Triple Negative Breast Cancer Lesions Research Articles

The potential role of breast MRI in evaluation of triple-negative breast cancer and fibroadenoma of less than 3 cm.

The majority of small-sized (<3 cm) triple-negative breast cancer (TNBC) exhibit smooth margins upon palpation and are often oval or rounded masses. Distinguishing these masses preoperatively from fibroadenomas (FAs) would be very meaningful for clinical practice. The aim of our study was to evaluate the magnetic resonance imaging (MRI) appearance of TNBC and differentiate it from FAs. In this retrospective single-center study, we included 37 patients with TNBCs and 36 patients with FAs who underwent breast MRI. We employed the χ2 test and t-test to compare the differences in morphological features, dynamic contrast-enhanced MRI (DCE-MRI) parameters, and apparent diffusion coefficient (ADC) values between the two groups. Additionally, we constructed non-parametric receiver operating characteristic (ROC) curves using ADC values, with pathological results serving as the gold standard. A total of 37 TNBC lesions and 39 FA lesions were included in the final analysis. TNBCs exhibited more frequent irregular shape, irregular margins, peritumoral edema, fast enhancement in the initial phase, rim enhancement, and time-signal intensity curve (TIC) type III compared to FAs (all P<0.05). Conversely, low-signal segregation in T2-weighted imaging (T2WI) and TIC type I were commonly found in FAs. The mean ADC value of TNBCs was significantly lower than that of FAs [(1.104±0.13)×10-3 vs. (1.613±0.16)×10-3 mm2/s, P<0.05]. The cutoff ADC for differentiating TNBCs from FAs was 1.239×10-3 mm2/s, yielding an area under the curve (AUC) of 0.997, a sensitivity of 94.6%, and a specificity of 100%. The morphological presentation of MRI, internal enhancement features of the mass, TIC curves, and ADC values provide valuable differential diagnostic information for TNBC and FA masses with a maximum diameter of less than 3 cm.

RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer

Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat due to the lack of targeted therapies. Cancer stem cells (CSCs) are strongly enriched in TNBC lesions and are responsible for the rapid development of chemotherapy resistance and metastasis. Ubiquitin-based epigenetic circuits are heavily exploited by CSCs to regulate gene transcription and ultimately sustain their aggressive behavior. Therefore, therapeutic targeting of these ubiquitin-driven dependencies may reprogram the transcription of CSC and render them more sensitive to standard therapies. In this work, we identified the Ring Finger Protein 40 (RNF40) monoubiquitinating histone 2B at lysine 120 (H2Bub1) as an indispensable E3 ligase for sustaining the stem-cell-like features of the growing mammary gland. In addition, we found that the RNF40/H2Bub1-axis promotes the CSC properties and drug-tolerant state by supporting the glycolytic program and promoting pro-tumorigenic YAP1-signaling in TNBC. Collectively, this study unveils a novel tumor-supportive role of RNF40 and underpins its high therapeutic value to combat the malignant behavior of TNBC.

Effect of microRNA-21 blockade in human triple negative breast cancer allografts on progression and immune checkpoint proteins.

e13097 Background: Triple negative breast cancer (TNBC) is an orphan disease that attacks &gt; 46,000 US women every year. TNBC recurs after standard-of-care surgery, chemotherapy and radiation, killing half its victims within 4 years. Poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates extend the survival of a fraction of TNBC patients for a few months. Thus, TNBC shows a critical need for molecularly-targeted therapy. Most TNBC cells show high microRNA 21 (miR-21), identical in humans and mice, which decreases tumor suppressor proteins that keep cell growth in check. Hypothesis: Short anti-miR-21 RNA analogs conjugated to a receptor ligand will direct TNBC cell uptake and slow the growth of TNBC orthotopic allografts with immune activation and minimal toxicity. Methods: We designed an anti-miR-21 RNA analog using aminomethyl bridged nucleic acid (BNA), exhibiting low toxicity, and serum stability. We lipofected a concentration ramp of the anti-miR-21 RNA analog into human TNBC cell lines (MDA-MB-231, MDA-MB-157, MDA-MB-436, MDA-MB-468, BT-20, BT-549, HCC1806, HCC1937, HCC1806) representing a spectrum of TNBC subtypes, then measured proliferation and biochemical markers. We also conjugated a peptide analog for endocytosis by the insulin-like growth factor 1 receptor (IGF1R), overexpressed in most TNBC lesions. The peptide ligand for IGF1R provides a unique strategy for delivering the anti-miR-21 RNA analog preferentially into TNBC cells. We administered 5 mg/kg of the anti-miR-21 RNA-peptide analog in sterile saline intraperitoneally twice a week for 14 days into immunocompetent female Balb/c mice bearing syngeneic EMT6 TNBC tumors in their mammary fat pads. Results: The anti-miR-21 RNA analog slowed proliferation, increased apoptosis, elevated tumor suppressor proteins, and suppressed immune checkpoint gene expression in multiple human TNBC lines. IC50 for inhibiting cell proliferation correlated with miR-21 copies/cell in 7 TNBC cell lines, but not in the non-tumorigenic breast epithelial cell line MCF-10A. The anti-miR-21 RNA-peptide analog stopped orthotopic TNBC allograft growth in syngeneic immunocompetent female Balb/c mice during 14 days of therapy, with apparent safety. Conclusions: This strategy optimizes cancer cell-specific delivery to block proliferation and immune checkpoints. The results support further preclinical proof-of-concept studies to enable an eIND submission for the anti-miR-21 RNA-peptide analog.

Expression of the Immunohistochemical Markers CK5, CD117, and EGFR in Molecular Subtypes of Breast Cancer Correlated with Prognosis.

Molecular-based subclassifications of breast cancer are important for identifying treatment options and stratifying the prognosis in breast cancer. This study aimed to assess the prognosis relative to disease-free survival (DFS) and overall survival (OS) in patients with triple-negative breast cancer (TNBC) and other subtypes, using a biomarker panel including cytokeratin 5 (CK5), cluster of differentiation 117 (CD117), and epidermal growth factor receptor (EGFR). This cohort-case study included histologically confirmed breast carcinomas as cohort arm. From a total of 894 patients, 572 patients with early breast cancer, sufficient clinical data, and archived tumor tissue were included. Using the immunohistochemical markers CK5, CD117, and EGFR, two subgroups were formed: one with all three biomarkers negative (TBN) and one with at least one of those three biomarkers positive (non-TBN). There were significant differences between the two biomarker subgroups (TBN versus non-TBN) in TNBC for DFS (p = 0.04) and OS (p = 0.02), with higher survival rates (DFS and OS) in the non-TBN subgroup. In this study, we found the non-TBN subgroup of TNBC lesions with at least one positive biomarker of CK5, CD117, and/or EGFR, to be associated with longer DFS and OS.

Calcification, Posterior Acoustic, and Blood Flow: Ultrasonic Characteristics of Triple-Negative Breast Cancer.

Previous studies suggest that triple-negative breast cancer (TNBC) may have unique imaging characteristics, however, studies focused on the imaging characteristics of TNBC are still limited. The aim of the present study is to analyze the ultrasonic characteristics of TNBC and to provide more reliable information on imaging diagnosis of TNBC. This retrospective study was performed including 162 TNBC patients with 184 TNBC lesions. 174 non-TNBC cases with 196 lesions were used as the control group. The median size of TNBC lesions and non-TNBC lesions were 23 mm × 16 mm and 21 mm × 15 mm, respectively. The shape of most breast cancer lesions was irregular. However, 15.30% (28/183) TNBC lesions and 16.84% (33/196) non-TNBC lesions were oval-shaped. Most breast cancer lesions (79.78% TNBC & 85.71% non-TNBC) were ill-defined. In comparison to non-TNBC, the distinctive ultrasonic characteristics of TNBC were summarized as three features: calcifications, posterior acoustic, and blood flow. Microcalcifications was less common in non-TNBC. The remarkable posterior acoustic characteristics on TNBC were no posterior acoustic features (136, 73.91%). Avascular pattern (21.74%) was also more common in TNBC. The other feature of TNBC was markedly hypoechoic lesions (23.91%). The above-mentioned differences between TNBC and non-TNBC were significant. 93.48% TBNC and 94.39% non–TNBC lesions were in BI-RADS-US category of 4A-5. The results indicate that TNBC has some distinctive ultrasound characteristics. Ultrasound is a useful adjunct in early detection of breast cancer. A combination of ultrasound with mammography is excellent for detecting breast cancer.

Folate receptor alpha expression associates with improved disease-free survival in triple negative breast cancer patients

Triple negative breast cancer (TNBC) comprises 15–20% of all invasive breast cancer and is associated with a poor prognosis. As therapy options are limited for this subtype, there is a significant need to identify new targeted approaches for TNBC patient management. The expression of the folate receptor alpha (FRα) is significantly increased in patients with TNBC and is therefore a potential biomarker and therapeutic target. We optimized and validated a FRα immunohistochemistry method, specific to TNBC, to measure FRα expression in a centrally confirmed cohort of 384 patients with TNBC in order to determine if expression of the protein is associated with invasive disease-free survival (IDFS) and overall survival (OS). The FRα IHC demonstrated exceptional performance characteristics with low intra- and interassay variability as well as minimal lot-to-lot variation. FRα expression, which varied widely from sample to sample, was detected in 274 (71%) of the TNBC lesions. In a multivariable model adjusted for baseline characteristics, FRα expression was associated with improved IDFS (HR = 0.63, p = 0.01) but not with OS. The results demonstrate the potential of targeting the FRα in the majority of TNBC patients and suggest that variable expression may point to a need to stratify on FRα expression in clinical studies.

Triple-negative breast cancer: Pretreatment magnetic resonance imaging features and clinicopathological factors associated with recurrence

PurposeWe aimed to investigate the magnetic resonance imaging (MRI) features and clinicopathologic factors with recurrence of triple-negative breast cancer (TNBC). Patients and methodsWe identified 281 patients with 288 surgically confirmed TNBC lesions who underwent pretreatment MRI between 2009 and 2015. The presence of intratumoral high signal on T2-weighted images, high-signal rim on diffusion-weighted images (DWI), and rim enhancement on the dynamic contrast-enhanced MRI and clinicopathological data were collected. Cox proportional analysis was performed. ResultsOf the 288 lesions, 36 (12.5%) recurred after a median follow-up of 18 months (range, 3.6–68.3 months). Rim enhancement (hazard ratio [HR] = 3.15; 95% confidence interval [CI] = 1.01, 9.88; p = .048), and lymphovascular invasion (HR = 2.73, 95% CI = 1.20, 6.23; p = .016) were independently associated with disease recurrence. While fibroglandular volume, background parenchymal enhancement, intratumoral T2 high signal, and high-signal rim on DWI, were not found to be risk factors for recurrence. ConclusionPretreatment MRI features may help predict a high risk of recurrence in patients with TNBC.

Applied value of routine ultrasound and shear wave elastography in the diagnosis of triple negative breast cancer

Objective To investigate the applied value of routine ultrasound and shear wave elastography (SWE) in the comparison between triple negative breast cancer (TNBC) and non-triple negative breast cancer (non-TNBC). Methods A total of 120 lesions in 120 patients with pathologically confirmed breast cancer were retrospectively reviewed, which were examined by routine ultrasound and SWE before surgery. According to immunohistochemical analysis, those were divided into TNBC group and non-TNBC group. The features of routine ultrasound(including shape, orientation, margin, boundary, echo pattern, microcalcification, posterior features of each lesion and blood flow characteristics) and the SWE index[including the average value of the lesion stiffness(Emean), the maximum value (Emax), the minimum value (Emin), the standard deviation (SD), and the ratio of the normal breast gland tissue (Eratio)] in the two groups were analyzed and compared. Results ①Two-dimensional ultrasound: TNBC lesions mostly showed the margin of microlobulated, abrupt boundaries and no internal microcalcification, but non-TNBC lesions were more likely to have an angular or spiculated margin, an echogenic halo of boundary and a few microcalcification in the interior, which were statistically different(P 0.05). ②The values of Emax, Emean and Eratio were statistically different(P<0.05), with AUCs of 0.811, 0.781, and 0.770, respectively. Conclusions Routine ultrasound and SWE can comprehensively analyze the morphology, blood flow and stiffness features of breast lesions, which provides more valid information to identify TNBC and non-TNBC. Key words: Ultrasonography; Shear wave elastography; Triple negative breast cancer

Dimeric Prodrug Self-Delivery Nanoparticles with Enhanced Drug Loading and Bioreduction Responsiveness for Targeted Cancer Therapy.

Efficient drug accumulation in tumor cells is essential for cancer therapy. Herein, we developed dimeric prodrug self-delivery nanoparticles (NPs) with enhanced drug loading and bioreduction responsiveness for triple negative breast cancer (TNBC) therapy. Specially designed camptothecin dimeric prodrug (CPTD) containing a disulfide bond was constructed to realize intracellular redox potential controlled drug release. Direct conjugation of hydrophobic CPTD to poly(ethylene glycol) PEG5000, a prodrug-based amphiphilic CPTD-PEG5000 co-polymer was synthesized, which could encapsulate parental CPTD prodrug spontaneously and form ultrastable NPs due to the highly analogous structure. Such dimeric prodrug self-delivery nanoparticles showed ultrahigh stability with critical micelle concentration as low as 0.75 μg/mL and remained intact during endocytosis. In addition, neurotensin (NT), a 13 amino acid ligand, was further modified on the nanoparticles for triple negative breast cancer (TNBC) targeting. Optimized NT-CPTD NPs showed improved pharmacokinetics profile and increased drug accumulation in TNBC lesions than free CPT, which largely reduced the systemic toxicity and presented an improved anticancer efficacy in vivo. In summary, with advantages of extremely high drug loading capacity, tumor microenvironmental redox responsiveness, and targeted TNBC accumulation, NT-CPTD NPs showed their potential for effective triple negative breast cancer therapy.

18F]FDG and [18F]FLT PET for the evaluation of response to neo-adjuvant chemotherapy in a model of triple negative breast cancer.

RationalePathological response to neo-adjuvant chemotherapy (NAC) represents a commonly used predictor of survival in triple negative breast cancer (TNBC) and the need to identify markers that predict response to NAC is constantly increasing. Aim of this study was to evaluate the potential usefulness of PET imaging with [18F]FDG and [18F]FLT for the discrimination of TNBC responders to Paclitaxel (PTX) therapy compared to the response assessed by an adapted Response Evaluation Criteria In Solid Tumors (RECIST) criteria based on tumor volume (Tumor Volume Response).MethodsNu/nu mice bearing TNBC lesions of different size were evaluated with [18F]FDG and [18F]FLT PET before and after PTX treatment. SUVmax, Metabolic Tumor Volume (MTV) and Total Lesion Glycolysis (TLG) and Proliferation (TLP) were assessed using a graph-based random walk algorithm.ResultsWe found that in our TNBC model the variation of [18F]FDG and [18F]FLT SUVmax similarly defined tumor response to therapy and that SUVmax variation represented the most accurate parameter. Response evaluation using Tumor Volume Response (TVR) showed that the effectiveness of NAC with PTX was completely independent from lesions size at baseline.ConclusionsOur study provided interesting results in terms of sensitivity and specificity of PET in TNBC, revealing the similar performances of [18F]FDG and [18F]FLT in the identification of responders to Paclitaxel.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

Assessing treatment response in triple-negative breast cancer from quantitative image analysis in perfusion magnetic resonance imaging.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is sensitive but not specific to determining treatment response in early stage triple-negative breast cancer (TNBC) patients. We propose an efficient computerized technique for assessing treatment response, specifically the residual tumor (RT) status and pathological complete response (pCR), in response to neoadjuvant chemotherapy. The proposed approach is based on Riesz wavelet analysis of pharmacokinetic maps derived from noninvasive DCE-MRI scans, obtained before and after treatment. We compared the performance of Riesz features with the traditional gray level co-occurrence matrices and a comprehensive characterization of the lesion that includes a wide range of quantitative features (e.g., shape and boundary). We investigated a set of predictive models ([Formula: see text]) incorporating distinct combinations of quantitative characterizations and statistical models at different time points of the treatment and some area under the receiver operating characteristic curve (AUC) values we reported are above 0.8. The most efficient models are based on first-order statistics and Riesz wavelets, which predicted RT with an AUC value of 0.85 and pCR with an AUC value of 0.83, improving results reported in a previous study by [Formula: see text]. Our findings suggest that Riesz texture analysis of TNBC lesions can be considered a potential framework for optimizing TNBC patient care.

The role of BRCA1-IRIS in the development and progression of triple negative breast cancers in Egypt: possible link to disease early lesion

BackgroundBreast cancer is the most globally diagnosed female cancer, with the triple negative breast cancer (TNBC) being the most aggressive subtype of the disease. In this study we aimed at comparing the effect of BRCA1-IRIS overexpression on the clinico-pathological characteristics in breast cancer patients with TNBC or non-TNBC in the largest comprehensive cancer center in Egypt.MethodsTo reach this goal, we conducted an observational study at the National Cancer Institute (NCI), Cairo University (Cairo, Egypt). The data on all diagnosed breast cancer patients, between 2009 and 2012, were reviewed. BRCA1-IRIS expression measured using real time RT/PCR in these patients’ tumor samples was correlated to tumor characteristics, such as to clinico-pathological features, therapeutic responses, and survival outcomes.Results96 patients were enrolled and of these 45% were TNBC, and 55% were of other subtypes (hereafter, non-TNBC). All patients presented with invasive ductal carcinomas. No significant difference was observed for risk factors, such as age and menopausal status between the TNBC and the non-TNBC groups except after BRCA1-IRIS expression was factored in. The majority of the tumors in both groups were ≤5 cm at surgery (p = 0.013). However, in the TNBC group, ≤5 cm tumors were BRCA1-IRIS-overexpressing, whereas in the non-TNBC group they were BRCA1-IRIS-negative (p = 0.00007). Most of the TNBC patients diagnosed with grade 1 or 2 were BRCA1-IRIS-overexpressing, whereas non-TNBCs were IRIS-negative (p = 0.00035). No statistical significance was measured in patients diagnosed with grade 3 tumors. Statistically significant difference between TNBCs and non-TNBCs and tumor stage with regard to BRCA1-IRIS-overexpression was observed. Presence of axillary lymph node metastases was positively associated with BRCA1-IRIS overexpression in TNBC group, and with BRCA1-IRIS-negative status in the non-TNBC group (p = 0.00009). Relapse after chemotherapy (p < 0.00001), and local recurrence/distant metastasis after surgery (p = 0.0028) were more pronounced in TNBC patients with BRCA1-IRIS-overexpressing tumors compared to non-TNBC patients. Finally, decreased disease-free survival in TNBC/BRCA1-IRIS-overexpressing patients compared to TNBC/BRCA1-IRIS-negative patients, and decreased overall survival in TNBC as well as non-TNBC patients was driven by BRCA1-IRIS overexpression.ConclusionsTNBC/BRCA1-IRIS-overexpressing tumors are more aggressive than TNBC/BRCA1-IRIS-negative or non-TNBC/BRCA1-IRIS-overexpressing or both negative tumors. Further studies are warranted to define whether BRCA1-IRIS drives the early TNBC lesions growth and dissemination and whether it could be used as a diagnostic biomarker and/or therapeutic target for these lesions at an early stage setting.

Brachyury, a vaccine target, is overexpressed in triple-negative breast cancer.

Patients diagnosed with triple-negative breast cancer (TNBC) have a high rate of tumor metastasis and a poor prognosis. The treatment option for these patients is currently chemotherapy, which results in very low response rates. Strategies that exploit the immune system for the treatment of cancer have now shown the ability to improve survival in several tumor types. Identifying potential targets for immune therapeutic interventions is an important step in developing novel treatments for TNBC. In this study, in silico analysis of publicly available datasets and immunohistochemical analysis of primary and metastatic tumor biopsies from TNBC patients were conducted to evaluate the expression of the transcription factor brachyury, which is a driver of tumor metastasis and resistance and a target for cancer vaccine approaches. Analysis of breast cancer datasets demonstrated a predominant expression of brachyury mRNA in TNBC and in basal vs luminal or HER2 molecular breast cancer subtypes. At the protein level, variable levels of brachyury expression were detected both in primary and metastatic TNBC lesions. A strong association was observed between nuclear brachyury protein expression and the stage of disease, with nuclear brachyury being more predominant in metastatic vs primary tumors. Survival analysis also demonstrated an association between high levels of brachyury in the primary tumor and poor prognosis. Two brachyury-targeting cancer vaccines are currently undergoing clinical evaluation; the data presented here provide rationale for using brachyury-targeting immunotherapy approaches for the treatment of TNBC.

Association between semiquantitative PET parameters and molecular subtypes of breast invasive ductal carcinoma.

Molecular subtypes of breast cancer have been proposed since 2012. The correlation between various baseline [18F]fluorodeoxyglucose ([18F]FDG) uptake parameters, including total lesion glycolysis (TLG), and molecular subtypes of primary breast cancer lesions in patients with invasive ductal cancer will be investigated. Staging [18F]FDG PET/CT for breast invasive ductal carcinoma were retrospectively evaluated. Breast lesions were examined for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and proliferation index (Ki-67). Breast tumors were classified into five molecular subtypes: Luminal A, Luminal B-HER2(-), Luminal B-HER2(+), HER2(+) and Basal or Triple Negative cancers. The correlations between tumor characteristics and PET semiquantitative data of primary breast lesion (SUVmean, SUVmax, Mean tumor volume (MTV), TLG) were assessed. Specific Breast Uptake Ratio (SBUR) is used as a new quantification method of breast uptake to correct for physiological background activity. Fifty-eight patients were included. TLG was significantly higher in triple negative group when compared with luminal A (P<0.01). Significantly higher uptake was found in triple negative lesions when compared with luminal B-HER2(-) and luminal B-HER2(+) categories using SUVmax, SUVmean and TLG (all P<0.05). Conversely, no statistically significant difference for [18F]FDG uptake was observed between all other molecular subtypes. No value of SBUR in terms of correlation with histopathological parameters was demonstrated. TLG was superior to SUVmax and SUVmean in differentiating between triple negative breast cancer lesions and all other molecular subtypes. SBUR was not different statistically between various molecular subtypes.

Abstract P2-03-04: Comparative analysis of somatic mutations in matched primary vs metastatic triple-negative breast cancers

Abstract Background. Treatment of triple-negative breast cancer (TNBC) is clinically challenging due to disease heterogeneity and aggressiveness which often result in high recurrence and mortality rates. We have previously reported on genomic alterations found in 14 metastatic TNBC tumors (Craig et al 2012) uncovered through whole-genome and transcriptome sequencing and found that majority of tumor changes in our cohort converged on RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways revealing potential therapeutic targets. In this study, we interrogated patients’ matched primary and metastatic cancers using exome sequencing. Differentiating between primary and metastasis-specific genomic alterations will further our understanding of TNBC recurrence. Methods. Tumor tissue was microdissected from 10uM FFPE sections from primary tumors of nine TNBC patients. DNA was extracted using Qiagen AllPrep kit. Whole-genome libraries were prepared from &amp;gt;250ng of DNA using Kapa-on–Bead method. Exomes were captured using All Exon V5 capture kit (Agilent Technologies) and sequenced on Illumina HiSeq. List of somatic variants (SNVs) in primary tissue was generated using Seurat variant caller and manually compared with the SNVs uncovered previously in metastatic tumors. Presence (or absence) of all reported somatic SNVs was visually confirmed in IGV genome viewer. Results. Our analysis revealed the majority of somatic mutations found in metastatic lesions were also present in the primary tissue (64.3% on average). Patient TNBC03 had an unusually high number of new mutations in the lung metastatic lesion (72.7%). All TP53 mutations in our cohort were an early event present in both primary and metastatic tissues. In patient TNBC01, acquired mutation in NF1 - an upstream negative regulator of RAS pathway - in addition to pre-existing PTEN deletion, CTNNA1 underexpression and RB1 mutation - suggests RAS pathway addiction which could have been responsible for disease recurrence in this patient. Interestingly, NEDD4 mutation was found in a lung metastasis of another patient whose disease was refractory to BEZ235 (PI3K/mTOR inhibitor) treatment. NEDD4 has recently been shown to be an upstream regulator of the PI3K pathway and a possible therapeutic target in cancers with downregulated PTEN. Additional select acquired mutations in metastatic lesions of our cohort included FGF14, ABCB10, MDM2, KIF1C, ATAD2B, PTPLAD2, MDM20, TDRD1, and USP6. Mutated CDH5 was found in metastases in 2 of 9 patients. Conclusion. Our findings demonstrate the complexity and variability in somatic events underlying TNBC recurrence, reinforcing the need to re-biopsy metastatic TNBC lesions and to employ combination targeted therapies wherever possible. Citation Format: Irene Cherni, Maren K Levin, Joyce A O"Shaughnessy, John D Carpten. Comparative analysis of somatic mutations in matched primary vs metastatic triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-04.

173P - Tle3 is a Useful Marker for Predicting the Therapeutic Effect of Eribulin Chemotherapy for Triple-Negative Breast Cancer

ABSTRACT Aim: The recently developed reagent, eribulin mesylate (eribulin), is a microtubule dynamics inhibitor with a mechanism of action that differs from that of taxanes and vinca alkaloids. This drug is considered to be a promising chemotherapeutic agent for the treatment of locally advanced or metastatic breast cancer (MBC). In this study, we investigated predictive factors with respect to the therapeutic effect of eribulin chemotherapy among variables such as b-tubulin class III, GSTP1 and TLE3, which have previously been reported to be predictive factors of the therapeutic effect of taxanes, in an aim to identify possible biomarkers for predicting the efficacy of eribulin. Methods: The subjects included 52 patients with MBC who underwent chemotherapy using eribulin. The median follow-up time was 431 days (range, 50-650 days). The overall response rate (ORR), clinical benefit rate (CBR), disease control rate (DCR), overall survival (OS), time to treatment failure (TTF) and progression-free survival (PFS) were calculated regarding the efficacy of this regimen. Moreover, breast cancer was classified into intrinsic subtypes according to the status of the ER, PR, HER2 and Ki67 expression. Results: The clinical effects were as follows: overall ORR= 34.6%, CBR = 44.2%, DCR= 51.9%; median OS = 334 days; median TTF = 81 days; and median PFS = 275 days. TLE3, b-tubulin class III and GSTP1 were expressed in 24 cases, 21 cases and 24 cases, respectively, among the 52 patients investigated. The expression of TLE3 was found significantly more frequently in the TNBC lesions than in the non-TNBC lesions (p = 0.030). The patients with TLE3-negative tumors experienced significantly poorer outcomes in terms of progression-free survival when comparing the prognosis within the group of patients with triple-negative breast cancer (TNBC) lesions (p = 0.011). Based on a multivariate logistic regression analysis including 22 patients with TNBC also showed that a positive TLE3 expression significantly correlated with a better progression-free survival (p = 0.037). Conclusions: Our findings suggest that TLE3 is a useful marker for predicting the therapeutic effect of eribulin chemotherapy for TNBC. Disclosure: All authors have declared no conflicts of interest.

Imaging of triple-negative breast cancer

Although triple-negative breast cancer (TNBC) has been studied extensively in the oncology and pathology literature, there are few reports on imaging features. Emerging data suggest that imaging features of TNBC are substantially different from other primary breast cancer immunotypes. In this work, we reviewed multi-modality imaging features of primary TNBC with emphasis on the appropriate niche for each technology in diagnosis, staging and management. TNBC lacks the typical suspicious mammographic features of breast cancer; namely irregular mass shape, spiculated margins and associated suspicious calcifications. Therefore, mammography alone is usually a sub-optimal tool for its initial diagnostic evaluation. Ultrasound has a much higher sensitivity, although its diagnostic capability may be impaired by associated benign features encountered in 21%-41% of TNBC lesions. Magnetic resonance imaging (MRI) consistently demonstrates the presence of all TNBC with a higher level of accuracy compared with other tumour sub-types, and provides a reliable baseline for neoadjuvant chemotherapy (NAC) follow-up. Preliminary studies also suggest that MRI may predict complete NAC response in TNBC more sensitively than other methods. [(18)F]2-fluoro-2-deoxy-D-glucose positron emission tomography ((18)F-FDG-PET) has a higher sensitivity for TNBC than estrogen-positive and human epidermal growth factor receptor 2-positive tumours, and axillary lymph node metastases are detected with more accuracy compared with other tumour molecular sub-types, although low FDG uptake of breast cancers in general may limit the routine clinical use of (18)F-FDG-PET in the initial diagnosis of TNBC patients and NAC follow-up.

CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer

The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb. CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided. CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis. This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

Abstract A36: ImmunoSPECT and ImmunoPET imaging of IGF-1R expression in a model for triple negative breast cancer

Abstract Introduction: Triple negative breast cancer has a poor prognosis and chemotherapy is currently the mainstay of treatment. A potential new target for the treatment of triple negative breast cancer is the insulin-like growth factor 1 receptor (IGF-1R). Patients with triple negative breast cancer lesions that express the IGF-1R (30 to 40%) may be most suited to be treated with IGF-1R antibodies. Therefore, the aim of the present study was to develop a noninvasive, in vivo imaging method, using radiolabeled R1507, to visualize IGF-1R expression. Material and methods: R1507, a monoclonal antibody directed against an epitope on the extracellular domain of the IGF-1R, was radiolabeled with 111Indium (111In), 125Iodine (125I) and 89Zirconium (89Zr). In vitro, the affinity and internalization kinetics of 111In-R1507 were determined using the IGF-1R expressing breast cancer cell line SUM149 (ER, PR and HER2 negative). In vivo studies were performed in BALB/c nude mice with subcutaneous SUM149 xenografts. To determine the pharmacodynamics of R1507, mice received an intravenous injection of 111In-R1507 and 125I-R1507. One, three and seven days post injection, mice were killed and ex vivo tumor uptake was measured. Non-specific uptake was determined by coinjection of an excess unlabeled R1507. A dose escalation study was performed with 111In-R1507 to determine the optimal protein dose of R1507 for in vivo imaging (dose range 1 – 1000 μg). Finally, SPECT and PET images were acquired of mice with subcutaneous SUM149 tumors one, three and seven days post injection of 111In-R1507 and 89Zr-R1507, respectively. Results: 111In-R1507 (IC50 = 0.1 nM) was slowly internalized by SUM149 cells. 111In-R1507 specifically and efficiently accumulated in the SUM149 xenografts: the specific tumor uptake was 20% ID/g, 33% ID/g and 31% ID/g at one, three and seven days post injection, respectively. The dose escalation study showed that the optimal dose for imaging experiments was ≤ 3 μg (tumor uptake: 35% ID/g). MicroSPECT and microPET imaging of mice clearly visualized the subcutaneous SUM149 xenograft with increasing contrast at later time points. Conclusion: 111In-R1507 SPECT and 89Zr-R1507 PET are excellent methods to visualize IGF-1R expression in vivo in triple negative breast cancer xenografts. In the future, these techniques may enable selection of patients with triple negative breast cancer for IGF-1R targeted therapy. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A36