miR-30e* is an independent subtype-specific prognostic marker in breast cancer.

BACKGROUND: Breast cancer clinical outcome is affected by tumor molecular features, and the identification of subtype-specific prognostic biomarkers is relevant for breast cancer translational research. Gene expression signatures proved to be able to complement prognostic information provided by classical clinico-pathological features. Recently, microRNAs (miRNAs) have been causally linked to tumorigenesis and cancer progression and have been associated with patient outcome, also in breast cancer. METHODS: MicroRNAs associated with the development of distant metastasis were identified in a cohort of 92 ESR1+/ERBB2- lymph node-negative breast cancers from patients not receiving adjuvant treatment. Results were confirmed and further investigated in a total of 1246 miRNA and gene expression profiles of the Molecular Taxonomy of Breast Cancer International Consortium data set. Moderated t-test, univariable and multivariable Cox regression models were used for statistical analyses. RESULTS: miR-30e* was identified as independent protective prognostic factor in lymph node-negative untreated patients with ESR1+/ERBB2- tumours and retained a significant association with a good prognosis in treated patients with the same tumor subtype as well as in the ERBB2+ subtype, but not in ESR1-/ERBB2- tumours. CONCLUSIONS: We highlighted a relevant and subtype-specific role in breast cancer for miR-30e* and demonstrated that adding miRNA markers to gene signatures and clinico-pathological features can help for a better prognostication.

Circulating tumor DNA for predicting recurrence in patients with operable breast cancer: a systematic review and meta-analysis.

BACKGROUND: The incorporation of circulating tumor DNA (ctDNA) into the management of operable breast cancer (BC) has been hampered by the heterogeneous results from different studies. We aimed to assess the prognostic value of ctDNA in patients with operable (non metastatic) BC. MATERIALS AND METHODS: A systematic search of databases (PubMed/Medline, Embase, and CENTRAL) and conference proceedings was conducted to identify studies reporting the association of ctDNA detection with disease-free survival (DFS) and overall survival (OS) in patients with stage I-III BC. Log-hazard ratios (HRs) were pooled at each timepoint of ctDNA assessment (baseline, after neoadjuvant therapy, and follow-up). ctDNA assays were classified as primary tumor-informed and non tumor-informed. RESULTS: Of the 3174 records identified, 57 studies including 5779 patients were eligible. In univariate analyses, ctDNA detection was associated with worse DFS at baseline [HR 2.98, 95% confidence interval (CI) 1.92-4.63], after neoadjuvant therapy (HR 7.69, 95% CI 4.83-12.24), and during follow-up (HR 14.04, 95% CI 7.55-26.11). Similarly, ctDNA detection at all timepoints was associated with worse OS (at baseline: HR 2.76, 95% CI 1.60-4.77; after neoadjuvant therapy: HR 2.72, 95% CI 1.44-5.14; and during follow-up: HR 9.19, 95% CI 3.26-25.90). Similar DFS and OS results were observed in multivariate analyses. Pooled HRs were numerically higher when ctDNA was detected at the end of neoadjuvant therapy or during follow-up and for primary tumor-informed assays. ctDNA detection sensitivity and specificity for BC recurrence ranged from 0.31 to 1.0 and 0.7 to 1.0, respectively. The mean lead time from ctDNA detection to overt recurrence was 10.81 months (range 0-58.9 months). CONCLUSIONS: ctDNA detection was associated with worse DFS and OS in patients with operable BC, particularly when detected after treatment and using primary tumor-informed assays. ctDNA detection has a high specificity for anticipating BC relapse.

A three-gene signature marks the time to locoregional recurrence in luminal-like breast cancer.

BACKGROUND: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival. PATIENTS AND METHODS: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value. RESULTS: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35). CONCLUSIONS: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence.

Early stage breast cancer follow-up in real-world clinical practice: the added value of cell free circulating tumor DNA.

PURPOSE: Physical examinations and annual mammography (minimal follow-up) are as effective as laboratory/imaging tests (intensive follow-up) in detecting breast cancer (BC) recurrence. This statement is now challenged by the availability of new diagnostic tools for asymptomatic cases. Herein, we analyzed current practices and circulating tumor DNA (ctDNA) in monitoring high-risk BC patients treated with curative intent in a comprehensive cancer center. PATIENTS AND METHODS: Forty-two consecutive triple negative BC patients undergoing neoadjuvant therapy and surgery were prospectively enrolled. Data from plasma samples and surveillance procedures were analyzed to report the diagnostic pattern of relapsed cases, i.e., by symptoms, follow-up procedures and ctDNA. RESULTS: Besides minimal follow-up, 97% and 79% of patients had at least 1 non-recommended imaging and laboratory tests for surveillance purposes. During a median follow-up of 5.1(IQR, 4.1-5.9) years, 13 events occurred (1 contralateral BC, 1 loco-regional recurrence, 10 metastases, and 1 death). Five recurrent cases were diagnosed by intensive follow-up, 5 by symptoms, and 2 incidentally. ctDNA antedated disseminated disease in all evaluable cases excepted two with bone-only and single liver metastases. The mean time from ctDNA detection to suspicious findings at follow-up imaging was 3.81(SD, 2.68), and to definitive recurrence diagnosis 8(SD, 2.98) months. ctDNA was undetectable in the absence of disease and in two suspected cases not subsequently confirmed. CONCLUSIONS: Some relapses are still symptomatic despite the extensive use of intensive follow-up. ctDNA is a specific test, sensitive enough to detect recurrence before other methods, suitable for clarifying equivocal imaging, and exploitable for salvage therapy in asymptomatic BC survivors.

Mammographic density to predict response to neoadjuvant systemic breast cancer therapy.

BACKGROUND: Mammographic density (MD) is a risk factor for breast cancer (BC) development, and recurrence. However, its predictive value has been less studied. Herein, we challenged MD as a biomarker associated with response in patients treated with neoadjuvant therapy (NAT). METHODS: Data on all NAT treated BC patients prospectively collected in the registry of Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy (2009-2019) were identified. Diagnostic mammograms were used to evaluate and score MD as categorized by the Breast Imaging-Reporting and Data System (BI-RADS), which identifies 4 levels of MD in keeping with relative increase of fibro-glandular over fat tissue. Each case was classified according to the following categories a (MD < 25%), b (26-50%), c (51-75%), and d (> 75%). The association between MD and pathological complete response (pCR), i.e., absence of BC cells in surgical specimens, was analyzed in multivariable setting used logistic regression models with adjustment for clinical and pathological variables. RESULTS: A total of 442 patients were analyzed, 120 of which (27.1%) attained a pCR. BI-RADS categories a, b, c, and d accounted for 10.0%, 37.8%, 37.1% and 15.2% of cases. Corresponding pCR were 20.5%, 26.9%, 30.5%, 23.9%, respectively. At multivariable analysis, when compared to cases classified as BI-RADS a, those with denser breast showed an increased likelihood of pCR with odds ratio (OR) of 1.70, 2.79, and 1.47 for b, c and d categories, respectively (p = 0.0996), independently of age, BMI [OR underweight versus (vs) normal = 3.76], clinical nodal and tumor status (OR T1/Tx vs T4 = 3.87), molecular subtype (HER2-positive vs luminal = 10.74; triple-negative vs luminal = 8.19). In subgroup analyses, the association of MD with pCR was remarkable in triple-negative (ORs of b, c and d versus a: 1.85, 2.49 and 1.55, respectively) and HER2-positive BC cases (ORs 2.70, 3.23, and 1.16). CONCLUSION: Patients with dense breast are more likely to attain a pCR at net of other predictive factors. The potential of MD to assist decisions on BC management and as a stratification factor in neoadjuvant clinical trials should be considered.

Radical metastasectomy followed by sorafenib versus observation in patients withclear cell renal cell carcinoma: extended follow -up of efficacy results from the randomized phase II RESORT trial.

Background: The RESORT trial showed no longer relapse free survival (RFS) with sorafenib following radical metastasectomy in metastatic renal cell carcinoma. We present the updated 42-month follow-up data.Methods: The phase II RESORT trial randomized patients to sorafenib or observation within 12 weeks from surgery. RFS was the primary endpoint.Results: We analyzed 68 patients (32 in sorafenib and 36 in the observation arm), randomized between November 2012 and November 2017. Eighty-one percent in the sorafenib arm and 80% in the observation arm had one metastasis . At a median follow-up of 42 months (interquartile range 31-58), in the observation arm the median RFS was 35 months, RFS probability was 57% (95% CI 42-76%) at 24 and 44% (95% CI 30-65%) at 48 months. In the sorafenib arm, median RFS was 21 months, RFS probability was 50% (95% CI 34-71%) at 24 and 32% (95% CI 18-57%) at 48 months (p = 0.342;HR 1.35;95% CI 0.72-2.54). Forty-seven percent and 37.5% of the patients in the two arms, respectively, are disease free. The site of relapses was independent of the previous metastasectomy site.Expert commentary: Sorafenib after metastasectomy did not improve RFS, but surgery in selected patients should be considered in order to potentially improve survival.Clinical trial registration: www.clinicaltrials.gov identifier is NCT0144480.

Blood-based genomics of triple-negative breast cancer progression in patients treated with neoadjuvant chemotherapy.

BACKGROUND: As neoadjuvant chemotherapy (NAC) is increasingly used in triple-negative breast cancer (TNBC), we investigated the value of circulating tumor DNA (ctDNA) for patient monitoring prior, during, and after NAC, and circulating tumor cells (CTCs) for disease characterization at clinical progression. MATERIALS AND METHODS: Forty-two TNBC patients undergoing NAC were prospectively enrolled. Primary tumor mutations identified by targeted-gene sequencing were validated and tracked in 168 plasma samples longitudinally collected at multiple time-points by droplet digital polymerase chain reaction. At progression, plasma DNA underwent direct targeted-gene assay, and CTCs were collected and analyzed for copy number alterations (CNAs) by low-pass whole genome sequencing. RESULTS: ctDNA detection after NAC was associated with increased risk of relapse, with 2-year event-free survival estimates being 44.4% [95% confidence interval (CI) 21.4%-92.3%] versus 77.4% (95% CI 57.8%-100%). ctDNA prognostic value remained worthy even after adjusting for age, residual disease, systemic inflammatory indices, and Ki-67 [hazard ratio (HR) 1.91; 95% CI 0.51-7.08]. During follow-up, ctDNA was undetectable in non-recurrent cases with the unique exception of one showing a temporary peak over eight samples. Conversely, ctDNA was detected in 8/11 recurrent cases, and predated the clinical diagnosis up to 13 months. Notably, recurrent cases without ctDNA developed locoregional, contralateral, and bone-only disease. At clinical progression, CTCs presented chromosome 10 and 21q CNAs whose network analysis showed connected modules including HER/PI3K/Ras/JAK signaling and immune response. CONCLUSION: ctDNA is not only associated with but is also predictive of prognosis in TNBC patients receiving NAC, and represents an exploitable tool, either alone or with CTCs, for personalized TNBC management.

Questioning the utility of pooling samples in microarray experiments with cell lines.

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

p53 as an independent prognostic marker in lymph node-negative breast cancer patients.

BACKGROUND: At present, most decisions concerning the use of adjuvant therapy in lymph node-negative breast cancer patients are made on the basis of traditional factors such as tumor size, nodal status, and histopathologic features. However, prognostic factors are being investigated that could identify high-risk groups and that could better address treatment efforts for those patients. Identification of more accurate prognostic markers, such as the expression of the mutant p53 protein encoded by the p53 (also known as TP53) tumor suppressor gene, that are reproducible, easily assessable, and independent in predicting clinical outcome would have a beneficial impact on cancer treatment decisions. PURPOSE: Our purpose was to analyze the predictive relevance of mutant p53 protein expression on 6-year relapse-free and overall survival in node-negative breast cancer patients in relation to menopausal status, tumor size, cell kinetics, and estrogen receptor status. METHODS: Expression of mutant p53 protein was detected by an immunohistochemical technique using a 1:50 dilution of PAb1801 monoclonal antibody on paraffin-embedded tumor specimens obtained from 256 axillary lymph node-negative breast cancer patients, with long-term follow-up (median, 72 months). The [3H]thymidine labeling index, a measure of cell kinetics, was evaluated on histologic sections after fresh tumor tissue was labeled with [3H]thymidine. Estrogen receptor status was determined by the dextran-coated charcoal absorption technique. Statistical comparisons were made for levels of p53 protein expression, [3H]thymidine labeling index, estrogen receptor status, tumor size, and menopausal status with respect to 6-year relapse-free survival and overall survival. RESULTS: Overexpression of the p53 protein, defined as the presence of more than 5% positive cells, was detected in 113 (44%) of 256 tumors. Odds ratios (ORs) for multiple regression analysis of 6-year relapse-free survival were significantly higher for p53 (OR = 3.24; 95% confidence limits [CL] = 2.01-5.23) and [3H]thymidine labeling index (OR = 1.92; 95% CL = 1.19-3.12), both of which appeared to be the most relevant indicators of relapse, than for tumor size (OR = 1.49; 95% CL = 0.94-2.38) and estrogen receptor status (OR = 0.91; 95% CL = 0.55-1.51). Overexpression was found to be unrelated to menopausal status. CONCLUSIONS: Immunohistochemically detected p53 overexpression is an independent marker for shortened 6-year relapse-free and overall survival in node-negative patients with resectable breast cancers. Based on these findings, p53 overexpression should be used with other established prognostic factors, such as [3H]thymidine labeling index and estrogen receptor status, to further refine the prognostic assessment of node-negative breast cancer.

Comparison of immunochemical and radioligand binding assays for estrogen receptors in human breast tumors.

We have compared a new enzyme immunoassay (EIA) for estrogen receptors (ER) with our conventional radioligand binding assays (multipoint dextran-coated charcoal assay for cytoplasmic ER and hydroxylapatite exchange assay for nuclear ER). Cytoplasmic ERs were measured in 76 human breast cancer specimens by EIA and by five-point Scatchard analysis. The correlation between the two assays yielded a straight line with a slope of 0.92 (r = 0.95; P less than 0.001); conversely, in 31 nuclear salt extracts, linear regression analysis of hydroxylapatite exchange assay data with EIA showed a clear correlation (r = 0.93; P less than 0.001) but a slope of 1.7, demonstrating that EIA detects more ER sites. The binding of the antibody to the cytoplasmic ER molecules was investigated by sucrose density gradient analysis, which showed that EIA recognizes both cytoplasmic forms (9 and 3S), but does not distinguish between them. Advantages and drawbacks of this method are discussed with respect to its application for routine receptor determination for clinical management of breast cancer patients.

Prognostic relevance of cathepsin D versus oestrogen receptors in node negative breast cancers.

The concentration of total cathepsin D in cytosols of 199 node negative women with primary breast cancer in a 10-year retrospective cohort was assayed. Cathepsin D status alone was unable to predict disease-free or overall survival. However, those patients with receptor positive tumours who were cathepsin D positive had shorter [corrected] disease-free (P = 0.02) and overall survival (P = 0.01) than cathepsin D negative patients. Therefore, measurement of cathepsin D appears to provide additional prognostic information for the prediction of disease-free and overall survival in patients with node negative breast cancer.

Interaction between hormone-dependent and hormone-independent human breast cancer cells.

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.

Prognostic relevance of pS2 status in association with steroid receptor status and proliferative activity in node-negative breast cancer.

Expression of the oestrogen-regulated pS2 protein was investigated on paraffin-embedded sections of primary breast tumours from 200 node-negative patients. Immunoreactivity was observed in 56% of the cases. pS2 expression was inversely correlated with tumour size and proliferative activity, whereas a direct correlation was observed with steroid receptor. 5-year relapse free survival was influenced by tumour size (P = 0.02), oestrogen receptor status (P less than 0.05), and proliferative activity (P less than 0.01). No difference in relapse-free survival was observed between patients subdivided according to pS2 expression alone. However, among patients with oestrogen-receptor-negative tumors, pS2 expression predicted a shorter relapse-free survival.

Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro.

Genistein significantly inhibited cell growth (IC50 around 10 microM) of MCF-7, MDAMB-231 and HBL-100 cell lines, but not of skin-derived fibroblasts and counteracted the growth-stimulatory effects exerted by estradiol and growth factors. It abolished the paracrine stimulation observed in MCF-7 cells in co-culture with MDAMB-231 or fibroblasts. Genistein-treated cells accumulated in the S and G2/M phases of the cell cycle and underwent apoptosis. Genistein decreased tyrosine phosphorylation induced upon treatment with transforming growth factor-alpha. Finally, genistein bound the estrogen receptor (ER) (relative affinity constant Kd = 4 nM), induced pS2 and cathepsin-D transcription and increased nuclear ER levels.

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells.

17 beta-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degree of estrogenic action in endocrine-dependent breast cancer cell lines.

Synthetic analogs of vitamin D3 have inhibitory effects on breast cancer cell lines.

BACKGROUND: 1,25-dihydroxycholecalciferol has been previously reported to negatively regulate human breast cancer cell growth. MATERIAL AND METHODS: The antiproliferative effect of 1,25-dihydroxycholecalciferol (Ro 21-5535) and of the two non hypercalcemic analogs (1a,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol+ ++, Ro 24-5531 and 1a,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholec alciferol, Ro 25-6760) was studied in MCF-7 and MDAMB-468 human breast cancer cell lines. Cell cycle distribution and apoptosis were evaluated by flow cytometry. Steroid receptor modulation was investigated by radioligand assay. RESULTS: The most effective drug was the Ro 25-6760 which at concentrations ranging between 1-100 nM caused a dose dependent growth inhibition apparently due to accumulation in G0/G1. Vitamin D3 analogs (10 nM) significantly counteracted the growth stimulation induced by TGF-a and IGF-I as well as the paracrine stimulation observed in co-cultures. They antagonized estradiol-promoted growth stimulation and progestrone receptor induction in MCF-7 cells. CONCLUSION: Vitamin D3 analogs represent a class of clinically attractive drugs for treatment of breast cancer due to their ability to counteract estradiol and growth factor-induced growth stimulation.

Activity of tamoxifen and new antiestrogens on estrogen receptor positive and negative breast cancer cells.

The effect of some new antiestrogens (ICI 164384, ICI 182780, Ly 133314 and Ly 117018) on the growth of a panel of breast cancer cell lines (MDA-MB 231, BT20, MCF7 and T47D) was studied. Their antiestrogenic activity was investigated in the presence of a physiologic concentration of estradiol and compared with the effect displayed by tamoxifen in the same experimental conditions. Tamoxifen confirmed its antagonistic activity in the presence of estradiol, whereas when given alone it did not exhibit a clear antiproliferative effect. All other compounds showed variable activity: ICI 164384 and ICI 182780 exerted a variable inhibitory effect in all cell lines, but only in the absence of estradiol; Ly 133314 and Ly 117018 did not show a clear antagonistic activity and sometimes had a synergistic effect with estradiol in increasing cell growth. These results indicate an extreme variability in terms of antagonistic effect of these new compounds and suggest their inadequacy to replace tamoxifen as an antiestrogen during endocrine treatment. Interestingly, ICI 164384 exerted an antiproliferative action also an estrogen receptor-negative cell lines, probably through an alternative mechanism non estrogen receptor-mediated, supporting the hypothesis that it could be effective on estrogen receptor-positive as well as estrogen receptor-negative tumors.

Effects of toremifene and its main metabolites on growth of breast cancer cell lines.

The effects of toremifene, an antiestrogen, and its two main metabolites (4-hydroxy- and N-desmethyltoremifene) on cell proliferation have been tested in a series of breast cancer cell lines, with positive (MCF7, ZR 75.1, T47D, 734B) or negative steroid receptor status (MDA-MB 231, BT20). Drug effects were evaluated at concentrations similar to those obtained in breast cancer patients on toremifene treatment, and in the presence or absence of estradiol. Results showed that, for both positive and negative cell lines, high concentrations of the antiestrogens (10(-6)M) were inhibitory, while lower doses (10(-7)M, 10(-8)M) were stimulatory. These data confirm the biphasic behaviour of toremifene and its derivatives. In terms of a clinical application of toremifene, these results seem to suggest that while high doses of toremifene and its derivatives might be therapeutic, lower concentrations might stimulate cell growth and enhance tumor progression.