PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim.

BACKGROUND: Protein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim. METHODS: To determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed. RESULTS: Enhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation. CONCLUSIONS: These studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

Evaluation of surgically excised breast tissue microstructure using wide-field optical coherence tomography.

BACKGROUND: Currently, positive margins at lumpectomy contribute to health care cost, patient anxiety, and treatment delay. Multiple technology solutions are being explored with the aim of lowering re-excision rates for breast-conserving surgery (BCS). We examined wide-field optical coherence tomography (WF-OCT), an innovative adjunct intraoperative imaging tool for tissue visualization of margins. METHODS: This IRB-approved pilot study included women with invasive or in situ carcinoma scheduled for primary BCS. Lumpectomy specimens and any final/revised margins were imaged by optical coherence tomography immediately prior to standard histological processing. The optical coherence tomography used provided two-dimensional, cross-sectional, real-time depth visualization of the margin widths around excised specimens. A volume of images was captured for 10 × 10 cm tissue surface at high resolution (sub-30 µm) to a depth of 2 mm. Integrated interpretation was performed incorporating final pathology linked with the optical image data for correlation. RESULTS: Wide-field optical coherence tomography was performed on 185 tissue samples (50 lumpectomy specimens and 135 additional margin shaves) in 50 subjects. Initial diagnosis was invasive ductal carcinoma (IDC) in 10, ductal carcinoma in situ (DCIS) in 14, IDC/DCIS in 22, invasive lobular carcinoma (ILC) in 2, ILC/DCIS in 1, and sarcoma in 1. Optical coherence tomography was concordant with final pathology in 178/185 tissue samples for overall accuracy of 86% and 96.2% (main specimen alone and main specimen + shave margins). Of seven samples that were discordant, 57% (4/7) were considered close (DCIS < 2 mm from margin) per final pathology. CONCLUSION: Wide-field optical coherence tomography demonstrated concordance with histology at tissue margins, supporting its potential for use as a real-time adjunct intraoperative imaging tool for margin assessment. Further studies are needed for comprehensive evaluation in the intraoperative setting.

Close and Positive Lumpectomy Margins are Associated with Similar Rates of Residual Disease with Additional Surgery.

BACKGROUND: Current guidelines state that "no ink on tumor" constitutes adequate surgical margins for lumpectomy specimens. However, there remains uncertainty when tumor is close (<1 mm) to multiple inked margins. METHODS: All local excisions for invasive breast cancer during 3 years at one center were reviewed. Tumor characteristics, margin status, patient age, reoperations, and pathology of reexcised specimen were recorded. Chi-square analysis and regression models were used to identify factors associated with residual disease upon reoperation. RESULTS: In 533 lumpectomies for invasive cancer, 60 (11 %) had at least one positive margin, and 106 (20 %) had one or more close margin. Multiple margins were either close or positive in 67 cases. Reoperation was performed in 125 of 533 cases (23 %) for close or positive margins. Positive margins were significantly more likely to undergo reoperation compared with close margins (p < 0.001). On reoperation, 73 of 125 (58 %) demonstrated residual cancer, including 39 of 68 (57 %) with close margins, and 34 of 57 (60 %) with positive margins (p = 0.52). When multiple margins were close or positive, residual cancer was found on reexcision in 45 of 59 (76 %) cases as opposed to 34 of 79 (43 %) cases with only one involved margin (p < 0.001). When controlling for other factors, positive margins were no more associated with residual disease than close margins (p = 0.32), whereas multiple close or positive margins were associated with significantly higher risk of residual disease (odds ratio 6.1; p = 0.002; 95 % confidence interval 2.6-14.45). CONCLUSIONS: The only significant predictor of residual tumor was multiple close or positive margins. It may be appropriate to recommend reexcision for patients with multiple close margins.

Breast pathology review: does it make a difference?

BACKGROUND: Breast pathology is a challenging field, and previous work has shown discrepancies in diagnoses, even among experts. We set out to determine whether mandatory pathology review changes the diagnosis or surgical management of breast disease. METHODS: Cases were referred for pathology review after patients presented for surgical opinion to the Dubin Breast Center at Mount Sinai Medical Center over the course of 2 years. Surgical pathologists with expertise in breast disease reviewed slides submitted from the primary institution and rendered a second opinion diagnosis. Comparison of these reports was performed for evaluation of major changes in diagnosis and definitive surgical management. RESULTS: A total of 306 patients with 430 biopsy specimens were reviewed. Change in diagnosis was documented in 72 (17 %) of 430 cases and change in surgical management in 41 (10 %). A change in diagnosis was more likely to occur in patients originally diagnosed with benign rather than malignant disease (31 vs. 7 %, p < 0.001). Twelve (7 %) of 169 specimens initially diagnosed as benign were reclassified as malignant. A malignant diagnosis was changed to benign in 4 (2 %) of 261 cases. Change in diagnosis was less common in specimens originating from commercial laboratories than community hospitals or university hospitals (8, 19, 21 %, p = 0.023). Change in management was not dependent on initial institution. Type of biopsy specimen (surgical or core) did not influence diagnostic or management changes. CONCLUSIONS: We recommend considering breast pathology review based on the individual clinical scenario, regardless of initial pathologic diagnosis or originating institution.

Surgery No More? Managing the Primary Tumor in Stage IV Breast Cancer.

The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors' suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice.

CALGB 40603 (Alliance): Long-Term Outcomes and Genomic Correlates of Response and Survival After Neoadjuvant Chemotherapy With or Without Carboplatin and Bevacizumab in Triple-Negative Breast Cancer.

PURPOSE: CALGB 40603 (NCT00861705), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS: The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS: Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION: Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease.

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

Predictors of completion axillary lymph node dissection in patients with positive sentinel lymph nodes.

BACKGROUND: Completion axillary lymph node dissection (CALND) is routinely performed in breast cancer patients with positive sentinel lymph nodes (SLN). We sought to determine the sociodemographic, pathologic, and therapeutic variables that were associated with CALND. METHODS: From 7/1997 to 7/2003, 1,470 patients with invasive breast cancer were SLN positive by intraoperative frozen section or final pathologic exam by hematoxylin-eosin and/or immunohistochemistry (IHC). A comorbidity score was assigned using Adult Comorbidity Evaluation-27 system. Fisher's exact, Wilcoxon tests, and multivariate logistic regression analysis were used. RESULTS: CALND was performed less often in patients with age >or= 70 years compared with age < 70 years, moderate or severe comorbidities compared with no or mild, IHC-only positive SLN and breast conservation therapy (BCT compared with mastectomy. Patients who did not undergo CALND were less likely than CALND patients to have grade III disease, lymphovascular invasion multifocal disease, tumor size > 2 cm or to receive adjuvant chemotherapy. However, they were more likely to undergo axillary radiotherapy (RT). On multivariate analysis, age >or= 70 years [odds ratio (OR) 0.4, 95% confidence interval (CI) 0.26-0.63], IHC-only positive SLN (OR 0.13, 95%CI 0.09-0.19), presence of moderate to severe comorbidities (OR 0.64, 95%CI 0.41-0.99), tumor size

HER-2/neu status is a determinant of mammary aromatase activity in vivo: evidence for a cyclooxygenase-2-dependent mechanism.

Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Given the significance of estrogen synthesis in hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 expression. The main objective of this study was to define the interrelationship between HER-2/neu, cyclooxygenase-2 (COX-2), and aromatase in mammary tissue. Mammary aromatase activity and prostaglandin E(2) (PGE(2)) levels were increased in mice with mammary-targeted expression of a COX-2 transgene. In vitro, overexpressing COX-2 caused both increased PGE(2) production and aromatase activity, effects that were suppressed by celecoxib, a selective COX-2 inhibitor. Previously, we found that overexpression of HER-2/neu was associated with increased levels of COX-2 in human breast cancers. Here, we show that overexpression of HER-2/neu is also associated with increased aromatase activity. These results suggested the possibility that COX-2 was the functional intermediate linking HER-2/neu and aromatase. Consistent with this idea, COX-2 deficiency led to a gene dose-dependent reduction in mammary aromatase activity in a HER-2/neu transgenic mouse model. Complementary in vitro studies showed that HER-2/neu-mediated induction of PGE(2) synthesis and aromatase activity were suppressed by inhibiting COX-2. Collectively, our data indicate that COX-2 is the functional intermediate linking HER-2/neu and aromatase and suggest that inhibitors of PGE(2) synthesis will suppress estrogen biosynthesis in breast tissue.

Priming the 'soil' for breast cancer metastasis: the pre-metastatic niche.

The long prevailing model of metastasis recognizes the importance of both "seed" and "soil" for metastatic progression [1]. Much attention has focused on understanding the molecular and genetic factors that confer an intrinsic metastatic advantage to certain tumor cells. Meanwhile, changes occurring within distant tissues, creating a "soil" conducive for tumor invasion, have been largely neglected. Bone marrow-derived hematopoietic progenitor cells (HPCs) recently emerged as key players in initiating these early changes, creating a receptive microenvironment at designated sites for distant tumor growth and establishing the "Pre-Metastatic Niche" [2]. This insight into the earliest stages in the metastatic cascade revises our concept of the metastatic "microenvironment" to include physiological cells recruited from the bone marrow. Moreover, the concept of pre-metastatic tissues as 'niches' similar to physiological stem cell niches establishes a paradigm in which disseminated tumor cells may reside within a highly defined microcosm, both supportive and regulatory, and which may confer specific functions on indwelling cells. Understanding the cellular and molecular cross-talk between "seed" and "soil" may further our understanding of the factors that govern both site-specific patterning in metastasis and the phenomenon of tumor dormancy. This may lead to therapeutic strategies to detect and prevent metastasis at its earliest inception.

Lymphedema Precautions: Time to Abandon Old Practices?

The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors' suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice.A 46-year-old premenopausal woman with a body mass index of 21 was found on screening mammography to have a new, approximately 1-cm spiculated mass with associated calcifications in the upper outer quadrant of the left breast. Stereotactic core biopsy showed a focus of invasive duct carcinoma, strongly positive for estrogen and progesterone receptors and negative for human epidermal growth factor receptor 2, with associated ductal carcinoma in situ. Clinical examination revealed no palpable mass or axillary lymphadenopathy. She underwent a left lumpectomy with seed localization and sentinel lymph node biopsy. Final pathology revealed an 8-mm well-differentiated invasive carcinoma without lymphovascular invasion and intermediate grade ductal carcinoma in situ. The margins were clear, and three sentinel lymph nodes were negative for metastasis. The 21-gene recurrence score was 10, suggesting a 7% risk of 10-year distant recurrence with adjuvant endocrine treatment. After the completion of adjuvant radiotherapy (42.50 Gy in 16 fractions to the breast), the patient has returned for a follow-up visit. She is a professional violinist and would like to know what she can do to prevent lymphedema on her upcoming flight to Vienna.

Decision-Making in Breast Cancer Surgery: Where Do Patients Go for Information?

Patient decision-making regarding breast cancer surgery is multifactorial, and patients derive information on surgical treatment options from a variety of sources which may have an impact on choice of surgery. We investigated the role of different information sources in patient decision-making regarding breast cancer surgery. Two hundred and sixty-eight patients with breast cancer, eligible for breast-conserving therapy were surveyed in the immediate preoperative period, and clinical data were also collected. This survey evaluated the scope and features of patient-driven research regarding their ultimate choice of surgical treatment. The two most common sources of information used by patients were written material from surgeons (199/268-74%) and the Internet (184/268-69%). There was a trend for women who chose bilateral mastectomy to use the Internet more frequently than those choosing unilateral mastectomy (P = 0.056). Number of surgeons consulted, genetic testing, and MRI were significant predictors of patient choice of mastectomy over breast-conserving therapy. Multivariate analysis showed that the number of surgeons consulted (P < 0.001) and genetic testing (P < 0.001) were independent predictors of choosing mastectomy, whereas MRI was not. In conclusions, understanding factors driving patient decision-making may promote more effective education for patients requiring breast cancer surgery.

Impact of the addition of carboplatin and/or bevacizumab to neoadjuvant once-per-week paclitaxel followed by dose-dense doxorubicin and cyclophosphamide on pathologic complete response rates in stage II to III triple-negative breast cancer: CALGB 40603 (Alliance).

PURPOSE: One third of patients with triple-negative breast cancer (TNBC) achieve pathologic complete response (pCR) with standard neoadjuvant chemotherapy (NACT). CALGB 40603 (Alliance), a 2 × 2 factorial, open-label, randomized phase II trial, evaluated the impact of adding carboplatin and/or bevacizumab. PATIENTS AND METHODS: Patients (N = 443) with stage II to III TNBC received paclitaxel 80 mg/m(2) once per week (wP) for 12 weeks, followed by doxorubicin plus cyclophosphamide once every 2 weeks (ddAC) for four cycles, and were randomly assigned to concurrent carboplatin (area under curve 6) once every 3 weeks for four cycles and/or bevacizumab 10 mg/kg once every 2 weeks for nine cycles. Effects of adding these agents on pCR breast (ypT0/is), pCR breast/axilla (ypT0/isN0), treatment delivery, and toxicities were analyzed. RESULTS: Patients assigned to either carboplatin or bevacizumab were less likely to complete wP and ddAC without skipped doses, dose modification, or early discontinuation resulting from toxicity. Grade ≥ 3 neutropenia and thrombocytopenia were more common with carboplatin, as were hypertension, infection, thromboembolic events, bleeding, and postoperative complications with bevacizumab. Employing one-sided P values, addition of either carboplatin (60% v 44%; P = .0018) or bevacizumab (59% v 48%; P = .0089) significantly increased pCR breast, whereas only carboplatin (54% v 41%; P = .0029) significantly raised pCR breast/axilla. More-than-additive interactions between the two agents could not be demonstrated. CONCLUSION: In stage II to III TNBC, addition of either carboplatin or bevacizumab to NACT increased pCR rates, but whether this will improve relapse-free or overall survival is unknown. Given results from recently reported adjuvant trials, further investigation of bevacizumab in this setting is unlikely, but the role of carboplatin could be evaluated in definitive studies, ideally limited to biologically defined patient subsets most likely to benefit from this agent.

Reoperative sentinel lymph node biopsy after previous mastectomy.

BACKGROUND: Sentinel lymph node (SLN) biopsy is the standard of care for axillary staging in breast cancer, but many clinical scenarios questioning the validity of SLN biopsy remain. Here we describe our experience with reoperative-SLN (re-SLN) biopsy after previous mastectomy. STUDY DESIGN: Review of the SLN database from September 1996 to December 2007 yielded 20 procedures done in the setting of previous mastectomy. SLN biopsy was performed using radioisotope with or without blue dye injection superior to the mastectomy incision, in the skin flap in all patients. In 17 of 20 patients (85%), re-SLN biopsy was performed for local or regional recurrence after mastectomy. RESULTS: Re-SLN biopsy was successful in 13 of 20 patients (65%) after previous mastectomy. Of the 13 patients, 2 had positive re-SLN, and completion axillary dissection was performed, with 1 having additional positive nodes. In the 11 patients with negative re-SLN, 2 patients underwent completion axillary dissection demonstrating additional negative nodes. One patient with a negative re-SLN experienced chest wall recurrence combined with axillary recurrence 11 months after re-SLN biopsy. All others remained free of local or axillary recurrence. Re-SLN biopsy was unsuccessful in 7 of 20 patients (35%). In three of seven patients, axillary dissection was performed, yielding positive nodes in two of the three. The remaining four of seven patients all had previous modified radical mastectomy, so underwent no additional axillary surgery. CONCLUSIONS: In this small series, re-SLN was successful after previous mastectomy, and this procedure may play some role when axillary staging is warranted after mastectomy.

Does the benefit of sentinel node frozen section vary between patients with invasive duct, invasive lobular, and favorable histologic subtypes of breast cancer?

BACKGROUND: Although many questions regarding sentinel lymph node (SLN) biopsy in breast cancer have been answered by observational studies and, increasingly, by prospective trials, the role of intraoperative SLN assessment remains a matter of debate. Here we report in detail the results of intraoperative SLN assessment by frozen section (FS), with particular attention to variations in sensitivity and yield by histologic subtype, by tumor size, and by other clinicopathologic parameters. METHODS: Five thousand two hundred ninety-eight consecutive patients with clinical stage T1-3N0 invasive breast carcinoma had SLN biopsy with intraoperative FS at Memorial Sloan Kettering Cancer Center between 1996 and 2004. We report the results of FS by sensitivity (the proportion of all positive SLN detected by FS) and by yield (the proportion of all FS procedures in which the FS was positive). RESULTS: The sensitivity of FS was 61% overall, was higher for invasive duct (ID) than for invasive lobular (IL) cancers (62% vs. 52%; P = 0.006), and was marginally lower for favorable subtypes (46%; P = 0.26). The yield of FS was 21% overall, with no difference between ID and IL cancers (22% vs. 21%; P = 0.49), and with a substantially lower yield for favorable subtypes (3%; P < 0.001). The yield of FS increased with tumor size for ID and IL cancers (P < 0.001), but not for favorable subtypes. For both ID and IL cancers, the sensitivity and yield of FS were significantly higher with younger patient age, increasing tumor size, and lymphovascular invasion. The yield of FS was <10% for all patients with ID or IL tumors < or =1 cm in size who were older than 60 years of age. Among all FS-positive patients, only 45% were identified by the first FS, whereas 91% were cumulatively identified by the first, second, or third FS. CONCLUSIONS: For patients with ID and IL cancers, the overall sensitivity of FS is >50%, but the yield of FS is <10% for individuals > or =60 years of age with T1a/b tumors. Intraoperative FS may not be worthwhile for this low-yield subset, especially for patients with invasive breast cancer of favorable type.