Uterine Sarcoma With EML4::NTRK3 Fusion: A Spectrum of Mesenchymal Neoplasms Harboring Actionable Gene Fusions.

NTRK gene fusions are part of a paradigm shift in oncology, arising as one of the main genomic alterations with actionability in the so-called "agnostic setting." In gynecologic pathology, the recent description of uterine sarcoma resembling fibrosarcoma and with NTRK rearrangements ( NTRK -rearranged uterine sarcoma) highlights the importance of recognizing clinicopathological cues that can lead to genomic profiling. Herein, we report the case of a 43-year-old woman presenting with vaginal bleeding and pelvic mass. Histopathology of the tumor showed moderately atypical spindle cells arranged in long fascicles reminiscent of fibrosarcoma, along with immunohistochemical positivity for S100, CD34, and pan-tropomyosin receptor kinase. This prompted RNA-sequencing and the finding of a rare EML4::NTRK3 fusion. Clinical, histologic, and molecular findings are described, in addition to discussions regarding differential diagnoses and possible implications of the findings in clinical practice.

Risk of metastasis in BRCA2 carriers diagnosed with triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the neoplasia most associated with BRCA1 germline pathogenic variants (PV) and is more likely to develop metastases than the other breast cancer (BC) subtypes, mainly in the lungs and the central nervous system (CNS). Recently, BRCA2 carriers were shown to have a higher risk for developing CNS metastases. However, the patterns of recurrence and metastases of BRCA2 carriers with TNBC are unknown. METHODS: TNBC patient data attending the A.C. Camargo Cancer Center, from 1998 through 2020, were verified either by medical records or by BRCA1/2 genetic testing carried out. Multivariable logistic regression models were fit to the data to assess the independent factors for bone and CNS metastases. Adjustment was done using all independent variables with p < 0.2 in the univariable Cox model to describe the relationship between the independent variables until time of death. RESULTS: A total of 388 TNBC patients were evaluated. We identified PV in BRCA1/2 genes in 21% (82/388), being 17.7% (69/388) in BRCA1 and only 3.3% (13/388) in BRCA2. A total of 120 patients (31%) developed distant metastases. Bone or CNS metastases were observed in 40% and 60% of BRCA2 PV carriers (p = 0.155), respectively. The BRCA2 carriers tended to have a higher likelihood of developing bone metastases (OR, 4.06; 95% CI, 0.82-20.01; p = 0.085), when compared to BRCA1 carriers (OR, 0.6; 95% CI, 0.12-2.87; p = 0.528). BRCA2 carriers had an OR of 1.75 (95% CI, 0.33-9.14; p = 0.503) for CNS metastasis development, while BRCA1 carriers had an OR of 0.72 (95% CI, 0.23-2.23; p = 0.574). CONCLUSIONS: Patients with TNBC and PV in the BRCA2 gene had higher frequencies of secondary bone involvement and CNS in the course of the disease. However, the BRCA2 PV did not represent an independent outcome predictor of metastases and overall survival. Efforts to increase the number of BRCA2 carriers among TNBC patients are crucial for determining their risk of developing bone and CNS metastases compared to BRCA2 noncarriers.

Systemic immune mediators reflect tumour-infiltrating lymphocyte intensity and predict therapeutic response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer (BC). Neoadjuvant chemotherapy has proven efficacy in its treatment, and a pathological complete response (pCR) to therapy is predictive of improved long-term survival. The immune response is key to successful neoadjuvant chemotherapy, as indicated by the relation between the percentage of stromal tumour-infiltrating lymphocytes (TILs) in pre-treated tumour tissue samples and the likelihood of achieving pCR. Here we studied systemic immune mediators from volunteer TNBC patients before undergoing neoadjuvant chemotherapy to determine the systemic response association with TIL intensity, treatment response and survival. Patients were classified into pCR responder or non-responder at time of surgery. We found higher levels of immune mediators before treatment began in patients that went on to be pCR responders versus non-pCR, with area under the curve (AUC) values of 0.64-0.80. We also observed a positive correlation between inflammatory systemic immune mediators and the percentage of TILs in pCR responder patients. Combining TILs and systemic immune mediator levels provided stronger AUC values (range of 0.72-0.82). Last, performing a progression-free survival analysis with several of the systemic cytokines that predict pCR, segregated the patients into long- and short-survival groups based on high and low production of the cytokines, respectively. Our study demonstrates that circulating cytokines, before treatment begins, predict pCR in TNBC patients treated with neoadjuvant chemotherapy. Moreover, they may act as a surrogate marker of high TILs or together with TILs to better predict pCR and survival.

In vitro and in vivo acute toxicity of a novel citrate-coated magnetite nanoparticle.

Magnetic nanoparticles (MNps) have become powerful tools for multiple biomedical applications such as hyperthermia drivers, magnetic resonance imaging (MRI) vectors, as well as drug-delivery systems. However, their toxic effects on human health have not yet been fully elucidated, especially in view of their great diversity of surface modifications and functionalizations. Citrate-coating of MNps often results in increased hydrophilicity, which may positively impact their performance as drug-delivery systems. Nonetheless, the consequences on the intrinsic toxicity of such MNps are unpredictable. Herein, novel magnetite (Fe3O4) nanoparticles covered with citrate were synthesized and their potential intrinsic acute toxic effects were investigated using in vitro and in vivo models. The proposed synthetic pathway turned out to be simple, quick, inexpensive, and reproducible. Concerning toxicity risk assessment, these citrate-coated iron oxide nanoparticles (IONps) did not affect the in vitro viability of different cell lines (HaCaT and HepG2). Moreover, the in vivo acute dose assay (OECD test guideline #425) showed no alterations in clinical parameters, relevant biochemical variables, or morphological aspects of vital organs (such as brain, liver, lung and kidney). Iron concentrations were slightly increased in the liver, as shown by Graphite Furnace Atomic Absorption Spectrometry and Perls Prussian Blue Staining assays, but this finding was considered non-adverse, given the absence of accompanying functional/clinical repercussions. In conclusion, this study reports on the development of a simple, fast and reproducible method to obtain citrate-coated IONps with promising safety features, which may be used as a drug nanodelivery system in the short run. (263 words).

Prognostic value of response evaluation based on breast MRI after neoadjuvant treatment: a retrospective cohort study.

OBJECTIVE: To investigate the impact of response evaluation after neoadjuvant chemotherapy (NAC) in breast cancer patients, assessed by both magnetic resonance imaging (MRI) and pathology, on disease-free survival (DFS). METHODS: This single-center, retrospective cohort study included consecutive breast cancer patients who underwent NAC and preoperative breast MRI. Resolution of invasive carcinoma in the breast and axilla was defined as complete pathological response (pCR). Radiological complete response (rCR) was defined as the absence of abnormal enhancement in the tumor site. Kaplan-Meier estimator was used to estimate the disease-free survival on 60 months. Cox regression analysis was used to estimate hazard ratio (HR) values. RESULTS: In total, 317 patients were included with a mean age of 47.3 years and a mean tumor size of 39.8 mm. The most common immunophenotype was luminal (44.9%), followed by triple-negative (26.8%). Overall, 126 patients (39.7%) had an rCR, while 119 (37.5%) had pCR; the radiological and pathological responses agreed in 252 cases (79.5%). During follow-up, patients who had rCR and pCR had a better DFS curve compared to patients with non-rCR and non-pCR, while those who had rCR or pCR presented an intermediate curve (Log-rank p = 0.003). Multivariate analysis showed a higher risk of recurrence in patients with non-rCR and non-pCR (HR: 5,626; p = 0.020) and those who had a complete response on MRI or pathology only (HR: 4,369; p = 0.067), when compared to patients with rCR and pCR. CONCLUSIONS: The association of MRI and pathological responses after NAC might better stratify the risk of recurrence and prognosis in breast cancer patients. KEY POINTS: • Association of response evaluation after neoadjuvant chemotherapy by pathology and MRI allows better stratification of prognosis. • Complete response to neoadjuvant chemotherapy on pathology and MRI was related to better disease-free survival. • Complete response on MRI or pathology only had a greater risk of recurrence.

Malignant Phyllodes Tumor of the Breast: A Practice Review.

INTRODUCTION: Phyllodes tumor (PT) of the breast, particularly malignant phyllodes tumor (mPT), is a rare fibroepithelial neoplasm. A complex diagnosis is based on pathologic, radiologic, and clinical findings, with controversies about what is the best therapeutic strategy. OBJECTIVE: Our objective was to provide an overview of the clinical, pathologic, and therapeutic aspects of this rare tumor. CONCLUSIONS: mPT is a rare presentation of breast cancer and a challenge in clinical practice. A multidisciplinary approach should take into account some aspects like pathogenic mutations and hereditary syndromes. Oncologic surgery is the fundamental approach, and the use of adjuvant therapies is still controversial due to the lack of clinical trials. Treatment recommendations should be individualized according to patient risk and preferences. Prospective studies are fundamental to clarifying the best treatment for these tumors.

Neuroendocrine Carcinomas of the Canine Mammary Gland: Histopathological and Immunohistochemical Characteristics.

Invasive mammary carcinomas with neuroendocrine differentiation are rare in women and were reported only once in female dogs. For the present study, ten cases of solid mammary carcinoma positive for chromogramin A in immunohistochemistry were selected. Histopathological characteristics of these tumors were described and immunohistochemical evaluation was performed with chromogranin A, synaptophysin, CD56, NSE, PGP 9.5, pancitokeratin, Ki67, estrogen receptor (ER), and progesterone receptor (PR). The average animal age was 13.2 years old and the average tumor size was 4.8 cm. In total, 70% of the neoplasms were classified as grade III and 30% as grade II by the Nottingham histological grade system. High mitotic index was observed with a mean of 27.5 mitoses in 10 high magnification fields. Only one case showed typical carcinoid tumor characteristics. In addition, vascular invasion was shown in 3 tumors. All carcinomas were positive for chromogran A, while only two cases were reactive to synaptophysin. For PGP 9.2, NSE and CD56, we observed positivity of 100, 90, and 70%, respectively, in the samples, being that no tumor was positive for all the neuroendocrine markers. All neoplasms showed ER and PR in at least 10% of neoplastic cells, while Ki67 varied from 29 to 95%, with mean mitotic index of 67%. Four of the ten animals died within 1 year of the tumor diagnosis. Neuroendocrine neoplasms occur in the canine mammary gland and are propably underdiagnosed. This is due to their non-specific morphological characteristics and the low use of neuroendocrine immunohistochemistric markers the diagnostic routine. More studies are necessary to determine the prognosis of this new histological type.

Breast implant-associated anaplastic large cell lymphoma in a Li-FRAUMENI patient: a case report.

BACKGROUND: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare malignancy, recently recognized as a provisional entity by the World Health Organization. Although increasing data have been published on this entity in recent years, a great number of patients and health professionals remain unaware of this diagnosis. CASE PRESENTATION: We herein report the case of a 56-year-old female with Li-FRAUMENI syndrome who presented with late right-sided recurrent breast swelling after prophylactic adenomastectomy with implant reconstruction. Imaging scans revealed an heterogeneous mass adjacent to the implant fibrous capsule. A biopsy of the lesion rendered the diagnosis of a BIA-ALCL. CONCLUSIONS: This case presents similarities with previous reports, but also some particularities, which should be stressed in order to make the diagnosis the earliest possible. The most distinct feature is that this is the second report of BIA-ALCL arising in the setting of Li-FRAUMENI syndrome.

Pleomorphic lobular carcinoma in situ of the breast: a single institution experience with clinical follow-up and centralized pathology review.

PURPOSE: The natural history of pleomorphic lobular carcinoma in situ (PLCIS) remains largely unknown. METHODS: A pathology database search (1995-2012) was performed to identify patients diagnosed with an LCIS variant. Patients with synchronous breast cancer and/or no evidence of pleomorphism were excluded. Original slides were re-evaluated by three pathologists to identify a consensus cohort of PLCIS. Borderline lesions with focal atypia were classified as LCIS with pleomorphic features (LCIS-PF). Clinical data were obtained from medical records. RESULTS: From 233 patients, we identified 32 with an LCIS variant diagnosis and no concurrent breast cancer. Following review, 16 cases were excluded due to lack of pleomorphism. The remaining 16 were classified as PLCIS (n = 11) and LCIS-PF (n = 5). 12/16 patients were treated with surgical excision ± chemoprevention. Patients with a prior breast cancer history and those having mastectomy were excluded from outcome analysis. Among the remaining 7 patients with PLCIS/LCIS-PF, 4/7 (57%) developed ipsilateral breast cancer at a median follow-up of 67 months. Median age at the time of breast cancer diagnosis was 56 years old and median time from PLCIS/LCIS-PF to cancer diagnosis was 59 months (range 45-66 months). The four cancers included 1 invasive lobular carcinoma (ILC), 1 microinvasive ILC, 1 invasive ductal carcinoma, and 1 ductal carcinoma in situ. CONCLUSIONS: We confirm that PLCIS in isolation is indeed a rare entity, further contributing to the difficulty in determining the actual risk conferred by this lesion. Long-term follow-up data on larger cohorts are needed to define standardized management and outcomes for patients with PLCIS.

Inositol 1, 4, 5-trisphosphate-dependent nuclear calcium signals regulate angiogenesis and cell motility in triple negative breast cancer.

Increases in nuclear calcium concentration generate specific biological outcomes that differ from those resulting from increased cytoplasmic calcium. Nuclear calcium effects on tumor cell proliferation are widely appreciated; nevertheless, its involvement in other steps of tumor progression is not well understood. Therefore, we evaluated whether nuclear calcium is essential in other additional stages of tumor progression, including key steps associated with the formation of the primary tumor or with the metastatic cascade. We found that nuclear calcium buffering impaired 4T1 triple negative breast cancer growth not just by decreasing tumor cell proliferation, but also by enhancing tumor necrosis. Moreover, nuclear calcium regulates tumor angiogenesis through a mechanism that involves the upregulation of the anti-angiogenic C-X-C motif chemokine 10 (CXCL10-IP10). In addition, nuclear calcium buffering regulates breast tumor cell motility, culminating in less cell invasion, likely due to enhanced vinculin expression, a focal adhesion structural protein. Together, our results show that nuclear calcium is essential for triple breast cancer angiogenesis and cell migration and can be considered as a promising strategic target for triple negative breast cancer therapy.

Clonal relationships between lobular carcinoma in situ and other breast malignancies.

BACKGROUND: Recent evidence suggests that lobular carcinoma in situ (LCIS) can be a clonal precursor of invasive breast cancers of both the ductal and lobular phenotypes. We sought to confirm these findings with an extensive study of fresh frozen breast specimens from women undergoing mastectomy. METHODS: Patients with a history of LCIS presenting for therapeutic mastectomy were identified prospectively. Frozen tissue blocks were collected, screened for lesions of interest, and subjected to microdissection and DNA extraction. Copy number profiling, whole-exome sequencing, or both were performed. Clonal relatedness was assessed using specialized statistical techniques developed for this purpose. RESULTS: After exclusions for genotyping failure, a total of 84 lesions from 30 patients were evaluated successfully. Strong evidence of clonal relatedness was observed between an LCIS lesion and the invasive cancer for the preponderance of cases with lobular carcinoma. Anatomically distinct in situ lesions of both ductal and lobular histology were also shown to be frequently clonally related. CONCLUSIONS: These data derived from women with LCIS with or without invasive cancer confirm that LCIS is commonly the clonal precursor of invasive lobular carcinoma and that distinct foci of LCIS frequently share a clonal origin, as do foci of LCIS and ductal carcinoma in situ.

Prognostic Impact of 21-Gene Recurrence Score in Patients With Stage IV Breast Cancer: TBCRC 013.

PURPOSE: The objective of this study was to determine whether the 21-gene Recurrence Score (RS) provides clinically meaningful information in patients with de novo stage IV breast cancer enrolled in the Translational Breast Cancer Research Consortium (TBCRC) 013. PATIENTS AND METHODS: TBCRC 013 was a multicenter prospective registry that evaluated the role of surgery of the primary tumor in patients with de novo stage IV breast cancer. From July 2009 to April 2012, 127 patients from 14 sites were enrolled; 109 (86%) patients had pretreatment primary tumor samples suitable for 21-gene RS analysis. Clinical variables, time to first progression (TTP), and 2-year overall survival (OS) were correlated with the 21-gene RS by using log-rank, Kaplan-Meier, and Cox regression. RESULTS: Median patient age was 52 years (21 to 79 years); the majority had hormone receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative (72 [66%]) or hormone receptor-positive/HER2-positive (20 [18%]) breast cancer. At a median follow-up of 29 months, median TTP was 20 months (95% CI, 16 to 26 months), and median survival was 49 months (95% CI, 40 months to not reached). An RS was generated for 101 (93%) primary tumor samples: 22 (23%) low risk (< 18), 29 (28%) intermediate risk (18 to 30); and 50 (49%) high risk (≥ 31). For all patients, RS was associated with TTP (P = .01) and 2-year OS (P = .04). In multivariable Cox regression models among 69 patients with estrogen receptor (ER)-positive/HER2-negative cancer, RS was independently prognostic for TTP (hazard ratio, 1.40; 95% CI, 1.05 to 1.86; P = .02) and 2-year OS (hazard ratio, 1.83; 95% CI, 1.14 to 2.95; P = .013). CONCLUSION: The 21-gene RS is independently prognostic for both TTP and 2-year OS in ER-positive/HER2-negative de novo stage IV breast cancer. Prospective validation is needed to determine the potential role for this assay in the clinical management of this patient subset.

Targeted capture massively parallel sequencing analysis of LCIS and invasive lobular cancer: Repertoire of somatic genetic alterations and clonal relationships.

PURPOSE: Lobular carcinoma in situ (LCIS) has been proposed as a non-obligate precursor of invasive lobular carcinoma (ILC). Here we sought to define the repertoire of somatic genetic alterations in pure LCIS and in synchronous LCIS and ILC using targeted massively parallel sequencing. METHODS: DNA samples extracted from microdissected LCIS, ILC and matched normal breast tissue or peripheral blood from 30 patients were subjected to massively parallel sequencing targeting all exons of 273 genes, including the genes most frequently mutated in breast cancer and DNA repair-related genes. Single nucleotide variants and insertions and deletions were identified using state-of-the-art bioinformatics approaches. RESULTS: The constellation of somatic mutations found in LCIS (n = 34) and ILC (n = 21) were similar, with the most frequently mutated genes being CDH1 (56% and 66%, respectively), PIK3CA (41% and 52%, respectively) and CBFB (12% and 19%, respectively). Among 19 LCIS and ILC synchronous pairs, 14 (74%) had at least one identical mutation in common, including identical PIK3CA and CDH1 mutations. Paired analysis of independent foci of LCIS from 3 breasts revealed at least one common mutation in each of the 3 pairs (CDH1, PIK3CA, CBFB and PKHD1L1). CONCLUSION: LCIS and ILC have a similar repertoire of somatic mutations, with PIK3CA and CDH1 being the most frequently mutated genes. The presence of identical mutations between LCIS-LCIS and LCIS-ILC pairs demonstrates that LCIS is a clonal neoplastic lesion, and provides additional evidence that at least some LCIS are non-obligate precursors of ILC.

Association between HER2 status in gastric cancer and clinicopathological features: a retrospective study using whole-tissue sections.

BACKGROUND: Gastric cancer is usually diagnosed in an advanced stage of disease and treatment options are sparse. Trastuzumab was recently approved for metastatic or locally advanced carcinomas arising in the stomach or in the gastroesophageal junction in patients with HER2-positive tumors. However, data on the frequency of HER2-positive cases among Brazilian patients are limited. Our aim was to characterize HER2 protein and gene status in a series of Brazilian patients with gastric cancer and to evaluate its association with clinicopathological data. METHODS: Histological slides from 124 primary gastrectomies were reviewed and their pathological reports were retrieved from the files at a Brazilian university hospital. Automated immunohistochemistry for HER2 was performed on whole-tissue sections from each tumor. HER2-equivocal cases by immunohistochemistry were submitted to automated dual in situ hybridization for gene amplification evaluation. HER2 status was confronted with clinicopathological parameters in order to assess statistically significant associations. RESULTS: Immunohistochemistry analysis revealed that 13/124 cases (10.5 %) were HER2 positive (3+), 10/124 cases (8.1 %) were equivocal (2+) and 101/124 cases (81.4 %) were negative, being 7 cases 1+. None of the equivocal cases showed gene amplification. The overall HER2 positivity rate was 10.5 %. There was an association between HER2 expression and Laurén's intestinal histological subtype (P = 0.048), well to moderately differentiated tumors (P = 0.004) and presence of lymphovascular invasion (P = 0.031). No association was found between HER2 status and tumor topography. CONCLUSIONS: Confronted with data published by other authors, the lower percentage of HER2-positive cases found in our series might be partially explained by the lower frequency of tumors arising at the gastroesophageal junction in comparison with distal gastric carcinomas in Brazilian patients. This could also account for the lack of statistically significant association between HER2 status and tumor topography in our study.

Lobular Carcinoma in Situ: A 29-Year Longitudinal Experience Evaluating Clinicopathologic Features and Breast Cancer Risk.

PURPOSE: The increased breast cancer risk conferred by a diagnosis of lobular carcinoma in situ (LCIS) is poorly understood. Here, we review our 29-year longitudinal experience with LCIS to evaluate factors associated with breast cancer risk. PATIENTS AND METHODS: Patients participating in surveillance after an LCIS diagnosis are observed in a prospectively maintained database. Comparisons were made among women choosing surveillance, with or without chemoprevention, and those undergoing bilateral prophylactic mastectomies between 1980 and 2009. RESULTS: One thousand sixty patients with LCIS without concurrent breast cancer were identified. Median age at LCIS diagnosis was 50 years (range, 27 to 83 years). Fifty-six patients (5%) underwent bilateral prophylactic mastectomy; 1,004 chose surveillance with (n = 173) or without (n = 831) chemoprevention. At a median follow-up of 81 months (range, 6 to 368 months), 150 patients developed 168 breast cancers (63% ipsilateral, 25% contralateral, 12% bilateral), with no dominant histology (ductal carcinoma in situ, 35%; infiltrating ductal carcinoma, 29%; infiltrating lobular carcinoma, 27%; other, 9%). Breast cancer incidence was significantly reduced in women taking chemoprevention (10-year cumulative risk: 7% with chemoprevention; 21% with no chemoprevention; P < .001). In multivariable analysis, chemoprevention was the only clinical factor associated with breast cancer risk (hazard ratio, 0.27; 95% CI, 0.15 to 0.50). In a subgroup nested case-control analysis, volume of disease, which was defined as the ratio of slides with LCIS to total number of slides reviewed, was also associated with breast cancer development (P = .008). CONCLUSION: We observed a 2% annual incidence of breast cancer among women with LCIS. Common clinical factors used for risk prediction, including age and family history, were not associated with breast cancer risk. The lower breast cancer incidence in women opting for chemoprevention highlights the potential for risk reduction in this population.

Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.

The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in The Cancer Genome Atlas project breast cancer methylome database. For cMethDNA, a fixed physiologic level (50 copies) of artificially constructed, standard nonhuman reference DNA specific for each gene is introduced in a constant volume of serum (300 µL) before purification of the DNA, facilitating a sensitive, specific, robust, and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in training (28 normal, 24 cancer) and test (27 normal, 33 cancer) sets of recurrent stage IV patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy two to 11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer.

Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer.

INTRODUCTION: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. METHODS: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 µCi of 124I-iodide. RESULTS: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time- and concentration-dependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( <10,000-fold increase from the initial viral dose ) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm3 versus 168 mm3 in untreated controls (P < 0.05). CONCLUSIONS: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.

Vaccinia virus GLV-1h153 is effective in treating and preventing metastatic triple-negative breast cancer.

OBJECTIVE: This study aimed to investigate the therapeutic impact of a new oncolytic vaccinia virus in a triple-negative breast cancer (TNBC) murine model and its potential for treating distant metastatic disease. BACKGROUND: TNBCs are aggressive tumors associated with a high metastatic rate. Their lack of targets for hormonal/biological therapy presents significant clinical challenges and a dire need for novel therapies. METHODS: GLV-1h153, a replication-competent vaccinia virus, was tested against multiple cell lines. Cytotoxicity and viral replication were determined. Intratumoral (IT) or intravenous (IV) injection of GLV-1h153 (1 × 10(7) plaque-forming units) or phosphate buffered saline was tested in an orthotopic murine model, which reliably produces systemic metastasis. Tumors, lymph nodes, and metastatic organs (lung, liver, and brain) were harvested 5 and 8 weeks after treatment and prepared for histopathological review. Demonstration of metastasis was performed using immunofluorescence and hematoxylin and eosin (H&E) staining. RESULTS: GLV-1h153 infected, replicated in, and killed all TNBC cell lines in vitro. In vivo, mean tumor volume 2 weeks after treatment was 22 (IT), 29 (IV) versus 245 mm(3) (control; P < 0.002). Five weeks after treatment, all harvested lymph nodes and organs showed no evidence of metastatic cells. All harvested tumors showed complete response to treatment, with only necrosis and fibrosis on H&E staining 8 weeks after treatment. CONCLUSIONS: This is the first study to demonstrate that TNBCs are killed by a novel vaccinia virus both in vitro and in vivo. Our results suggest that GLV-1h153 is a promising therapeutic agent for preventing and treating metastatic TNBC and warrants further clinical testing in patients.

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions.

INTRODUCTION: Lobular carcinoma in situ (LCIS) has been accepted as a marker of risk for the development of invasive breast cancer, yet modern models of breast carcinogenesis include LCIS as a precursor of low-grade carcinomas. We provide evidence favoring a clonal origin for LCIS and synchronous estrogen receptor-positive malignant lesions of the ductal and lobular phenotype. METHODS: Patients with prior LCIS undergoing mastectomy were identified preoperatively from 2003 to 2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and were subject to microdissection. Samples from 65 patients were hybridized to the Affymetrix SNP 6.0 array platform. Cases with both an LCIS sample and an associated ductal carcinoma in situ (DCIS) or invasive tumor sample were evaluated for patterns of somatic copy number changes to assess evidence of clonal relatedness. RESULTS: LCIS was identified in 44 of the cases, and among these a DCIS and/or invasive lesion was also identified in 21 cases. A total of 17 tumor pairs had adequate DNA/array data for analysis, including nine pairs of LCIS/invasive lobular cancer, four pairs of LCIS/DCIS, and four pairs of LCIS/invasive ductal cancer. Overall, seven pairs (41%) were judged to be clonally related; in five (29%) evidence suggested clonality but was equivocal, and five (29%) were considered independent. Clonal pairs were observed with all matched lesion types and low and high histological grades. We also show anecdotal evidence of clonality between a patient-matched triplet of LCIS, DCIS, and invasive ductal cancer. CONCLUSION: Our results support the role of LCIS as a precursor in the development of both high-grade and low-grade ductal and lobular cancers.

Prognostic impact of the cancer stem cell related markers ALDH1 and EZH2 in triple negative and basal-like breast cancers.

AIMS: We assessed the expression of ALDH1 and EZH2, cancer stem cell (CSC) related markers, in triple negative and basal-like breast cancers, investigating their association with clinicopathological features and outcome. METHODS: Clinicopathological data were obtained from 140 cases of triple negative breast cancer. A tissue microarray was constructed and immunohistochemistry for ER, PR, HER2, ALDH1, EZH2, CK5, CK14, EGFR, p63, caveolin, and p53 was performed. Tumour cell and stromal expression of ALDH1 were evaluated. Multivariate analysis was conducted, including all significant variables. RESULTS: The majority of triple negative breast cancers were invasive ductal carcinomas of no special type (NST) (116/140). Tumour cells exhibited cytoplasmic expression of ALDH1 in 26 of 140 cases, while stromal expression was detected in 117 of 140 cases. Tumour cell expression did not correlate with any of the parameters. Conversely, stromal expression was associated with better overall survival (p=0.044). Assessment by Cox Regression Model showed a HR of 2.80 (HR = 1/0.357 = 2.80; 95%CI 0.178-0.714; p = 0.004) for breast cancer death when ALDH1 was not found in the stromal compartment of tumours, independent of age, histological type/grade, nodal status, stage, relapse, and expression of basal markers. High EZH2 expression was noted in 120 of 140 triple negative breast cancers and was not associated with other variables. Basal-like cancers comprised 75% (105/140) of triple negative breast cancers. Interestingly, we found association between EZH2 and CK14 expression (p = 0.041). CONCLUSIONS: ALDH1 expression is frequent in tumour-associated stromal cells of triple negative breast cancer and is associated with better outcome. Tumour microenvironment should be considered when studying prognostic impact of CSCs in breast cancer.