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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.

Breast cancer is a heterogeneous tumor type characterized by a complex spectrum of molecular aberrations, resulting in a diverse array of malignant features and clinical outcomes. Deciphering the molecular mechanisms that fuel breast cancer development and act as determinants of aggressiveness is a primary need to improve patient management. Among other alterations, aberrant expression of microRNAs has been found in breast cancer and other human tumors, where they act as either oncogenes or tumor suppressors by virtue of their ability to finely modulate gene expression at the post-transcriptional level. In this study, we describe a new role for miR-181a/b as negative regulators of the DNA damage response in breast cancer, impacting on the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). We report that miR-181a and miR-181b were overexpressed in more aggressive breast cancers, and their expression correlates inversely with ATM levels. Moreover we demonstrate that deregulated expression of miR-181a/b determines the sensitivity of triple-negative breast cancer cells to the poly-ADP-ribose-polymerase1 (PARP1) inhibition. These evidences suggest that monitoring the expression of miR-181a/b could be helpful in tailoring more effective treatments based on inhibition of PARP1 in breast and other tumor types.

Sodium butyrate (NaB), a product of colonic fermentation of dietary fibre, has been shown to inhibit cell proliferation by blocking cells in the G(0)/G(1) phase of the cell cycle through a mechanism of action still not completely understood. We investigated the effect of NaB on the level of some G(1) phase‐related proteins in a colon carcinoma cell line (HT29). In particular, we addressed our attention to cyclin D1 (the key regulator of G(1)S progression), p21(waf1/cip1) (the main inactivator of the cyclin D/cdk complex), and p53 (the most important regulator of p21(waf1/cip1) gene transcription). At inhibitory concentrations (higher than 1 mM) NaB reduced cyclin D1 and p53 level in a dose‐dependent manner and sustained the synthesis of p21(waf1/cip1), probably in a p53‐independent way, accounting for the G(0)/G(1) block observed by flow cytometry. Present results provide further evidence on the molecular mechanism at the basis of the physiological role of NaB and support the hypothesis that an unbalanced diet, poor in carbohydrates and therefore in NaB, could result in functional alterations with clinical and carcinogenic implications.

Cholesteryl-butyrate chosen as lipid matrix of solid lipid nanospheres (SLNs) could be a suitable pro-drug to deliver butyric acid and overcome one of the most limiting disadvantages of the compound: the short half-life due to a rapid metabolism.We evaluated the antiproliferative effect, with respect to that of sodium butyrate, of four SLNs (SLN1, SLN2, SLN3 and SLN4) characterized by a different concentration of cholesteryl-butyrate (range, 1.7-30 mM) on NIH-H460, a non-small cell lung carcinoma cell line.After 6 days of treatment, all SLN preparations induced a dose-dependent inhibition of NHI-H460 cell growth: the most effective SLN preparation (SLN1) was able to induce a complete growth inhibition already at 0.25 mM, a concentration at which sodium butyrate induced only a 55% inhibition. Fluorescence microscopy showed that 6-coumarin-tagged SLNs were almost completely internalized by cells after 5 min of treatment.The present results indicate that owing to their peculiar characteristics, SLNs could be an interesting alternative approach for butyric acid delivery into tumor cells.

The recent availability of the monoclonal antibody MIB‐1 (which is able to detect the human nuclear cell proliferation‐associated antigen Ki‐67 even on formalin‐fixed, paraffin‐embedded sections, microwave‐treated and routinely processed for immunohistochemistry) could open new avenues for validation of the clinical role of tumour cell proliferation on large, consecutive and unselected series of human tumours. However, the routine use of such a marker requires a methodological standardization as well as the comparative assessment of some technical and biological aspects. The MIB‐1 index was determined in parallel samples from 50 consecutive invasive breast carcinomas processed with different fixatives for different times. The median values of MIB‐1 indices following 2, 6 and 24 h of formalin fixation were similar (29.4%, 30.6% and 29.7%, respectively) and consistent with those reported in the literature; squared linear regression coefficients were 0.99. The median values of MIB‐1 indices were markedly lower in Bouin‐fixed, paraffin‐embedded, and in frozen samples (20.0% and 19.8%, respectively), with a poor correlation coefficient with the values detected following formalin fixation (R (2)=0.456). Moderate and poor correlations were observed between Ki‐67 index and MIB‐1 detected on frozen (r (s), 0.78) or formalin‐fixed, paraffin‐embedded samples (r (s), 0.47) and a minimal concordance was observed between TLI and MIB‐1 or Ki‐67 (r (s), 0.25 and 0.22, respectively). Our results indicate interference of the fixative type on immunoreactivity to MIB‐1 and also suggest that Ki‐67 and MIB‐1 reacted with different epitopes of the same antigen.

Most studies are in favor of a prognostic relevance of p53 accumulation determined by immunohistochemistry in breast cancer, but negative results are not lacking. On a series of 1400 patients with lymph node-negative cancers treated with local-regional therapy alone until relapse and with a median follow-up of 10 years, we validated the prognostic relevance for overall relapse and death of p53 accumulation observed in a pilot study and analyzed its predictivity on different adverse events. p53 protein accumulation was immunocytochemically detected using PAb1801. The case series had also been previously characterized for hormone receptor content [estrogen receptors (ERs) and progesterone receptors (PgRs)] and for cell proliferation [[3H]thymidine labeling index ([3H]dT LI)]. p53 expression, considered as a dichotomous variable with a cutoff value of 5% positive cells, significantly predicted the occurrence of overall relapse, distant metastasis, and death with an interaction with cell proliferation. p53 accumulation, cell proliferation, and their interaction term, along with tumor size and patient age, retained a predictive role for overall relapse, and together with tumor size and PgR, also for overall survival. When considered as continuous variables, we observed that the hazard of metastasis increased linearly with the increase of [3H]dT LI and decreased linearly with the increase of ER and PgR. Conversely, the hazard increased with the increase of p53-positive cells only for tumors with a [3H]dT LI lower than 7.5%. In multivariate analysis, the same prognostic factors for distant metastasis were identified when the biomarkers were considered as continuous or dichotomous variables.

From December 1980 to September 1985, a total of 90 eligible patients with stage T1-3a, node-negative, and estrogen receptor-negative (less than or equal to 10 fmol/mg of cytosol protein) tumors were entered into a randomized study to assess the effectiveness of adjuvant intravenous (IV) cyclophosphamide, methotrexate, and fluorouracil (CMF) in a subset of patients at high risk of early disease relapse. High values of [3H]thymidine labeling index were documented in two thirds of 62 assessed specimens from the patient population, and one half of the patients had histologically undifferentiated tumors. Patients were allocated to either local-regional modality alone (control group, 45 women) or to CMF (45 patients) after surgery. A full dose of CMF (600 mg/m2 each of cyclophosphamide and fluorouracil, and 40 mg/m2 of methotrexate) was administered IV on day 1, and then repeated every 3 weeks for a total of 12 treatments. After a median follow-up of 80 months, the 7-year results confirmed the superiority of adjuvant CMF compared to local-regional modality alone (relapse-free survival 85% vs 42%, P = .0001; total survival 86% vs 58%, P = .006). A benefit from adjuvant CMF was observed in all subgroups, and the rates of both local-regional and distant failure were decreased. Treatment was fairly well tolerated and devoid of life-threatening toxicity. Present results confirm our previous observation concerning the dismal prognosis of node-negative and estrogen receptor-negative breast cancer patients as well as the beneficial effect of adjuvant chemotherapy in this selected subset.

The last few years have shown an extraordinary development of laboratory and clinical research in breast cancer. The areas which appear particularly important for possible clinical application are many and diversified. In this review we will update the present knowledge regarding four major issues: 1) biologic and molecular tests of prognostic significance in operable breast cancer patients; 2) detection of breast cancer cells in the bone marrow; 3) the endocrine aspects of breast cancer treatment; and 4) the indication of conservative treatments.

The enzymatic pattern of five enzymes involved in the purine salvage pathway, namely purine nucleoside phosphorylase (EC 2.4.2.1), adenosine deaminase (EC 3.5.4.4), 5'-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been evaluated both in human intestinal and breast carcinomas and compared to that of normal tissues. A higher level of hypoxanthine-guanine phosphoribosyltransferase was associated with tumor tissues. This metabolic alteration should lead to an elevated synthesis of nucleotides in cancer cells, might confer selective growth advantages to neoplastic tissues, and account, at least in part, for the difficulties encountered in the chemotherapy of human tumors, by using compounds affecting only the purine de novo biosynthesis.

Tumor cellular proliferative activity was evaluated by 3H-thymidine labeling index (LI) determination in 73 lesions from 62 patients with ovarian cancers. The median LI value for the overall series was 5.8% and places this tumor type in a position of intermediate proliferative activity. In this case series, the relationship between proliferative activity and different morphologic and pathologic characteristics of the disease was analyzed. Similar median LI values were found for ascitic effusions and solid tumors and, among these, between primary tumors and metastases. No significant relation was observed between proliferative activity and histologic type, whereas a definite direct correlation was found between the kinetic variable and prognostic features such as pathologic stage and histologic grading. In fact median LI was higher for stage IV for stage I and for grade 3 than for grade 1 tumors. The strong association between tumor proliferative rate and conventional prognostic factors suggests that also on this tumor type the kinetic variable could play a role as a prognostic indicator and modulator of aggressiveness.

The study analyzes the prognostic implication of cell kinetics on 52 locally advanced breast cancers. All patients were subjected to a multimodal treatment, including primary chemotherapy with doxorubicin and vincristine, surgery, or radiotherapy followed by additional chemotherapy with the same drug regimen. Pretreatment labeling index (LI) is not related to response to primary chemotherapy, whereas it provides information on the course of the disease. In fact, high LI significantly predicts a higher progression rate at the end of the entire combined treatment, a shorter time to disease progression, and a poorer probability of 4-year survival in comparison to low LI. From the present findings, LI appears to be a useful tool to select women with locally advanced breast cancer that would require very aggressive treatment.

The proliferative activity of breast cancer has been analyzed in relation to the hormonal characteristics of the host and of the tumor for 199 patients. The analyses of labeling index frequency distributions of estrogen receptor positive (ER+) and negative (ER-) cancers from premenopausal and postmenopausal patients have allowed us to identify three different kinetic groups. A first group, with a very low proliferative activity, includes ER+ cancers from postmenopausal patients; a second group, with an intermediate proliferative activity, includes ER+ cancers from premenopausal and ER- cancers from postmenopausal patients; and a third group, with a very high proliferative activity, includes ER- cancers from premenopausal patients. Generally, the amount of estrogen receptors in ER+ cancers is inversely correlated with the proliferative activity. Lower levels of ER in premenopausal in comparison to postmenopausal patients were found in low proliferative activity tumors.

The feasibility and reliability of an in vitro assay that evaluates drug interference on nucleic acid precursor incorporation were investigated on 135 previously untreated locally advanced breast cancers. The assay, which was carried out on tumor fragments incubated for 3 hours with drugs, proved to be feasible on a sufficiently high percentage of biopsy specimens (70%) for routine clinical use. In vitro drug activity evaluated with this assay appeared to reproduce the clinical patterns of sensitivity of the tumor type as well as of the individual tumors. In fact, in vitro response rates to conventional agents resembled the clinical response rates reported for the same agents used in monochemotherapy. From a retrospective--correlative study carried out on 41 patients treated in vitro and in vivo with the same drugs (Adriamycin [doxorubicin] and vincristine), in vitro effect of Adriamycin on 3H-uridine incorporation appeared significantly correlated with clinical response (overall agreement, 78%; P = 0.0032) with specific agreements of sensitivity and resistance of 75% and 81%, respectively.

The relevance of tumor proliferative activity as an indicator of biologic aggressiveness was analyzed on a series of 506 patients with primary breast cancer. In 258 patients with operable tumors without nodal and distant metastases, none of whom was subjected to postoperative irradiation or systemic adjuvant therapy, proliferative activity was significantly correlated with prognosis; 6-year relapse-free survival (RFS) and overall survival (OS) were higher for patients with slowly proliferating tumors for patient with fast-proliferating tumors (RFS: 80.5% vs 59.6%, p = 0.00004; OS: 90.8% vs 74.4%, p = 0.002). On a series of 196 patients with node-positive operable tumors, subjected to 6 or 12 cycles of cyclophosphamide, methotrexate and 5-fluorouracil, a trend in favor of longer 6-year RFS was observed for patients with slowly proliferating tumors than for patients with fast-proliferating tumors (62.5% vs 48.3%, p = 0.08), whereas proliferative activity did not influence OS. In 52 patients with locally advanced disease treated with a multimodality approach, including chemotherapy (adriamycin and vincristine), surgery or radiotherapy, tumor proliferative activity was a strong indicator of biologic aggressivity, since women with slowly proliferating cancers had a higher 4-year probability of OS than women with fast-proliferating tumors (68.1% vs 36.7%, p = 0.02).

The stimulation of RNA synthesis induced by estradiol has been studied on 60 breast cancers in short-term cultures. In the same tumors the estrogen receptor (ER) assay was carried out. In 35% of the cases studied the stimulating effect by estradiol in vitro was observed. The frequency of response to in vitro hormone treatment was higher (42%) in ER+ cancers and lower (25%) in ER- cancers. The specificity of the in vitro test was assessed by the use of tamoxifen.

The labeling index has been determined in 34 non-Hodgkin malignant lymphomas. The kinetic parameter has been analyzed in relation to the different histologic types, according to the Kiel calssification, and a kinetic classification with three main groups at low, intermediate, and high proliferative activity has been proposed. The analysis of the survival of the patients in relation to the labeling index of the malignant lymphoma cell population has shown that the potential proliferative activity has an important prognostic significance.

Several in vivoand in vitro data demonstrated that the DNA damage response (DDR) plays a crucial role in tumorigenesisacting as an anti-cancer barrier. Indeed, cells respond to DNA breaks caused by both exogenous (e.g. chemicals, chemotherapeutics) and endogenous (e.g. deregulated function of oncogenes) sources of DNA damages by properly triggering the activation of the DDR. The latter involves the recruitment of DNA repair proteins to sites of damage and the activation of checkpoint mechanisms to slow down cell cycle progression, thus allowing DNA damage repair or, if the damage could not be repaired, leading to apoptosis or cellular senescence. As a consequence, alterations of DDR functionality have a deep impact on cell proliferation, survival, genomic instability and tumorprogression. microRNAs(miRNAs) are small non coding RNAs that finely regulate gene expression by binding the 3’UTR of their target mRNAs, thus altering theirtranslation, stability and localization. It has been shown that several miRNAsmodulate critical cellular processes deregulated in cancer, acting either as oncogenesor tumorsuppressors. In the present study, we focused on the identification of miRNAsas new modulators of DNA damage activated signalingin several normal and transformed breast cancer cells. In particular, we discovered that miR-181 family acts as a modulator of the levels of ATM (ataxia telangiectasiamutated) kinase, one of the most important proteins involved in sensing DNA double strand breaks and activating DDR. The role of miR-181 family in the DDR pathway, its involvement in the induction of cellular senescence triggered by oncogenicstress (OIS) and its relevance in breast cancer will be presented.