down in mammary epithelial cells. Suppression of BTG2 enhances the motility of cells in vitro and tumor growth and metastasis in vivo. The effects of BTG2 knockdown are mediated through stabilization of the human epidermal growth factor receptor (HER) ligands neuregulin and epiregulin and activation of the HER2 and HER3 receptors, leading to elevated AKT phosphorylation. Suppression of HER activation using the tyrosine kinase inhibitor lapatinib abrogates the effects of BTG2 knockdown, including the increased cell migration observed in vitro and the enhancement of tumorigenesis and metastasis in vivo. These results link BTG2-dependent effects on tumor progression to ErbB receptor signaling, and raise the possibility that targeted inhibition of this pathway may be relevant in the treatment of breast cancers that have reduced BTG2 expression.

Purpose To examine the relationship between lymphovascular invasion (LVI) and regional nodal failure (RNF) in breast cancer patients with zero to three positive nodes treated with breast-conservation therapy (BCT). Methods and Materials The records of 1,257 breast cancer patients with zero to three positive lymph nodes were reviewed. All patients were treated with BCT at Massachusetts General Hospital from 1980 to December 2003. Lymphovascular invasion was diagnosed by hematoxylin and eosin–stained sections and in some cases supported by immunohistochemical stains. Regional nodal failure was defined as recurrence in the ipsilateral supraclavicular, axillary, or internal mammary lymph nodes. Regional nodal failure was diagnosed by clinical and/or radiologic examination. Results The median follow-up was 8 years (range, 0.1–21 years). Lymphovascular invasion was present in 211 patients (17%). In univariate analysis, patients with LVI had a higher rate of RNF (3.32% vs. 1.15%; p = 0.02). In multivariate analysis, only tumor size, grade, and local failure were significant predictors of RNF ( p = 0.049, 0.013, and 0.0001, respectively), whereas LVI did not show a significant relationship with RNF (hazard ratio=2.07; 95% CI, 0.8–5.5; p = 0.143). The presence of LVI in the T2/3 population did not increase the risk of RNF over that for those with no LVI ( p = 0.15). In addition, patients with Grade 3 tumors and positive LVI did not have a higher risk of RNF than those without LVI ( p = 0.96). Conclusion These results suggest that LVI can not be used as a sole indicator for regional nodal irradiation in breast cancer patients with zero to three positive lymph nodes treated with BCT.

Antigen presentation plays a major role in tumor cell recognition and targeting by immune cells, and is critical to the success of many cancer immunotherapies. How the abnormal tumor microenvironment affects tumor cell antigen presentation is unclear. Hypoxia is a prevalent feature of the tumor microenvironment. Here, we showed that the expression of major histocompatibility complex class I (MHCI) is associated with regions of hypoxia in human breast tumors. The association between hypoxia and MHCI is independent of the breast tumor hormone receptor and HER2 expression status. In vitro studies revealed that hypoxia directly regulates the expression levels of MHCI along with other components of the antigen presentation machinery. Multiple kinase regulators of MHCI expression are responsive to hypoxia. These results suggest that hypoxia effects on cancer cell antigen presentation may be a potential mechanism of tumor immune evasion and treatment resistance. Citation Format: Mei Rosa Ng, Francesco Sabbatino, Mark Duquette, Kamila Naxerova, Mark Badeaux, Gino B. Ferraro, Shan M. Chin, Divya Bezwada, Elena F. Brachtel, Soldano Ferrone, Rakesh K. Jain. Hypoxia regulation of antigen presentation machinery expression in breast cancer [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 LB-057.

e12605Background: Obtaining clear margins in breast cancer lumpectomy surgery is made difficult by complex tumor geometry and lack of rapid, accurate approaches for intraoperative margin assessment...

An 85-year-old woman had poorly differentiated invasive ductal cancer (positive for estrogen and progesterone receptors and HER2) with no clinical evidence of lymph-node or distant metastases. She had hypercholesterolemia, hypertension, chronic obstructive pulmonary disease, hypothyroidism, arthritis, and a history of pleural and pericardial effusions of unknown cause. She lived with a daughter and performed all activities of daily living. A management decision was made.

The mechanisms underlying tumoral secretion of signaling molecules into the microenvironment, which modulates tumor cell fate, angiogenesis, invasion, and metastasis, are not well understood. Aberrant expression of transcription factors, which has been implicated in the tumorigenesis of several types of cancers, may provide a mechanism that induces the expression of growth and angiogenic factors in tumors, leading to their local increase in the tumor microenvironment, favoring tumor progression. In this report, we demonstrate that the transcription factor HOXB9 is overexpressed in breast carcinoma, where elevated expression correlates with high tumor grade. HOXB9 induces the expression of several angiogenic factors (VEGF, bFGF, IL-8, and ANGPTL-2), as well as ErbB (amphiregulin, epiregulin, and neuregulins) and TGF-ß, which activate their respective pathways, leading to increased cell motility and acquisition of mesenchymal phenotypes. In vivo, HOXB9 promotes the formation of large, well-vascularized tumors that metastasize to the lung. Thus, deregulated expression of HOXB9 contributes to breast cancer progression and lung metastasis by inducing several growth factors that alter tumor-specific cell fates and the tumor stromal microenvironment.

Epithelial-to-mesenchymal transition (EMT) plays an important role during normal embryogenesis, and it has been implicated in cancer invasion and metastasis. Here, we report that Ladybird homeobox 1 (LBX1), a developmentally regulated homeobox gene, directs expression of the known EMT inducers ZEB1, ZEB2, Snail1, and transforming growth factor β2 (TGFB2). In mammary epithelial cells, overexpression of LBX1 leads to morphological transformation, expression of mesenchymal markers, enhanced cell migration, increased CD44high/CD24low progenitor cell population, and tumorigenic cooperation with known oncogenes. In human breast cancer, LBX1 is up-regulated in the unfavorable estrogen receptor (ER)/progesterone (PR)/HER2 triple-negative basal-like subtype. Thus, aberrant expression of LBX1 may lead to the activation of a developmentally regulated EMT pathway in human breast cancer.

A 62-year-old woman with a history of breast cancer was found to have a mass in the contralateral breast on magnetic resonance imaging performed for follow-up of the first cancer. Excision of the mass and examination of the biopsy specimen showed infiltrating and in situ ductal carcinoma, grade 3 of 3, positive for human epidermal growth factor receptor 2. Options for management are discussed in a multidisciplinary setting.

Background Precise anatomical relationships between ducts and vasculature within the nipple remain unknown. This study investigated nipple microvessels and their position relative to ducts. Methods Nipple and duct bundle cross-sectional areas were measured in 48 specimens. Vessels located within the central duct bundle or within a peripheral rim were counted in 7 non-irradiated and 5 irradiated nipples. Results Mean nipple diameter was 11.1 mm and duct bundle diameter 5.2 mm. A 2-mm and a 3-mm peripheral rim of nipple tissue would result in complete duct excision in 96% and 87% of sections, respectively. Twenty-nine percent of vessels are located in the duct bundle. A 2-mm rim contains 50%; a 3-mm rim contains 66%. Similar proportions were seen in irradiated nipples. Conclusions This study describes a strategy to balance duct removal with vascular preservation. Ducts can be excised leaving a rim of nipple tissue that contains a large proportion of microvessels.

The initial diagnosis of lung carcinoma is frequently made by fine-needle aspiration biopsy. Novel therapeutic strategies of this disease include tyrosine kinase inhibitors (TKI), such as gefitinib (Iressa) or erlotinib (Tarceva), which target the kinase domain of epidermal growth factor receptor (EGFR). Somatic mutations of this region have been shown to predict a therapeutic response of lung carcinomas to TKI. EGFR mutations have been described in adenocarcinomas of the lung, especially the bronchioloalveolar subtype, which has both cytopathologic and histopathologic definitions. This study investigates whether tumors with EGFR mutations display a characteristic phenotype on fine-needle aspiration biopsy. We identified 37 fine-needle aspiration biopsy of lung masses on which molecular analysis for EGFR mutations was available. Molecular analysis was performed on DNA isolated from formalin-fixed, paraffin-embedded, or frozen tissue from the corresponding core biopsies/cell blocks or resection specimens followed by PCR with primers for the tyrosine kinase region exons 18-24 and nucleotide sequence analysis by gel electrophoresis. Two observers who were blinded to the mutational data assessed several cytomorphological parameters. A semiquantitative analysis included predominant tissue pattern (flat or overlapping), nuclear features (nucleoli, intranuclear inclusions, grooves), cytoplasmic qualities, and extracellular material. All cases were adenocarcinomas primary in the lung. Thirteen cases showed EGFR mutations in exons 18, 19, 20, or 21 of the tyrosine kinase domain. The 24 cases negative for the relevant mutation served as the control group. Tumors with EGFR mutations were statistically more likely to demonstrate a predominantly flat, monolayer architecture (P=0.04) with nuclear inclusions (P=0.014) and the absence of macronucleoli (P=0.001). The predominance of flat monolayers in conjunction with the absence of extracellular mucin and macronucleoli indicated the presence of EGFR mutations with a positive predictive value of 69% and a negative predictive value of 92%. All four cases with extracellular mucin were negative for the examined mutations. Some of the traditional cytomorphological features of bronchioloalveolar carcinoma, i.e., flat monolayers, intranuclear inclusions, and the absence of macronucleoli, statistically correlated with the presence of mutations within the tyrosine kinase region of EGFR. Cytomorphological features could serve as an adjunctive predictive marker of response to TKIs and possibly to other new therapies in development.

Background: The use of digital whole slide imaging for human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) could create improvements in workflow and performance, allowing for central archiving of specimens, distributed and remote interpretation, and the potential for additional computerized automation. Procedures: The accuracy, precision, and reproducibility of manual digital interpretation for HER2 IHC were determined by comparison to manual glass slide interpretation. Inter- and intra-pathologist reproducibility and precision between the glass slide and digital interpretations of HER2 IHC were determined in 5 studies using DAKO HercepTest-stained breast cancer slides with the Philips Digital Pathology System. In 2 inter-method studies, 3 pathologists interpreted glass and digital slides in sequence or in random order with a minimum of 7 days as a washout period. These studies also measured inter-observer reproducibility and precision. Another two studies measured intra-pathologist reproducibility on cases read 10 times by glass and digital methods. One additional study evaluated the effects of adding IHC control slides with each run, using 1 pathologist interpreting glass and digital slides randomized from the sets above along with appropriate controls for each slide in the set. Results: The overall results show that there is no statistical difference between the variance of performance when comparing glass and digital HER2 interpretations; and there were no effects noted when control tissues were evaluated in conjunction with the test slides. Conclusions: The results show that there is an equivalence of result when interpreting HER2 IHC slides in breast cancer by either glass slides or digital images. Digital interpretation can therefore be safely and effectively used for this purpose.

Some low-grade endometrioid carcinomas arise from a background of endometrioid tumours of borderline malignancy. To determine the molecular mechanisms involved in the initiation of endometrioid carcinoma, the present study investigated whether the genetic alterations reported in these tumours (mutations in PTEN, KRAS, and beta-catenin genes, and microsatellite instability) are already present in endometrioid tumours of borderline malignancy. Eight endometrioid tumours of borderline malignancy were studied. By immunohistochemistry, beta-catenin was expressed in the nuclei of all tumours, suggesting the presence of stabilizing beta-catenin mutations. By mutational analysis, five different beta-catenin mutations were found in seven of eight cases (90%), affecting codons 32, 33, and 37. In contrast, only one tumour harboured a PTEN mutation, which affected codon 130. Neither KRAS mutations nor microsatellite instability was detected. A review of the literature indicated that beta-catenin mutations are characteristic of well-differentiated endometrioid carcinomas, since they were present in nearly 60% of grade I but in less of 3% of grade III tumours. In conclusion, the present study identifies beta-catenin mutation as a nearly constant molecular alteration in borderline endometrioid tumours, whereas PTEN and KRAS mutations and microsatellite instability are very infrequent. The findings in the present study, and previously reported data, strongly suggest that beta-catenin mutation is an early event in endometrioid ovarian carcinogenesis, and that it is involved in the development of low-grade endometrioid tumours.

The categorization of intraductal proliferative lesions of the breast based on routine light microscopic examination of histopathologic sections is in many cases challenging, even for experienced pathologists. The development of computational tools to aid pathologists in the characterization of these lesions would have great diagnostic and clinical value. As a first step to address this issue, we evaluated the ability of computational image analysis to accurately classify DCIS and UDH and to stratify nuclear grade within DCIS. Using 116 breast biopsies diagnosed as DCIS or UDH from the Massachusetts General Hospital (MGH), we developed a computational method to extract 392 features corresponding to the mean and standard deviation in nuclear size and shape, intensity, and texture across 8 color channels. We used L1-regularized logistic regression to build classification models to discriminate DCIS from UDH. The top-performing model contained 22 active features and achieved an AUC of 0.95 in cross-validation on the MGH data-set. We applied this model to an external validation set of 51 breast biopsies diagnosed as DCIS or UDH from the Beth Israel Deaconess Medical Center, and the model achieved an AUC of 0.86. The top-performing model contained active features from all color-spaces and from the three classes of features (morphology, intensity, and texture), suggesting the value of each for prediction. We built models to stratify grade within DCIS and obtained strong performance for stratifying low nuclear grade vs. high nuclear grade DCIS (AUC = 0.98 in cross-validation) with only moderate performance for discriminating low nuclear grade vs. intermediate nuclear grade and intermediate nuclear grade vs. high nuclear grade DCIS (AUC = 0.83 and 0.69, respectively). These data show that computational pathology models can robustly discriminate benign from malignant intraductal proliferative lesions of the breast and may aid pathologists in the diagnosis and classification of these lesions.

ContextAssessment of accuracy and feasibility of whole slide imaging (WSI) for interinstitutional consultation in surgical pathology.ObjectivesTo train technical and pathologist staff in WSI technology, establish and evaluate a WSI workflow using training cases and second-opinion consultations, and assess diagnostic accuracy.DesignFirst, WSI training and evaluation using selected subspecialty service cases were performed and compared with the clinical glass slide (GS) diagnosis. Second, WSI and GS diagnoses of consecutive, second-opinion consultation cases were compared. Discrepancies underwent adjudication to determine a reference diagnosis. Participant observations on WSI initiation to practice were gathered.ResultsThere were 130 cases evaluated, with 123 correlations (94.6%) and 6 minor (4.6%) and 1 major (0.8%) discrepancies. The 74 consultation cases interpreted had 52 correlations (70.3%), and 18 minor (24.3%) and 4 major (5.4%) discrepancies. The WSI and GS adjusted major discrepancy rates in second-opinion consultations were 2.7% (2 of 74) and 4.1% (3 of 74), respectively. Statistical analysis showed that WSI was not inferior to GS interpretation. Pathologists agreed the software was easy to use and the images were adequate, but more time was spent rendering WSI interpretations.ConclusionsA significant learning curve was observed in the transition from the training set to clinical consultation cases associated both with WSI interpretation and adjustments to the digital analogs of routine GS workflow. Results from second-opinion consultations indicated that WSI interpretation was as accurate as GS interpretation among properly trained and experienced users. Overall, WSI-based practice appears feasible for second-opinion consultations.

A 58-year-old man was being evaluated for an abdominal aortic aneurysm when a pancreatic mass was incidentally found; in retrospect, he had experienced vague abdominal discomfort and weight loss for several months. Further evaluation disclosed a pancreatic adenocarcinoma that encased the superior mesenteric and portal veins. Treatment options are discussed.

A 58-year-old man with esophageal cancer had nausea, spontaneous vomiting, and intractable hiccups. The discussants review the role of palliative care in the management of distressing symptoms in patients with cancer, emphasizing the importance of the differential diagnosis in identifying the cause and selecting appropriate treatment for each symptom.

BTG2, a p53-inducible antiproliferative gene, is stimulated in breast cancer cells by activation of nuclear factor kappa B (NF-kappaB). In rat mammary glands, BTG2 is expressed in epithelial cells and levels decreased during pregnancy and lactation but recovered during involution. Estrogen and progestin suppress BTG2 expression, suggesting that these steroids, which stimulate proliferation and lobuloalveolar development of mammary epithelial cells, may downregulate BTG2 in the mammary gland during pregnancy. Consistent with the report that BTG2 inhibits cyclin D1 expression, suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and progestin, correlated with stimulation of cyclin D1. Ectopic expression of BTG2 inhibited breast cancer cell growth by arresting cells in the G1 phase, an effect reversed by cyclin D1. BTG2 expression was very low or undetectable in human breast cancer cell lines compared with nontumorigenic mammary epithelial cells, and nuclear expression of BTG2 was absent in 65% of human breast tumors compared with adjacent matched normal glands. Spontaneous mammary tumors arising in a mouse model with targeted expression of the early region of the SV40 large tumor Ag demonstrated loss of BTG2 protein very early during the tumorigenic process. Thus deregulation of BTG2 may be an important step in the development of mammary tumors.

Although fine-needle aspiration biopsy (FNAB) is a highly accurate tool for the diagnosis of pleomorphic adenomas, even this common salivary gland neoplasm can be diagnostically challenging and cause pitfalls in cytodiagnosis. In particular, the presence of either cystic degeneration or squamous and mucinous metaplasia can lead to a false positive diagnosis of malignancy. Here we present the case of a 16-year-old female who presented with a painless, slowly growing mass in the superficial lobe of the right parotid gland. Magnetic resonance imaging of the parotid demonstrated a mass with heterogeneous postcontrast enhancement and a central, nonenhancing area suggestive of necrosis. FNAB of the lesion yielded proteinaceous debris and numerous whorls of keratin, small cohesive clusters of basaloid and squamoid epithelial cells, and many vacuolated and foamy cells. Initially and after consultation at an outside institution, the FNAB was reported as an "atypical neoplasm, cannot exclude mucoepidermoid carcinoma." A total, nerve-sparing parotidectomy and level II neck dissection revealed a pleomorphic adenoma with central cystic degeneration and extensive mixed appendageal differentiation. While foci of squamous metaplastic changes may occur in pleomorphic adenomas, the combination of cystic degeneration and extensive appendageal differentiation with numerous keratotic cysts is rare, and it presents the potential for misinterpretation of the FNAB as indicative of malignancy in general, and mucoepidermoid carcinoma in particular.

Brain metastases represent a devastating progression of luminal breast cancer. While targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade PI3K inhibition despite efficient drug delivery. In comparison to extracranial disease, there is increased HER3 expression and phosphorylation in the brain lesions. HER3 blockade overcomes the resistance of both HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, leading to striking tumor growth delay and significant improvement of mouse survival. Collectively, these data provide a mechanistic basis underlying therapeutic resistance in the brain microenvironment and identify rapidly translatable treatment strategiesfor HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases. Citation Format: Gino B. Ferraro, David P. Kodack, Vasileios Askoxylakis, Qing Sheng, Mark Badeaux, Shom Goel, Xiaolong Qi, Ram Shankaraiah, Alexander Z. Cao, Rakesh R. Ramjiawan, Divya Bezwada, Bhushankumar Patel, Youngchul Song, Carlotta Costa, Kamila Naxerova, Christina Wong, Jonas Kloepper, Rita Das, Angela Tam, Jantima Tanboon, Dan G. Duda, Ryan C. Miller, Marni B. Siegel, Carey K. Anders, Melinda Sanders, Valeria M. Estrada, Robert Schlegel, Carlos L. Arteaga, Elena Brachtel, Alan Huang, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation [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 5008. doi:10.1158/1538-7445.AM2017-5008

TPS1126 Background: Stage 2-3 triple negative breast cancer (TNBC) remains at high risk for recurrence despite modern adjuvant therapy. An important role for the immune system in TNBC has recently emerged. Tumor infiltrating lymphocytes (TILs) are correlated with improved prognosis and several PD-1/PD-L1 checkpoint inhibitors, including Durvalumab (DUR), demonstrated activity in metastatic TNBC. Vaccines are a promising approach to further enhance the immune response in many cancers including TNBC. PVX-410 (PVX) is a novel tetra-peptide vaccine against XBP1 (2 splice variants), CD138 and CS1 that was safe and induced immune responses in a phase 1b study in smoldering myeloma. XBP1 and CD138 are also over-expressed in TNBC. Methods: This Phase 1b multi-center, single arm study will enroll 20 HLA-A2+ female patients (pts) following completion of all adjuvant therapy for stage 2-3 TNBC. Pts will receive 6 doses of 800ug PVX (emulsified in Montanide (SC) and co-administered with Hiltonol (IM)) at 2-week intervals, and 2 doses of DUR 1500mg IV at the 4th and 6th vaccine visits. Eligible pts must be between 1-6 months from completing adjuvant therapy, have no prior autoimmune disease, and have residual disease if neoadjuvant therapy was used. The primary objective is to determine the safety and tolerability of the combination, and the key secondary objective is to determine the immune response to PVX + DUR. If ≤1 pt in the first 6 has a protocol defined dose limiting toxicity within 4 weeks after the first DUR dose, accrual will continue to 20 pts. Immune response will be assessed at baseline, pre-dose 4 PVX/dose 1 DUR, and 4 weeks after completing protocol therapy. Paired data in 20 pts provides 90% power to see a shift of 0.75 standardized units from baseline to 4 weeks post treatment with the signed rank test. Immune response will be determined by a FACs based assay of antigen specific CD3+CD8+ T lymphocyte response and IFN-γ production (intracellular staining) in patient PBMCs. Additional correlative studies, including T-cell PD-1 and tissue PD-L1, XBP1, and CD138, are planned. Currently 4 pts are enrolled. Clinical trial information: NCT02826434.

This abstract was withdrawn by the authors.

Background: Triple negative breast cancer (TNBC) often presents as high grade, poorly differentiated tumors resulting in a more aggressive disease for which accurate and timely diagnosis is critical to treatment selection or clinical trial enrollment. Furthermore, the high rate of distant metastases and absence of breast-specific immunohistochemical markers that contribute to diagnostic uncertainty may delay or limit treatment modalities that can lead to poorer outcomes. The 92-gene assay is an RT-PCR-based cancer classifier that previously demonstrated 80% accuracy for the diagnosis of breast cancer. In this study, blinded validation of an optimized algorithm and assay specifically developed to improve performance in TNBC is described. Methods: To increase clinical scope for the diagnosis of TNBC, formalin fixed paraffin embedded specimens (N=103) representing a range of breast tumor histologies (e.g. TNBC, adenoid cystic, neuroendocrine, metaplastic, lobular, mucinous, DCIS) were added to the tumor reference database. A revised computational algorithm was constructed by the integration of machine learning techniques. For validation, tumor specimens (N=160) of TNBC (57%) and non-breast tumors (43%) were blindly tested using a 92-gene cancer classifier (CancerTYPE ID®, Biotheranostics, Inc). Tumor type predictions were reported as rank-order probabilities based on the degree of similarity to the tumor reference database. Assay sensitivity based on concordance of the main tumor type prediction with the reference diagnosis established by clinicopathologic review was analyzed. Results: Assay results included 85 breast carcinomas (TNBC) (53%), 23 Salivary gland carcinomas (14%), and 52 carcinomas (33%) representing 11 other tumor types. For performance in TNBC, the 92-gene assay demonstrated an overall sensitivity of 93% (CI, 86-98), and sensitivities of 96% [95% CI, 89-99] and 80% [95% CI, 52-96], in primary and metastatic tumors, respectively (P=0.085). Additional performance characteristics are shown in Table 1. Table 1Pathology subsetN, Validation setN, Correct 92-gene assay predictionsSensitivity (95% CI)All TNBC91850.93 (0.86-0.98)TNBC-primary76730.96 (0.89-0.99)TNBC-metastatic15120.80 (0.52-0.96)All Non-breast69550.80 (0.68-0.88)Salivary gland carcinoma25230.92 (0.74-0.99)Overall performance1601400.88 (0.81-0.92) Conclusions: An optimized 92-gene assay specifically modified to increase performance for the molecular diagnosis of TNBC showed strong accuracy in this blinded study. These findings support use of the 92-gene cancer classifier to aid in the diagnosis of primary or metastatic TNBC. With more refined tumor characterization, TNBC-specific chemotherapy regimens or clinical trial therapies may be pursued with the potential for improved patient outcomes. Citation Format: Sullivan PS, Soifer HS, Liu J, Zhang Y, Schnabel CA, Brachtel EF. An optimized 92-gene assay for the molecular diagnosis of triple-negative breast cancer [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 P5-03-01.

// Yangyang Wang 1,10 , Wende Li 2,10 , Shalin S. Patel 3,10 , Juan Cong 1,10 , Nan Zhang 1,10 , Francesco Sabbatino 1,10 , Xiaoyan Liu 1 , Yuan Qi 1 , Peigen Huang 2,10 , Hang Lee 4,10 , Alphonse Taghian 2,10 , Jian-Jian Li 5 , Albert B. DeLeo 6 , Soldano Ferrone 1,3,10 , Michael W. Epperly 7 , Cristina R. Ferrone 8,10 , Amy Ly 9,10 , Elena F. Brachtel 9,10 and Xinhui Wang 1,10 1 Division of Surgical Oncology, Department fo Surgery, Massachusetts General Hospital, Boston, MA 2 Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 3 Department of Orthopaedics, Massachusetts General Hospital, Boston, MA 4 Department of Biostatistics Center, Massachusetts General Hospital, Boston, MA 5 Department of Radiation Oncology, University of California, Davis, Sacramento, CA 6 Department of Pathology, University of Pittsburgh, Pittsburgh, PA 7 Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA 8 Division of General and Gastrointestinal Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA 9 Department of Pathology, Massachusetts General Hospital, Boston, MA 10 Harvard Medical School, Boston, MA Correspondence: Xinhui Wang, email: // Keywords : Breast Cancer, Radiation, Cancer Stem Cells, NF-kappaB, Stemness genes Received : February 4, 2014 Accepted : May 18, 2014 Published : May 20, 2014 Abstract The goal of adjuvant (post-surgery) radiation therapy (RT) for breast cancer (BC) is to eliminate residual cancer cells, leading to better local tumor control and thus improving patient survival. However, radioresistance increases the risk of tumor recurrence and negatively affects survival. Recent evidence shows that breast cancer stem cells (BCSCs) are radiation-resistant and that relatively differentiated BC cells can be reprogrammed into induced BCSCs (iBCSCs) via radiation-induced re-expression of the stemness genes. Here we show that in irradiation (IR)-treated mice bearing syngeneic mammary tumors, IR-induced stemness correlated with increased spontaneous lung metastasis (51.7%). However, IR-induced stemness was blocked by targeting the NF-κB- stemness gene pathway with disulfiram (DSF)and Copper (Cu 2+ ). DSF is an inhibitor of aldehyde dehydrogenase (ALDH) and an FDA-approved drug for treating alcoholism. DSF binds to Cu 2+ to form DSF-Cu complexes (DSF/Cu), which act as a potent apoptosis inducer and an effective proteasome inhibitor, which, in turn, inhibits NF-κB activation. Treatment of mice with RT and DSF significantly inhibited mammary primary tumor growth (79.4%) and spontaneous lung metastasis (89.6%) compared to vehicle treated mice. This anti-tumor efficacy was associated with decreased stem cell properties (or stemness) in tumors. We expect that these results will spark clinical investigation of RT and DSF as a novel combinatorial treatment for breast cancer.

We evaluated management of positive sub-areolar/nipple duct margins in nipple-sparing mastectomies (NSM) at our institution. Retrospective chart review of all NSM from January 2007 to April 2012 was performed and patient, tumor, and treatment information was collected. Sub-areolar/nipple duct margins included ductal tissue from within the nipple. Of 438 NSM, 22 (5%) had positive sub-areolar/nipple duct margins; 21 of 220 cancer-bearing breasts (10%) and 1 of 218 prophylactic mastectomies (0.5%). Positive margins included four with invasive lobular carcinoma and 18 with ductal carcinoma in situ (DCIS). Management included removal of eight nipples and nine nipple areola complexes (NAC). Four of 17 nipple/NAC specimens had evidence of residual DCIS and none had residual invasive cancer. The majority of nipple/NAC specimens excised for a positive margin had no residual malignancy. Future studies are needed to determine the extent of NAC tissue removal required for positive margins.

Intratumor genetic heterogeneity reflects the evolutionary history of a cancer and is thought to influence treatment outcomes. Here we report that a simple PCR-based assay interrogating somatic variation in hypermutable polyguanine (poly-G) repeats can provide a rapid and reliable assessment of mitotic history and clonal architecture in human cancer. We use poly-G repeat genotyping to study the evolution of colon carcinoma. In a cohort of 22 patients, we detect poly-G variants in 91% of tumors. Patient age is positively correlated with somatic mutation frequency, suggesting that some poly-G variants accumulate before the onset of carcinogenesis during normal division in colonic stem cells. Poorly differentiated tumors have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We generate poly-G mutation profiles of spatially separated samples from primary carcinomas and matched metastases to build well-supported phylogenetic trees that illuminate individual patients' path of metastatic progression. Our results show varying degrees of intratumor heterogeneity among patients. Finally, we show that poly-G mutations can be found in other cancers than colon carcinoma. Our approach can generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Context.—Breast cancer is increasingly treated with neoadjuvant chemotherapy to improve surgical resectability and evaluate tumor response, which is assessed histopathologically. Several histopathologic classification systems have been previously described for assessment of treatment response.Objectives.—To test performance in a side-by-side comparison of several histopathologic classification systems after neoadjuvant chemotherapy with clinical outcome.Design.—Sixty-two patients were enrolled in a randomized trial receiving sequential neoadjuvant chemotherapy with doxorubicin and paclitaxel. Histologic sections from the patients' tumors sampled before (core biopsy) and after treatment (excision or mastectomy) were reviewed. Histologic response was assessed following National Surgical Adjuvant Breast and Bowel Project protocol B18, Miller-Payne grading, Sataloff tumor and nodes, Residual Cancer Burden (RCB), and Residual Disease in Breast and Nodes (RDBN). Pathologic classification results were correlated with survival using Kaplan-Meier and Cox hazards regression with a median follow-up of 93 months.Results.—RDBN was associated with distant disease-free survival by univariate and multivariate analysis (P = .01 and .004, respectively), as were lymph node metastases (P = .02 and .01, respectively). Five patients (8%) had complete pathologic response after neoadjuvant chemotherapy, and none of them relapsed during the study period. Survival was shorter among patients with higher Residual Cancer Burden scores, but the associations were not significant. Miller-Payne grading and Sataloff tumor scores were not correlated with survival.Conclusions.—Evaluation of breast specimens after neoadjuvant chemotherapy by the composite index RDBN correlates with long-term outcome. The residual disease in breast and nodes system is suitable for routinely processed pathology cases. This study confirms the importance of lymph node status after neoadjuvant chemotherapy and favorable outcome in patients with pathologic complete response.

Microscopically clear lumpectomy margins are essential in breast conservation, as involved margins increase local recurrence. Currently, 18-50% of lumpectomies have close or positive margins that require re-excision. We assessed the ability of micro-computed tomography (micro-CT) to evaluate lumpectomy shaved cavity margins (SCM) intraoperatively to determine if this technology could rapidly identify margin involvement by tumor and reduce re-excision rates. Twenty-five SCM from six lumpectomies were evaluated with a Skyscan 1173 table top micro-CT scanner (Skyscan, Belgium). Micro-CT results were compared to histopathological results. We scanned three SCM at once with a 7-minute scanning protocol, and studied a total of 25 SCM from six lumpectomies. Images of the SCM were evaluated for radiographic signs of breast cancer including clustered microcalcifications and spiculated masses. SCM were negative by micro-CT in 19/25 (76%) and negative (≥2 mm) by histopathology in 19/25 (76%). Margin status by micro-CT was concordant with histopathology in 23/25 (92%). Micro-CT overestimated margin involvement in 1/25 and underestimated margin involvement in 1/25. Micro-CT had an 83.3% positive predictive value, a 94.7% negative predictive value, 83.3% sensitivity, and 94.7% specificity for evaluation of SCM. Evaluation of SCM by micro-CT is an accurate and promising method of intraoperative margin assessment in breast cancer patients. The scanning time required is short enough to permit real-time feedback to the operating surgeon, allowing immediate directed re-excision.

Intraoperative radiographic examination of breast specimens is commonly performed to confirm excision of image-detected breast lesions, but it is not reliable for assessing margin status. A more accurate method of intraoperative breast specimen imaging is needed. Micro-CT provides quantitative imaging parameters, image rotation, and virtual "slicing" of intact breast specimens. We explored the use of micro-CT for assessment of a variety of clinical breast specimens. Specimens were evaluated with a table top micro-CT scanner, Skyscan 1173 (Skyscan, Belgium), with a 40-130 kV, 8 W X-ray source. Skyscan software for 3D image analysis (Dataviewer and CTVox) was employed to review 3D graphics of specimens. Scanning for 7 min and another 7 min for image reconstruction provided the desired resolution for breast specimens. Breast lumpectomy specimens, shaved cavity margins, mastectomy specimens, and axillary lymph nodes were imaged by micro-CT. The micro-CT images could be rotated in all directions and cross sections of internal portions of specimens could be visualized from any angle. This provided information about spatial orientation of masses and calcifications relative to margins in intact lumpectomy specimens. Micro-CT is a potentially useful tool for assessment of breast cancer specimens, allowing real-time analysis of tumor location in breast lumpectomy specimens or shaved cavity margins. Micro-CT may also be useful for assessing sentinel lymph nodes and mastectomy specimens.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.