Davide Balmativola

Reliability of whole sentinel lymph node analysis by one-step nucleic acid amplification for intraoperative diagnosis of breast cancer metastases.

Objective: To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. Background: Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. Methods: Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. Results: Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. Conclusions: Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.

Gene Status in HER2 Equivocal Breast Carcinomas: Impact of Distinct Recommendations and Contribution of a Polymerase Chain Reaction-Based Method

BACKGROUND The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco’s Modified Eagle’s Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.

The impact of malignant nipple discharge cytology (NDc) in surgical management of breast cancer patients

Background The role of nipple discharge cytology (NDc) in the surgical management of breast cancer patients is unclear. We aimed: (i) to evaluate the effect of malignant NDc on the surgical approach to the nipple-areola complex, and (ii) to verify the association between malignant NDc and nipple malignancy. Methods We retrospectively analyzed a case series of 139 patients with NDc who underwent breast surgery. The clinical and histological findings, types of surgery with emphasis on nipple-areola complex amputation, immunohistochemical phenotypes of the carcinomas and measurements of the tumor-nipple distance were recorded. Additionally, in patients who showed HER2-positive lesions on definitive surgery, we evaluated the HER2 immunocytochemistry of the NDc smears. Results Thirty-two malignant and 107 benign/borderline NDc diagnoses were identified. All 32 malignant-NDc cases were histologically confirmed as malignant. Thirty borderline/benign-NDc cases were histologically diagnosed as malignant (sensitivity 58%). The majority of the patients with malignant NDc were treated with nipple-areola complex amputations in both the mastectomy and conservative surgery groups (P<0.001, χ251.77). Nipple involvement was strongly associated with HER2-positive ductal carcinoma in-situ (P<0.001, χ211.98). HER2 immunocytochemistry on the NDc revealed a 100% correlation with the immunocytochemistry performed on the surgical tissues. Conclusions Malignant NDc influenced surgical management. The association of malignant NDc with nipple involvement is highly related to ductal carcinoma in-situ with HER2 overexpression. In case of HER2 positive NDc, nipple-areola complex involvement is more likely than in HER2 negative cases.

Is Regression after Neoadjuvant Chemotherapy for Locally Advanced Breast Cancer Different in Sentinel and Non-sentinel Nodes?

Tumor draining sentinel lymph nodes (SLNs) are the sites of selective changes as compared to non-SLNs. They show features of tumor-reactive lymphadenopathy, including increased total number of functional blood vessels, but a relative immunosuppressed status has also been described in them. We explored the hypothesis of a selective regression or non-regression in SLNs versus non-SLNs in 142 patients with 110 estrogen receptor-positive and 32 estrogen receptor-negative tumors undergoing both SLN biopsy and axillary lymph node dissection after neoadjuvant therapy by assessing the tumoral (metastatic) and regression statuses of SLNs and non-SLNs separately. Of the 89 cases with signs of nodal regression, 22 cases (25%) were in favor of a selective non-regression in SLNs, 18 cases (20%) were supportive of a selective and more pronounced regression in the SLNs and the remaining showed equal degrees of regression or non-regression in SLNs and non-SLNs. The results indicate that there is no obvious difference in the degree of regressive histological changes shown by SLNs and NSLNs. Therefore, this phenomenon may not be a major contributor to the higher false negative rate of SLN biopsy after neoadjuvant treatment.

FOXA1 and AR in invasive breast cancer: new findings on their co-expression and impact on prognosis in ER-positive patients

BackgroundThe role of forkhead-box A1 (FOXA1) and Androgen receptor (AR) in breast cancer (BC) has been extensively studied. However, the prognostic role of their co-expression in Estrogen receptor positive (ER+) BC has not been investigated so far. The aim of the present study was thus to assess the co-expression (protein and mRNA) of FOXA1 and AR in BC patients, in order to evaluate their prognostic impact according to ER status.MethodsImmunohistochemical expression of AR and FOXA1 was evaluated on 479 consecutive BC, with complete clinical-pathological and follow up data. Fresh-frozen tissues from 65 cases were available. The expression of AR and FOXA1 with ER was validated using mRNA analyses. Survival and Cox proportional hazard analyses were used to evaluate the relationship between FOXA1, AR and prognosis.ResultsExpression of ER, AR and FOXA1 was observed in 78, 60 and 85% of cases respectively. Most AR+ cases (97%) were also FOXA1+. The level of FOXA1 mRNA positively correlated with level of both AR mRNA (r = 0.8975; P < 0.001) and ER mRNA (r = 0.7326; P < 0.001). In ER+ BC, FOXA1 was associated with a good prognosis independently of AR expression in the three subgroups analyzed (FOXA1+/AR+; FOXA1+/AR-; FOXA1−/AR-). Multivariate analyses confirmed that FOXA1 may provide more information than AR in Disease-Free Interval (DFI) of ER+ BC patients.ConclusionOur results suggest that in BC the expression of FOXA1 is directly related to the expression of AR. Despite that, FOXA1 is found as superior predicting marker of recurrences compared to AR in ER+ BC patients.

Abstract P1-07-27: Neutrophil elastase modulates breast cancer progression by fostering collective cell detachment and tumor emboli dissemination

Proteases constitute a large family of enzymes involved in several processes, including degradation and remodeling of the extracellular matrix to drive dissemination of cancer cells into adjacent tissue. Within this large family, the serine protease neutrophil elastase (NE) has been proven clinically meaningful in breast cancer. Indeed, high levels of NE correlate with poor outcome and endocrine resistance in breast cancer patients. In an experimental model it has been demonstrated that estrogen receptor positive (ER+) MCF7 breast cancer cells grow in suspension as 3D-spheroids in NE-addicted medium, thus resembling the micropapillae of a human breast cancer characterized by high propensity to metastasize, i.e. the micropapillary carcinoma. We hypothesized that NE may produce disarrangement of tumor cell adhesion to the substrate fostering neoplastic lymphovascular invasion (LVI) and metastasis. ER+/E-Cadherin (E-CAD)+/HER2- (MCF7, T-47D, ZR-75-1), ER+/E-CAD+/HER2+ (BT-474), ER-/E-CAD-/HER2+ (SK-BR-3) and ER-/E-CAD-/HER2- (MDA-MB-231) cells were grown with serine proteases (NE, cathepsin-G), hyaluronidase and collagenase. NE and cathepsin-G led to 3D-spheroid formation of ER+/E-CAD+ cells only. In 3D-spheroids from MCF7 cells grown with NE the luminal Epithelial Membrane Antigen (EMA) lined the external border of cell clusters, which faced cancer associated fibroblasts in co-cultures experiments, thus recapitulating the inverted polarity of MPCs. MCF7 3D-spheroids were tamoxifen resistant. Injection of NE in tumors of MCF7 cells in SCID mice triggered neoplastic cluster detachment, micropapillae, vascular emboli similar to 3D-spheorids and metastases. In a cohort of human breast carcinomas with LVI the MCF7-3D-spheroid-alike pattern was the most prevalent in tumor emboli. Immunohistochemical NE expression was mainly detected in polymorphous neutrophilic granulocytes (PMNs) within vessels and in the stroma and PMNs were significantly higher in breast carcinomas with LVI. In fully developed metastases within lymph-nodes, which reverted the EMA expression as in the primary tumor, no NE+ PMNs were observed. Our results may explain why high levels of NE negatively act on patient prognosis by creating a favorable environment for breast cancer invasion and metastasis. Citation Format: Caterina Marchio, Laura Annaratone, Davide Balmativola, Maria Stella Scalzo, Stefania Bolla, Silvia Grasso, Isabella Russo, Federica Fusella, Luigia Macri, Mara Brancaccio, Paola Defilippi, Anna Sapino. Neutrophil elastase modulates breast cancer progression by fostering collective cell detachment and tumor emboli dissemination [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 P1-07-27.

Gastric Cancer After Restrictive Bariatric Surgery: A Clinical Pitfall

Although vertical banded gastroplasty is rarely performed at present, most bariatric surgery departments continue to follow up patients who underwent this procedure in the past few decades. In view of this, it is advisable for bariatric and general surgeons to know how to diagnose the very rare event of the development of a gastric cancer after this restrictive procedure. In this report, 2 cases of gastric cancer occurring years after vertical banded gastroplasty are presented, and clinical presentation and diagnostic difficulties are discussed.