Review: Peering through a keyhole: liquid biopsy in primary and metastatic central nervous system tumours.

Tumour molecular profiling by liquid biopsy is being investigated for a wide range of research and clinical purposes. The possibility of repeatedly interrogating the tumour profile using minimally invasive procedures is helping to understand spatial and temporal tumour heterogeneity, and to shed a light on mechanisms of resistance to targeted therapies. Moreover, this approach has been already implemented in clinical practice to address specific decisions regarding patients' follow-up and therapeutic management. For central nervous system (CNS) tumours, molecular profiling is particularly relevant for the proper characterization of primary neoplasms, while CNS metastases can significantly diverge from primary disease or extra-CNS metastases, thus compelling a dedicated assessment. Based on these considerations, effective liquid biopsy tools for CNS tumours are highly warranted and a significant amount of data have been accrued over the last few years. These results have shown that liquid biopsy can provide clinically meaningful information about both primary and metastatic CNS tumours, but specific considerations must be taken into account, for example, when choosing the source of liquid biopsy. Nevertheless, this approach is especially attractive for CNS tumours, as repeated tumour sampling is not feasible. The aim of our review was to thoroughly report the state-of-the-art regarding the opportunities and challenges posed by liquid biopsy in both primary and secondary CNS tumours.

Practice parameters for the diagnosis and treatment of anal intraepithelial neoplasia (AIN) on behalf of the Italian Society of Colorectal Surgery (SICCR).

Squamous cell carcinoma (SCC) of the anus is a human papilloma virus (HPV) related malignancy that is preceded by anal intraepithelial neoplasia (AIN) making this cancer, at least theoretically, a preventable disease. In the past 10 years the diagnosis, management and nomenclature of AIN has dramatically changed. Increased life expectancy in human immunodeficiency virus (HIV) positive patients due to highly active antiretroviral therapy (HAART) has caused an increase in the incidence of SCC of the anus. While many experts recommend screening and treatment of anal high-grade squamous intraepithelial lesion (HSIL), there is no consensus on the optimal management these lesions. Therefore, there is a need to review the current evidence on diagnosis and treatment of AIN and formulate recommendations to guide management. Surgeons who are members of the Italian Society of Colorectal Surgery (SICCR) with a recognized interest in AIN were invited to contribute on various topics after a comprehensive literature search. Levels of evidence were classified using the Oxford Centre for Evidence-based Medicine of 2009 and the strength of recommendation was graded according to the United States (US) preventive services task force. These recommendations are among the few entirely dedicated only to the precursors of SCC of the anus and provide an evidence-based summary of the current knowledge about the management of AIN that will serve as a reference for clinicians involved in the treatment of patients at risk for anal cancer.

Favourable prognostic role of histological regression in stage III positive sentinel lymph node melanoma patients.

BACKGROUND: Sentinel lymph node (SLN)-positive melanoma patients are a heterogeneous group of patients with survival rates ranging from ∼20 to over 80%. No data are reported concerning the role of histological regression on survival in stage III melanoma. METHODS: The study included 365 patients with positive SLN from two distinct hospitals. The model was developed on patients from 'AOU Città della Salute e della Scienza di Torino', and externally validated on patients from IRCCS of Candiolo. Survival analyses were carried out according to the presence of regression and adjusted for all other prognostic factors. RESULTS: Among patients followed at 'AOU Città della Salute e della Scienza di Torino' (n=264), the median follow-up time to death or censoring (whatever two events occurred earlier) was 2.7 years since diagnosis (interquartile range: 1.3-5.8). In all, 79 patients died from melanoma and 11 from other causes. Histological regression (n=43) was associated with a better prognosis (sub-HR=0.34, CI 0.12-0.92), whereas the other factors above showed an inverse association. In the external validation, the concordance index was 0.97 at 1 year and decreased to 0.66 at 3 years and to 0.59 at 5 years. Adding histological regression in the prognostic model increased the discriminative ability to 0.75 at 3 years and to 0.62 at 5 years. Finally, using a cutoff of 20% for the risk of death led to a net re-classification improvement of 15 and 11% at 3 and 5 years after diagnosis, respectively. CONCLUSIONS: Histological regression could lead to an improvement in prognostic prediction in patients with stage III-positive SLN melanoma.

Detection rate of serrated polyps and serrated polyposis syndrome in colorectal cancer screening cohorts: a European overview.

OBJECTIVE: The role of serrated polyps (SPs) as colorectal cancer precursor is increasingly recognised. However, the true prevalence SPs is largely unknown. We aimed to evaluate the detection rate of SPs subtypes as well as serrated polyposis syndrome (SPS) among European screening cohorts. METHODS: Prospectively collected screening cohorts of ≥1000 individuals were eligible for inclusion. Colonoscopies performed before 2009 and/or in individuals aged below 50 were excluded. Rate of SPs was assessed, categorised for histology, location and size. Age-sex-standardised number needed to screen (NNS) to detect SPs were calculated. Rate of SPS was assessed in cohorts with known colonoscopy follow-up data. Clinically relevant SPs (regarded as a separate entity) were defined as SPs ≥10 mm and/or SPs >5 mm in the proximal colon. RESULTS: Three faecal occult blood test (FOBT) screening cohorts and two primary colonoscopy screening cohorts (range 1.426-205.949 individuals) were included. Rate of SPs ranged between 15.1% and 27.2% (median 19.5%), of sessile serrated polyps between 2.2% and 4.8% (median 3.3%) and of clinically relevant SPs between 2.1% and 7.8% (median 4.6%). Rate of SPs was similar in FOBT-based cohorts as in colonoscopy screening cohorts. No apparent association between the rate of SP and gender or age was shown. Rate of SPS ranged from 0% to 0.5%, which increased to 0.4% to 0.8% after follow-up colonoscopy. CONCLUSIONS: The detection rate of SPs is variable among screening cohorts, and standards for reporting, detection and histopathological assessment should be established. The median rate, as found in this study, may contribute to define uniform minimum standards for males and females between 50 and 75 years of age.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Value of staging squamous cell carcinoma of the anal margin and canal using the sentinel lymph node procedure: an update of the series and a review of the literature.

BACKGROUND: Inguinal metastases in patients affected by anal cancer are an independent prognostic factor for local failure and overall mortality. Since 2001, sentinel lymph node biopsy was applied in these patients. This original study reports an update of personal and previous published series, which were compared with Literature to value the incidence of inguinal metastases T-stage related and the overall incidence of false negative inguinal metastases at sentinel node. METHODS: In all, 63 patients diagnosed with anal cancer submitted to inguinal sentinel node. Furthermore a research in the Pub Med database was performed to find papers regarding this technique. RESULTS: In our series, detection rate was 98.4%. Inguinal metastases were evidentiated in 13 patients (20.6%). Our median follow-up was 35 months. In our series, no false negative nodes were observed. CONCLUSION: Sentinel node technique in the detection of inguinal metastases in patients affected by anal cancer should be considered as a standard of care. It is indicated for all T stages in order to select patients to be submitted to inguinal radiotherapy, avoiding related morbidity in negative ones. An overall 3.7% rate of false negative must be considered acceptable.

A simple and reproducible prognostic index in luminal ER-positive breast cancers.

BACKGROUND: The group of estrogen receptor (ER)-positive breast cancers (both luminal-A and -B) behaves differently from the ER-negative group. At least in early follow-up, ER expression influences positively patients' prognosis. This low aggressive biology flattens out the differences of clinical management. Thus we aimed to produce a prognostic index specific for ER-positive (ERPI) cancers that could be of aid for clinical decision. PATIENTS AND METHODS: The test set comprised 495 consecutive ER-positive breast cancers. Tumor size, number of metastatic lymph nodes and androgen receptor expression were the only independent variables related to disease-specific survival. These variables were used to create the ERPI, which was applied to the entire test set and to selected subpopulations (grade 2 (G2)-tumors, luminal-A and -B breast cancers). A series of 581 ER-positive breast cancers, collected from another hospital, was used to validate ERPI. RESULTS: In the test population, 96.9% of patients classified as ERPI-good showed a good prognosis compared with 79.6% classified as ERPI-poor (P < 0.001). ERPI effectively discriminated outcome in luminal-A and luminal-B and in G2-tumors. In the validation series, the ERPI maintained its value. CONCLUSION: ERPI is a practical tool in refining the prediction of outcome of patients with ER-positive breast cancer.

A novel biomarker-based analysis reliably predicts nodal metastases in anal carcinoma: preliminary evidence of therapeutic impact.

AIM: Routine prophylactic inguinal irradiation in anal cancer may cause significant toxicity associated with overtreatment bias. The aim of this study was to determine the risk of regional node metastases in anal carcinoma by identifying predictive molecular biomarkers. METHOD: Clinicohistopathological data from 50 pretreatment anal carcinoma biopsies were collected. Immunohistochemical analyses with antibodies against Ki67, p53, epidermal growth factor receptor (EGFR) and YKL-40 were performed. Statistical correlations between biomarkers and clinicopathological features and outcomes were studied. Sentinel lymph node biopsy was performed in a subset of 36 patients. RESULTS: All patients had undergone synchronous radiochemotherapy; tumour recurrence had developed in 26%, and 16% had died. YKL-40 tumour expression correlated with lymph node metastasis, whereas no inguinal node metastases were found in any of the (14%) patients presenting with a YKL-40/EGFR-negative tumour. YKL-40 expression and node metastasis were both significantly associated with shorter overall and disease-free survival. Tumour grade significantly correlated with disease-free survival only. HIV, tumour histological type, Ki67, p53 and EGFR were not associated with outcome. CONCLUSION: YKL-40 expression in anal carcinoma is correlated with a poor outcome and can predict lymph node metastases. The combined absence of YKL-40 and EGFR expression in a first biopsy of anal carcinoma reliably selects a subset of patients without inguinal metastases. Such patients could be spared sentinel lymph node biopsy and/or inguinal radiotherapy.

Prognostic value of EGFR expression in de novo and progressed atypical and anaplastic meningiomas: an immunohistochemical and fluorescence in situ hybridization pilot study.

AIM: The aim of this study was to assess both the epidermal growth factor receptor (EGFR) protein expression by immunohistochemistry and the EGFR gene amplification by fluorescence in situ hybridization in meningiomas of different grade, in order to evaluate their possible role in the development of the disease. EGFR protein belongs to the family of tyrosine kinase growth factor receptors, which also includes HER2, HER3 and HER4. Elevated expression or activity of EGFR has been reported in several cancers, including brain tumours. EGFR activation can enhance the malignant potential of epithelial tissues. METHODS: We investigated whether there was a difference in the EGFR protein expression and the EGFR gene amplification between the so called de novo malignant meningiomas and recurrent meningiomas with or without malignant progression from a previously lower grade tumor. Our goal was to evaluate if EGFR expression was a useful marker to select patients affected by meningioma with a major risk of recurrences. We also assessed the prognostic value of the EGFR expression on overall survival. RESULTS: Progression from benign meningiomas to atypical or anaplastic meningiomas correlated with an increase in the expression of EGFR protein. Our study shows that EGFR immunostaining in meningiomas directly correlates to the tumor's grade. The EGFR expression did not correlate with the overall survival and the recurrence-free survival of the patients affected by meningioma (de novo, recurrent and progressed). CONCLUSION: We submit that the EGFR expression is not a useful prognostic element to identify patients with a major risk of meningioma recurrence.

Safe transportation of formalin-fixed liquid-free pathology specimens.

Diagnostic pathology activities are largely based on fixation of tissues in 4% formaldehyde, which has recently been re-classified as a carcinogenic compound and banned in several countries. Hospitals that do not have in-house pathology services need to send surgical and biopsy specimens to referral centers. These are generally transferred in liquid containers, under suboptimal safety conditions, as accidental spillage of potentially dangerous substances may occur. A safe, innovative, two-step procedure for pathology sample transportation is presented. Formalin-fixed material from ten surgical cases was dissected (including surrogate biopsies) and preserved in liquid-free plastic bags under vacuum for up to 30 days and subsequently processed for conventional histology, several immunohistochemical markers, and molecular tests (e.g., RAS mutation). The data were compared with the corresponding routine analyses. Formalin-fixed specimens after up to 30 days under vacuum storage gave equivalent results compared to standard histopathological slides and molecular tests, regarding both hematoxylin-eosin, immuno-stained slides and also nucleic acid extracted for molecular tests. The proposal of under-vacuum sealing pathology specimens that were previously formalin fixed can be adopted to transfer liquid-free biopsy and surgical specimens to referral pathology services. In fact, it is easy to perform, less expensive (both plastic bags and domestic-type vacuum chamber machines are at affordable costs), and above all is fully safe and adequate in the pre-analytical processing of pathology specimens.

The post-surgical era of GBM: How molecular biology has impacted on our clinical management. A review.

Glioblastoma (GBM) is the most common glioma in adults, with incidence increasing by 3% per year. According to the World Health Organization Classification of Central Nervous System Tumors, GBM is considered a grade IV tumor due to its malignant behavior. The aim of this review is to summarize the main biological aspects of GBM. In particular, we focused our attention on those alterations which have been proven to have an impact on patients' outcome, mainly in terms of overall survival (OS), or on the tumor response to therapies. We have also analyzed the cellular biology and the interactions between GBM and the surrounding environment.

Dosimetric predictors of acute hematologic toxicity during concurrent intensity-modulated radiotherapy and chemotherapy for anal cancer.

PURPOSE: This study aimed at investigating whether the irradiated volume of pelvic bone marrow (PBM) and specific subsites may predict the occurrence of acute hematologic toxicity (HT) in anal cancer patients undergoing concurrent chemo-radiation. METHODS: 50 patients, submitted to IMRT and concurrent chemotherapy, were analyzed. Several bony structures were defined on planning-CT: PBM and lumbar-sacral (LSBM), lower pelvis (LPBM) and iliac (IBM) bone marrow. On dose-volume histograms, dosimetric parameters were taken. Endpoints included white blood-cell-count (WBC), absolute-neutrophil-count (ANC), hemoglobin (Hb) and platelet nadirs and acute hematologic toxicity (HT) according to RTOG scoring scale. Generalized linear modeling was used to find correlations between dosimetric variables and blood cell nadirs, while logistic regression analysis was used to test correlation with ≥G3 HT. Receiver Operating Characteristic (ROC) curve analysis was used to evaluate the optimal cut-off points for predictive dosimetric variables with the Youden method. RESULTS: Maximum detected acute HT comprised 38 % of ≥G3 leukopenia and 32 % of ≥G3 neutropenia. Grade 2 anemia was observed in 4 % of patients and ≥G3 thrombocytopenia in 10 %. On multivariate analysis a higher PBM-V 20 was associated with lower WBC nadir. Increased LSBM-V 40 was correlated with a higher likelihood to develop ≥G3 HT. A cut-off point at 41 % for LSBM-V 40 was found. Patients with LSBM-V 40 ≥41 % were more likely to develop ≥G3 HT (55.3 vs. 32.4 %; p < 0.01). CONCLUSIONS: Increased low-dose to pelvic bony structures significantly predicted for WBC decrease. Medium-high dose to specific osseous subsites was associated with a higher probability of HT. LSBM-V 40 was a strong predictor of ≥G3 HT. A threshold at 41 % for LSBM-V 40 could be used to limit HT.

Ultrasound-based follow-up does not increase survival in early-stage melanoma patients: A comparative cohort study.

INTRODUCTION: Different protocols have been used to follow up melanoma patients in stage I-II. However, there is no consensus on the complementary tests that should be requested or the appropriate intervals between visits. Our aim is to compare an ultrasound-based follow-up with a clinical follow-up. PATIENTS AND METHODS: Analysis of two prospectively collected cohorts of melanoma patients in stage IB-IIA from two tertiary referral centres in Barcelona (clinical-based follow-up [C-FU]) and Turin (ultrasound-based follow-up [US-FU]). Kaplan-Meier curves were used to evaluate distant metastases-free survival (DMFS), disease-free interval (DFI), nodal metastases-free survival (NMFS) and melanoma-specific survival (MSS). RESULTS: A total of 1149 patients in the American Joint Committee on Cancer stage IB and IIA were included in this study, of which 554 subjects (48%) were enrolled for a C-FU, and 595 patients (52%) received a protocolised US-FU. The median age was 53.8 years (interquartile range [IQR] 41.5-65.2) with a median follow-up time of 4.14 years (IQR 1.2-7.6). During follow-up, 69 patients (12.5%) in C-FU and 72 patients (12.1%) in US-FU developed disease progression. Median time to relapse for the first metastatic site was 2.11 years (IQR 1.14-4.04) for skin metastases, 1.32 (IQR 0.57-3.29) for lymph node metastases and 2.84 (IQR 1.32-4.60) for distant metastases. The pattern of progression and the total proportion of metastases were not significantly different (P = .44) in the two centres. No difference in DFI, DMFS, NMFS and MSS was found between the two cohorts. CONCLUSION: Ultrasound-based follow-up does not increase the survival of melanoma patients in stage IB-IIA.

Dual MET/EGFR therapy leads to complete response and resistance prevention in a MET-amplified gastroesophageal xenopatient cohort.

Amplification of the MET oncogene occurs in 2-4% of gastroesophageal cancers and defines a small and aggressive subset of tumors. Although in vitro studies have given very promising results, clinical trials with MET inhibitors have been disappointing, showing few and short lasting responses. The aim of the work was to exploit a MET-amplified patient-derived xenograft model to optimize anti-MET therapeutic strategies in gastroesophageal cancer. We found that despite the high MET amplification level (26 gene copies), in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, whereas dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. Similar results were also obtained in a MET-addicted, established gastric cancer cell line. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined MET/EGFR treatment completely shut off the signaling. Previously reported cases, as well as those described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy-even in the absence of EGFR genetic alterations-induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients.

Ghrelin and cortistatin in lung cancer: expression of peptides and related receptors in human primary tumors and in vitro effect on the H345 small cell carcinoma cell line.

Ghrelin, a natural GH secretagogue (GHS) acylated peptide, and cortistatin (CST), a natural SRIF-like peptide, interfere with neoplastic growth in different cancers. We tested forty-one lung carcinomas and the H345 small cell lung carcinoma (SCLC) cell line by RT-PCR to investigate the presence of ghrelin and CST and related receptors, including type 1a GHS receptor (GHS-R1a), all SRIF-receptor subtypes (sst 1-5) and MRGX2. Moreover, the presence of ghrelin and CST peptides was studied in both tumors and H345 cells. Ghrelin and CST mRNA were present in the majority of tested tumors, but ghrelin and CST proteins were revealed only in tumors with a neuroendocrine phenotype. All the receptors mRNA had a heterogeneous expression without correlation between ghrelin (or CST) and their receptor distribution. All the transcripts, but not GHS-R1a, were expressed in H345 cells. However, ghrelin and desacyl ghrelin induced in vitro a dose-dependent inhibition on the H345 cell proliferation and increased apoptosis. Conversely, neither CST nor SRIF affected H345 cell growth, despite the presence of their specific receptors. The anti-proliferative and the pro-apoptotic effects of ghrelin were consistent with binding experiments on H345 cell, where either acylated or des-acylated ghrelin recognized a common binding site. In conclusion, the present study indicates that: a) ghrelin and CST mRNAs are expressed in lung cancers, although some neuroendocrine tumors contain detectable amounts of the peptides; b) GHSR-1a mRNA is present exclusively in neuroendocrine tumors, whereas MRGX2 mRNA (but not peptide) is expressed in all histological types; c) both ghrelin forms inhibit H345 cell proliferation, both directly and enhancing apoptosis, despite the absence of GHS-R1a, whereas CST and its receptors do not interfere with cell growth.

111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-lys(8)-vasotocin: a new powerful radioligand for oxytocin receptor-expressing tumors.

We developed a radioactive ligand for tumors expressing oxytocin receptors (OTRs) by linking the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) to Lys(8)-vasotocin (LVT), an analogue of oxytocin with high affinity for OTRs. The new reagent (DOTA-LVT) retained high affinity for human OTRs, as proved by in vitro affinity binding to cells endogenously expressing OTRs, such as MCF7 breast carcinoma and MOG-U-V-W glioblastoma cells lines, as well as to transiently transfected COS7 cells. In in vivo experiments, DOTA-LVT carrying (111)In showed specific binding activity to OTR-positive TS/A mouse mammary tumors. The present study opens new perspectives for imaging and, possibly, therapy of OTR-positive human tumors such as breast and endometrial carcinomas, neuroblastomas, and glioblastomas.

A plasmin-derived hexapeptide from the carboxyl end of osteocalcin counteracts oxytocin-mediated growth inhibition [corrected] of osteosarcoma cells.

We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1-44) and COOH-terminal RFYGPV hexapeptide (44-49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OT-mediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OC-OTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.