Is routine axillary lymph node dissection needed to tailor systemic treatments for breast cancer patients in the era of molecular oncology? A position paper of the Italian National Association of Breast Surgeons (ANISC).

BACKGROUND: De-escalation of axillary surgery in breast cancer (BC) management began when sentinel lymph node biopsy (SLNB) replaced axillary lymph node dissection (ALND) as standard of care in patients with node-negative BC. The second step consolidated ALND omission in selected subgroups of BC patients with up to two macrometastases and recognized BC molecular and genomic implication in predicting prognosis and planning adjuvant treatment. Outcomes from the recent RxPONDER and monarchE trials have come to challenge the previous cut-off of two SLN in order to inform decisions on systemic therapies for hormone receptor-positive (HR+), human epidermal growth factor receptor type-2 (HER2) negative BC, as the criteria included a cut-off of respectively three and four SLNs. In view of the controversy that this may lift in surgical practice, the Italian National Association of Breast Surgeons (Associazione Nazionale Italiana Senologi Chirurghi, ANISC) reviewed data regarding the latest trials on this topic and proposes an implementation in clinical practice. MATERIAL AND METHODS: We reviewed the available literature offering data on the pathological nodal status of cN0 breast cancer patients. RESULTS: The rates of pN2 status in cN0 patients ranges from 3.5 % to 16 %; pre-surgical diagnostic definition of axillary lymph node status in cN0 patients by ultrasound could be useful to inform about a possible involvement of ≥4 lymph nodes in this specific sub-groups of women. CONCLUSIONS: The Italian National Association of Breast Surgeons (ANISC) considers that for HR + HER2-/cN0-pN1(sn) BC patients undergoing breast conserving treatment the preoperative workup should be optimized for a more detailed assessment of the axilla and the technique of SLNB should be optimized, if considered appropriate by the surgeon, not considering routine ALND always indicated to determine treatment recommendations according to criteria of eligibility to RxPONDER and monarch-E trials.

Hsa-miR-155-5p Up-Regulation in Breast Cancer and Its Relevance for Treatment With Poly[ADP-Ribose] Polymerase 1 (PARP-1) Inhibitors.

miR-155-5p is a well-known oncogenic microRNA, showing frequent overexpression in human malignancies, including breast cancer. Here, we show that high miR-155-5p levels are associated with unfavorable prognostic factors in two independent breast cancer cohorts (CSS cohort, n = 283; and TCGA-BRCA dataset, n = 1,095). Consistently, miR-155-5p results as differentially expressed in the breast cancer subgroups identified by the surrogate molecular classification in the CSS cohort and the PAM50 classifier in TCGA-BRCA dataset, with the TNBC and HER2-amplified tumors carrying the highest levels. Since the analysis of TCGA-BC dataset also demonstrated a significant association between miR-155-5p levels and the presence of mutations in homologous recombination (HR) genes, we hypothesized that miR-155-5p might affect cell response to the PARP-1 inhibitor Olaparib. As expected, miR-155-5p ectopic overexpression followed by Olaparib administration resulted in a greater reduction of cell viability as compared to Olaparib administration alone, suggesting that miR-155-5p might induce a synthetic lethal effect in cancer cells when coupled with PARP-1-inhibition. Overall, our data point to a role of miR-155-5p in homologous recombination deficiency and suggest miR-155-5p might be useful in predicting response to PARP1 inhibitors in the clinical setting.

Hsa-miR-210-3p expression in breast cancer and its putative association with worse outcome in patients treated with Docetaxel.

MicroRNA-210-3p is the most prominent hypoxia regulated microRNA, and it has been found significantly overexpressed in different human cancers. We performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 76 months (n = 283). An association between higher levels of miR-210-3p and risk of disease progression (HR: 2.13, 95%CI: 1.33-3.39, P = 0.002) was found in the subgroup of patients treated with Epirubicin and Cyclophosphamide followed by Docetaxel. Moreover, a cut off value of 20.966 established by ROC curve analyses allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3- (AUC: 0.870, 95%CI: 0.690-1.000) and 83% at 5-years follow up (AUC: 0.832, 95%CI: 0.656-1.000). Whereas the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC: 0.804, 95%CI: 0.517-1.000) and 10-years (AUC: 0.804. 95%CI: 0.517-1.000) follow-up. In silico analysis of miR-210-3p and Docetaxel targets provided evidence for a putative molecular cross-talk involving microtubule regulation, drug efflux metabolism and oxidative stress response. Overall, our data point to the miR-210-3p involvement in the response to therapeutic regimens including Docetaxel in sequential therapy with anthracyclines, suggesting it may represent a predictive biomarker in breast cancer patients.

Microinvasive breast carcinoma: An analysis from ten Senonetwork Italia breast centres.

BACKGROUND AND OBJECTIVES: We studied a large series of ductal carcinoma in situ with microinvasion (MIDC) an infrequent disease whose diagnosis and management are not well defined. METHODS: 17,431 cases of breast carcinoma were treated between 2011 and 2016 by ten Italian Breast Units. Our analysis included diagnostic and clinic-pathological characteristics, surgical management, and the use of adjuvant therapies. RESULTS: 15,091 cases (86.6%) were infiltrating carcinomas (IC), 2107 (12.1%) ductal carcinoma in situ (DCIS), and 233 (1.3%) MIDC. Age at diagnosis did not differ between DCIS and MIDC. MIDC were usually larger and expressed more frequently biologically aggressive features (higher Ki67 values, hormone receptor negativity and HER2/neu over-expression) (p < 0.01). Axillary lymph nodes were involved in 25 MIDC cases (12%), but >3 lymph nodes were involved in two cases only (1%). At multivariable analysis, only lymphovascular invasion (LVI) was associated with lymph node status (p < 0.01). Hormone therapy was prescribed in 388/1462 DCIS cases (26.5%), in 84/200 MIDC cases (42%), and in 11,086/14,188 IC cases (84.7%) (p < 0.01). Chemotherapy was administered in 28/190 MIDC cases (14.7%), and in 4080/11,548 IC cases (35.3%) (p < 0.001). CONCLUSIONS: This is one of the largest studies of MIDC reported in the literature. Approximately 10% of DCIS harbor one or more foci of MIDC, and the latter often expresses aggressive biological features. LVI is a predictor of axillary node involvement, but this is infrequent and usually limited. Conservative surgery is performed less often than in DCIS, and adjuvant chemotherapy is less frequently utilized compared to IC.

Stepwise analysis of MIR9 loci identifies miR-9-5p to be involved in Oestrogen regulated pathways in breast cancer patients.

miR-9 was initially identified as an epigenetically regulated miRNA in tumours, but inconsistent findings have been reported so far. We analysed the expression of miR-9-5p, miR-9-3p, pri-miRs and MIR9 promoters methylation status in 131 breast cancer cases and 12 normal breast tissues (NBTs). The expression of both mature miRs was increased in tumours as compared to NBTs (P < 0.001) and negatively correlated with ER protein expression (P = 0.005 and P = 0.003, for miR-9-3p and miR-9-5p respectively). In addition, miR-9-5p showed a significant negative correlation with PgR (P = 0.002). Consistently, miR-9-5p and miR-9 3p were differentially expressed in the breast cancer subgroups identified by ER and PgR expression and HER2 amplification. No significant correlation between promoter methylation and pri-miRNAs expressions was found either in tumours or in NBTs. In the Luminal breast cancer subtype the expression of miR-9-5p was associated with a worse prognosis in both univariable and multivariable analyses. Ingenuity Pathway Analysis exploring the putative interactions among miR-9-5p/miR-9-3p, ER and PgR upstream and downstream regulators suggested a regulatory loop by which miR-9-5p but not miR-9-3p is induced by steroid hormone receptor and acts within hormone-receptor regulated pathways.

Current trends and outcomes of breast reconstruction following nipple-sparing mastectomy: results from a national multicentric registry with 1006 cases over a 6-year period.

BACKGROUND: Reconstruction options following nipple-sparing mastectomy (NSM) are diverse and not yet investigated with level IA evidence. The analysis of surgical and oncological outcomes of NSM from the Italian National Registry shows its safety and wide acceptance both for prophylactic and therapeutic cases. A further in-depth analysis of the reconstructive approaches with their trend over time and their failures is the aim of this study. METHODS: Data extraction from the National Database was performed restricting cases to the 2009-2014 period. Different reconstruction procedures were analyzed in terms of their distribution over time and with respect to specific indications. A 1-year minimum follow-up was conducted to assess reconstructive unsuccessful events. Univariate and multivariate analyses were performed to investigate the causes of both prosthetic and autologous failures. RESULTS: 913 patients, for a total of 1006 procedures, are included in the analysis. A prosthetic only reconstruction is accomplished in 92.2 % of cases, while pure autologous tissues are employed in 4.2 % and a hybrid (prosthetic plus autologous) in 3.6 %. Direct-to-implant (DTI) reaches 48.7 % of all reconstructions in the year 2014. Prophylactic NSMs have a DTI reconstruction in 35.6 % of cases and an autologous tissue flap in 12.9 % of cases. Failures are 2.7 % overall: 0 % in pure autologous flaps and 9.1 % in hybrid cases. Significant risk factors for failures are diabetes and the previous radiation therapy on the operated breast. CONCLUSIONS: Reconstruction following NSM is mostly prosthetic in Italy, with DTI gaining large acceptance over time. Failures are low and occurring in diabetic and irradiated patients at the multivariate analysis.

[Added value of SPECT/CT over planar imaging in improving sentinel node detection in breast cancer patients]. / Il valore aggiunto della SPECT/CT rispetto all'imaging planare nel migliorare la ricerca del linfonodo sentinella in pazienti con carcinoma mammario.

UNLABELLED: The aim of the study was to assess the diagnostic contribution of hybrid SPECT/CT lymphoscintigraphy compared to planar imaging for the correct identification of sentinel lymph nodes (SLN) in breast cancer (BC) patients. MATERIALS AND METHODS: 73 planar and SPECT/CT lymphoscintigraphies were performed in 70 consecutive patients with BC (70 women, mean age 55.7±12.0 years, range 26-84) for pre-surgical SLNs research. Cohen'K was performed to evaluate the agreement between both techniques; their diagnostic capability was evaluated by the Student's t-test for paired data. RESULTS: In 54/73 (73.9%) lymphoscintigraphies, SLNs were detected both on planar and SPECT/CT images. In 19/73 (26.1%) discordant cases, planar technique showed a higher number of SLNs in 4/19 lymphoscintigraphies, while SPECT/CT in 15/19. Radio-guided surgery confirmed SPECT/CT findings. Concordance between the two techniques was poor (K=-0.095). Overall, SPECT/CT detected 13 SLNs more than planar imaging (p=0.07). Furthermore, in 17/73 (23.2%) lymphoscintigraphies, SPECT/CT defined the exact SLNs anatomical localization, equivocal on planar images. CONCLUSIONS: SPECT/CT demonstrated added value over planar imaging, providing more information for the best surgical approach. It is useful especially in patients with negative or doubt planar imaging.

Nipple sparing mastectomy: Surgical and oncological outcomes from a national multicentric registry with 913 patients (1006 cases) over a six year period.

BACKGROUND: Nipple sparing mastectomy is deemed surgically and oncologically safe based on a long lasting literature data from reviews of single institution series. This study aims at evaluating surgical and oncological outcomes of NSM on a large multi-institutional scale, by means of the Italian National registry. METHODS: In July 2011 a panel of Italian specialists agreed upon and designed a National database of NSM. Centers with at least 150 cancers per year and following the National follow-up schedule guidelines could participate inserting any NSM case performed, retrospectively and prospectively from that moment on. In March 2015 analysis of data was accomplished. Dataset for this study consists of cases performed in the period between January 1st 2009 and December 31st 2014. RESULTS: 913 Women were included in the analysis, for a total of 1006 procedures. Prophylactic mastectomies were 124 (12.3%). MRI utilization increased over time. NSM failure rate, with NAC removal for any reason was 11.5%. NAC necrosis rate was 4.8%. Larger skin-flap necrosis rate was 2.3%. Major surgical complications rate was 4.4%. Oncological outcomes were calculated among primitive EBC cases only: locoregional recurrences rate was 2.9%, NAC recurrence 0.7%. Systemic recurrence rate was 1.0%. Five deaths (0.7%) were registered. CONCLUSIONS: More than 10% of NSM procedures are prophylactic mastectomies. MRI is gaining more importance over time. Surgical and oncological results show that NSM is effective. This National multicentric analysis enables a comparison of results with no geographical differences and a "safe" state of the art of NSM in Italy.

Evaluation of microRNA-10b prognostic significance in a prospective cohort of breast cancer patients.

BACKGROUND: MicroRNA-10b (miR-10b) has a prominent role in regulating tumor invasion and metastasis by targeting the HOXD10 transcriptional repressor and has been found up-regulated in several tumor types. METHODS: We evaluated the expression of miR-10b in paired tumor and normal specimens obtained from a prospective cohort of breast cancer patients with at least 36 months follow-up enrolled according to the REMARK guidelines (n = 150). RNA quality was measured and only samples with RNA Integrity Number (RIN) ≥7.0 were analyzed. RESULTS: The relative expression of miR-10b in tumor as compared to its normal counterpart (RER) was determined by RT-qPCR. miR-10b RERs were higher in the subgroup of patients with synchronous metastases (n = 11, Median 0.25; IQR 0.11-1.02) as compared with patients without metastases (n = 90, Median 0.09; IQR 0.04-0.29) (p = 0.028). In the subgroup of patients without synchronous metastases (n = 90), higher miR-10b RERs were associated with increased risk of disease progression and death in both univariable (HR 1.16, p = 0.021 and HR 1.20, p = 0.015 respectively for 0.10 unitary increase of miR-10b RERs levels) and multivariable (HR1.30, p < 0.001, and HR 1.31, p = 0.003 respectively for 0.10 unitary increase of miR-10b RERs levels) Cox regression models. The addition of miR-10b RERs to the Nottingham Prognostic Index (NPI) provided an improvement in discrimination power and risk reclassification abilities for the clinical outcomes at 36 months. Survival C-indices significantly increased from 0.849 to 0.889 (p = 0.009) for OS and from 0.735 to 0.767 (p = 0.050) for DFS. CONCLUSIONS: Our results provide evidences that the addition of miR-10b RERs to the prognostic factors used in clinical routine could improve the prediction abilities for both overall mortality and disease progression in breast cancer patients.

Nipple-areola-complex sparing mastectomy: five years of experience in a single centre.

This study aimed at assessing the outcomes of nipple-areola-complex-sparing mastectomy (NSM) of breast cancer in a single-centre and single-surgeon series after 5 years of experience. From June 2007 to January 2012, 58 female patients with breast cancer were admitted for NSM at our unit and 55 of these underwent NSM. All patients underwent a preoperative clinical and instrumental evaluation. The clinical evaluation consisted of physical examination by taking measurements of the prosthesis and through anamnesis. The instrumental evaluation used were mammography, chest X-ray, abdominal and breast ultrasound, blood test and, if necessary, magnetic resonance imaging. Specific inclusion criteria have been met in recruiting patients: clinically negative axillas, tumours < 3 cm in any of the quadrants, tumours more than 1 cm away from the NAC. The patients underwent a surgical and oncological follow-up. The surgical follow-up consisted in treating wounds, managing drainages and tissue expanders and then planning the replacement surgeries. The oncological follow-up consisted of periodical medical (such as blood tests including cancer markers) and radiological evaluations (such as mammography, chest X-ray, abdominal and transvaginal ultrasound scan and/or thorax-abdomen computed tomography) to assess the disease progression, and, if necessary the patients underwent chemotherapy or hormonal therapy. Out of 58 recruited patients undergoing NSM, only 3 patients (5.1 %) had an occult tumour in the NAC at intraoperative extemporaneous histological examination and required the NAC removal. The other 55 patients (94.9 %) did not have neoplastic involvement of NAC and at an average follow-up period of 21.7 months (range 3-55 months) only 4 (7.2 %) had disease progression. Nipple-Sparing Mastectomy is a highly specialized and oncologically sure procedure to be performed only in reference centres on selected patients.

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features.

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65-59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007-0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

High RAD51 mRNA expression characterize estrogen receptor-positive/progesteron receptor-negative breast cancer and is associated with patient's outcome.

Mutations in DNA double-strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)-positive/progesteron receptor (PR)-negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER-positive/PR-negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER-positive/PR-negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Changes in CpG islands promoter methylation patterns during ductal breast carcinoma progression.

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens.

Comparison between real-time quantitative PCR detection of HER2 mRNA copy number in peripheral blood and ELISA of serum HER2 protein for determining HER2 status in breast cancer patients.

BACKGROUND: The development of non-invasive procedure to determine HER2 status may represent a powerful method for monitoring disease progression and response to the treatment. METHODS: Serum samples and RNA from peripheral blood were evaluated in 85 breast cancer patients (49 HER2 positive and 36 HER2 negative) and 22 healthy controls. HER2 mRNA levels were measured by real-time quantitative PCR (QPCR) and serum HER2 protein by immunoenzimatic assay (EIA). ROC curve analyses were used to determine the optimal cut off values. RESULTS: A statistically significant difference was detected for both QPCR and EIA in HER2 positive patients as compared with both healthy controls and HER2 negative tumours. QPCR showed a 91% (CI95%: 84%-98%) specificity and a 78% (CI95%: 68%-88%) sensitivity for an optimal cut off value of 4.74. The optimal cut off value for EIA was 22 ng/ml yielding a 95% (CI95%: 90%-100%) specificity and a 59% (CI95%: 48%-70%) sensitivity. The QPCR assay was slightly less specific than EIA in discriminating HER2 positive breast cancers from HER2 negative tumours (78% CI95%: 69%-87% versus 86% CI95%: 79%-93%), but it was more sensitive (76% CI95%: 67%-85% versus 55% CI95%: 44%-66%). CONCLUSIONS: Our results indicate that QPCR performs better than EIA in the determination of HER2 status of breast cancer patients and could be useful in monitoring the disease during follow up.

Development of real-time quantitative reverse transcription-PCR for Her2 detection in peripheral blood from patients with breast cancer.

BACKGROUND: Amplification of the Her2neu oncogene is a well known indicator of poor prognosis in breast cancer patients. The implementation into the clinical setting of therapeutical strategies directly targeting the Her2neu gene product, has create the need for the development of non-invasive analytical techniques in order to monitoring minimal residual disease and response to the treatment. METHODS: To detect the expression of Her2neu mRNA in peripheral blood, we developed a specific real-time quantitative reverse transcription-PCR (QPCR) assay. The analyses were performed on blood samples obtained from 30 breast cancer patients positive for Her2neu overexpression by immunohistochemical analysis (IHC), 10 breast cancer patients negative for Her2neu overexpression, and 24 healthy controls. RESULTS: Her2neu positive tumors showed a significant increase in mRNA transcripts as compared with both healthy controls (n=24) and Her2neu negative patients (n=10). After establishing a cut-off value, 18 out of the 30 Her2neu positive patient scored positive for Her2neu expression, whereas only 1 out of the 10 Her2neu negative patients was weakly positive. CONCLUSIONS: Her2neu QPCR is suitable method for Her2neu overexpression detection in peripheral blood from clinical samples of breast cancer patients. QPCR could be used to identify breast cancer patients with poor prognosis and for monitoring response to the therapy.