Sara Ravaioli

Cell-free DNA detected by “liquid biopsy” as a potential prognostic biomarker in early breast cancer

As conventional biomarkers for defining breast cancer (BC) subtypes are not always capable of predicting prognosis, search for new biomarkers which can be easily detected by liquid biopsy is ongoing. It has long been known that cell-free DNA (CF-DNA) could be a promising diagnostic and prognostic marker in different tumor types, although its prognostic value in BC is yet to be confirmed. This retrospective study evaluated the prognostic role of CF-DNA quantity and integrity of HER2, MYC, BCAS1 and PI3KCA, which are frequently altered in BC. We collected 79 serum samples before surgery from women at first diagnosis of BC at Forlì Hospital (Italy) from 2002 to 2010. Twenty-one relapsed and 58 non-relapsed patients were matched by subtype and age. Blood samples were also collected from 10 healthy donors. All samples were analyzed by Real Time PCR for CF-DNA quantity and integrity of all oncogenes. Except for MYC, BC patients showed significantly higher median values of CF-DNA quantity (ng) than healthy controls, who had higher integrity and lower apoptotic index. A difference nearing statistical significance was observed for HER2 short CF-DNA (p = 0.078, AUC value: 0.6305). HER2 short CF-DNA showed an odds ratio of 1.39 for disease recurrence with p = 0.056 (95% CI 0.991-1.973). Our study suggests that CF-DNA detected as liquid biopsy could have great potential in clinical practice once demonstration of its clinical validity and utility has been provided by prospective studies with robust assays.

Multiple myeloma-derived Jagged ligands increases autocrine and paracrine interleukin-6 expression in bone marrow niche

Multiple myeloma cell growth relies on intrinsic aggressiveness, due to a high karyotypic instability, or on the support from bone marrow (BM) niche. We and other groups have provided evidences that Notch signaling is related to tumor cell growth, pharmacological resistance, localization/recirculation in the BM and bone disease. This study indicates that high gene expression levels of Notch signaling members (JAG1, NOTCH2, HES5 and HES6) correlate with malignant progression or high-risk disease, and Notch signaling may participate in myeloma progression by increasing the BM levels of interleukin-6 (IL-6), a major player in myeloma cell growth and survival. Indeed, in vitro results, confirmed by correlation analysis on gene expression profiles of myeloma patients and immunohistochemical studies, demonstrated that Notch signaling controls IL-6 gene expression in those myeloma cells capable of IL-6 autonomous production as well as in surrounding BM stromal cells. In both cases Notch signaling activation may be triggered by myeloma cell-derived Jagged ligands. The evidence that Notch signaling positively controls IL-6 in the myeloma-associated BM makes this pathway a key mediator of tumor-directed reprogramming of the bone niche. This work strengthens the rationale for a novel Notch-directed therapy in multiple myeloma based on the inhibition of Jagged ligands.

Cell-Free DNA Integrity Analysis in Urine Samples.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals. A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

PSMA expression: a potential ally for the pathologist in prostate cancer diagnosis

Prostate cancer (PCa) patients are risk-stratified on the basis of clinical stage and PSA level at diagnosis and the Gleason Score (GS) in prostate biopsy. However, these parameters are not completely accurate in discriminating between high- and low-risk disease, creating a need for a reliable marker to determine aggressiveness. Prostate-specific membrane antigen (PSMA) appears to fulfill this need. We analyzed 79 prostate biopsies and 28 prostatectomies to assess whether PSMA expression detected by immunohistochemistry is related to GS. PSMA expression was correlated with GS in both sample types (biopsies, P < 0.0001 and prostatectomy samples, P = 0.007). We observed lower PSMA expression in Gleason pattern 3 than Gleason pattern 4, suggesting that this biomarker could be useful to distinguish between these entities (p < 0.0001). The best cut-off value of 45% immunopositivity was determined by receiver operating characteristic (ROC) curve analysis. In Gleason pattern 3 vs. Gleason pattern 4 and 5, PSMA sensitivity was 84.1% (95% CI 76.5%-91.7%) and specificity was 95.2% (95% CI 90.6%-99.8%), with an area under the curve of 93.1 (95% CI 88.8–97.4). Our results suggest that PSMA represents a potential ally for the pathologist in the diagnostic work-up of PCa to overcome long-standing morphological classification limits.

Easily detectable cytomorphological features to evaluate during ROSE for rapid lung cancer diagnosis: from cytology to histology

In lung cancer patients, the only available diagnostic material often comes from biopsy or from cytological samples obtained by fine needle aspiration (FNA). There is a lack of easily detectable cytomorphological features for rapid on-site evaluation (ROSE) to orient lung cancer diagnosis towards a specific tumor histotype. We studied the cytological features evaluated on site to define tumor histotype and to establish the number of specimens to be taken. Cytological specimens from 273 consecutive patients were analyzed with ROSE: bronchoscopy with transbronchial needle aspiration (TBNA) had been performed in 72 patients and with endobronchial ultrasound (EBUS)-TBNA in 201. Cytomorphological features were correlated with the final diagnosis and diagnostic accuracy was measured. Analysis of the different cytomorphological parameters showed that the best sensitivity and specificity were obtained for adenocarcinoma by combining the presence of nucleoli and small/medium cell clusters, and for squamous cell carcinoma by considering the presence of necrosis ≥50% and large cell clusters. For small cell carcinoma, the best diagnostic accuracy was obtained by combining moderate necrosis (<50%) and the presence of single cells. Overall accuracy ranged from 90% to 97%. We showed that it was possible to establish the histotype of the most frequent lung cancers during ROSE using only a few easily identifiable cytomorphological parameters. An accurate diagnosis during ROSE could help endoscopists to decide how many tumor samples must be taken, e.g.a higher number of samples is needed for the biomolecular characterization of adenocarcinoma, whereas one sample may be sufficient for squamous cell carcinoma.

Are fine-needle aspiration biopsy-derived cell blocks a useful surrogate for tissue samples in breast cancer?

The diagnosis of breast cancer (BC) is based on clinical examination in combination with imaging, and confirmed by pathological assessment of core needle biopsy or fine‐needle aspiration biopsy (FNAB). The biological profile of the lesion is needed to define the prognosis and establish therapy. Given the importance of an early and minimally invasive diagnosis, we aimed to verify whether the biological features detected in FNAB‐derived cytological material reflect the biological characteristics of surgical samples.

Androgen and oestrogen receptors as potential prognostic markers for patients with ductal carcinoma in situ treated with surgery and radiotherapy

Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has been investigated less extensively than invasive breast cancer. Women with DCIS are mainly treated with conservative surgery almost exclusively followed by radiotherapy. However, as radiation treatment is not always effective, the search for biomarkers capable of identifying DCIS lesions that could progress to invasive cancer is ongoing. Although conventional biomarkers have been thoroughly studied in invasive tumours, little is known about the role played by androgen receptor (AR), widely expressed in DCIS. A series of 42 DCIS patients treated with quadrantectomy and radiotherapy were followed for a period of up to 95 months. Of these, 11 had recurrent DCIS or progressed to invasive cancer. All tumours were analysed for clinical pathological features. Conventional biomarkers and androgen receptor expression were determined by immunohistochemistry. Our results showed that AR was higher in tumours of relapsed patients than non‐relapsed patients (P value: 0.0005). Conversely, oestrogen receptor (ER) was higher, albeit not significantly, in non‐relapsed patients than in relapsed patients. AR/ER ratio was considerably different in the two subgroups (P value: 0.0033). Area under the curve (AUC) values were 0.85 for AR and 0.80 for the AR/ER ratio. These preliminary results highlight the potentially important role of both AR and the AR/ER ratio as prognostic markers in DCIS.

Are Androgen and Estrogen Receptors in DCIS Patients Prognostic Indicators of Relapse Independently of Treatment

Ductal carcinoma in situ (DCIS) is a highly heterogenous tumor that is now more frequently diagnosed because of the increased number of screening programs. Women with DCIS are mainly treated with conservative surgery almost always followed by radiotherapy. Although conventional biomarkers, i.e. ER, PgR, Ki67, and HER2, have been extensively investigated in invasive tumors, little is known about their role in DCIS, especially that of the androgen receptor (AR). In the present study, the expression of conventional biomarkers and AR was determined by immunohistochemistry in 85 DCIS samples from patients monitored for a maximum of 13 years: 43 patients were treated with quadrantectomy and 42 patients underwent quadrantectomy and radiotherapy. Of these, 5 and 11 patients relapsed, respectively. Our findings showed that ER and PgR were higher in nonrelapsed than in relapsed patients (P=0.025 and 0.0038). In contrast, AR expression and the AR/ER ratio were higher in relapsed patients than in the nonrelapsed group (P=0.0069 and 0.0012). At the best cut-off value of 1.1, the AR/ER ratio showed an overall accuracy of 92% and 80% in predicting in situ relapse or progression to invasive carcinoma in DCIS patients treated with surgery and those treated with surgery plus radiotherapy, respectively. AR would therefore appear to be an independent prognostic biomarker in the latter group. Our preliminary results highlight the potentially important role of the AR/ER ratio as a predictive indicator of DCIS relapse, independently of treatment.