The use of platelets as a clinical tool in oncology: opportunities and challenges.

Platelets are small circulating anucleated cells mainly involved in thrombosis and hemostasis processes. Moreover, platelets play an active role in tumorigenesis and cancer progression, stimulating angiogenesis and vascular remodelling, and protecting circulating cancer cells from shear forces and immune surveillance. Several reports indicate that platelet number in the blood circulation of cancer patients is associated with prognosis and response to treatment. However, the mechanisms of platelets "education" by cancer cells and the crosstalk between platelets and tumor are still unclear, and the role of "tumor educated platelets" (TEPs) is achieving growing interest in cancer research. TEPs are a biological source of cancer-derived biomarkers, especially RNAs that are protected by platelets membrane from circulating RNases, and could serve as a non-invasive tool for tumor detection, molecular profiling and evolution during therapy in clinical practice. Moreover, short platelet lifespan offers the possibility to get a snapshot assessment of cancer molecular profile, providing a real-time tool. We review and discuss the potential and the clinical utility, in terms of cancer diagnosis and monitoring, of platelet count together with other morphological parameters and of the more recent and innovative TEP profiling.

p53/TP53 Status Assessment in Gastroesophageal Adenocarcinoma.

Chromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized by TP53 deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with "high" staining levels to be positive. Aiming to improve aberrant TP53 detection, droplet digital PCR (ddPCR) was used to evaluate TP53 deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutational TP53 profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining "low" and "high" IHC staining level groups, the proportion of deletion events was significantly higher compared to the "intermediate" group (72.9% vs. 47.5%, p-value = 0.002). The ddPCR TP53 deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen's kappa = 0.48) with the assay performed with FFPE-DNA of the "low-level" group. NGS analysis confirmed that, in the "low-level" group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.

Putative Clinical Potential of ERBB2 Amplification Assessment by ddPCR in FFPE-DNA and cfDNA of Gastroesophageal Adenocarcinoma Patients.

Anti-HER2 monoclonal antibody trastuzumab improves the survival of those patients with advanced gastroesophageal adenocarcinoma (GEA) exhibiting HER2/ERBB2 overexpression/amplification. The current gold standard methods used to diagnose the HER2 status in GEA are immunohistochemistry (IHC) and silver or fluorescence in situ hybridization (SISH or FISH). However, they do not permit spatial and temporal tumor monitoring, nor do they overcome intra-cancer heterogeneity. Droplet digital PCR (ddPCR) was used to implement the assessment of HER2 status in formalin-fixed paraffin-embedded (FFPE) tumor DNA from a retrospective cohort (86 patients) and in cell-free DNA (cfDNA) samples from a prospective cohort (28 patients). In comparison to IHC/SISH, ddPCR assay revealed ERBB2 amplification in a larger patient fraction, including HER2 2+ and 0-1+ of the retrospective cohort (45.3% vs. 15.1%). In addition, a considerable number of HER2 2+ and 0-1+ prospective patients who were negative in FFPE by both IHC/SISH and ddPCR, showed ERBB2 amplification in the cfDNA collected just before surgery. cfDNA analysis in a few longitudinal cases revealed an increasing ERBB2 trend at progression. In conclusion, ddPCR in liquid biopsy may improve the detection rate of HER2 positive patients, preventing those patients who could benefit from targeted therapy from being incorrectly excluded.

MSI Analysis in Solid and Liquid Biopsies of Gastroesophageal Adenocarcinoma Patients: A Molecular Approach.

Gastroesophageal adenocarcinoma (GEA) patients with the microsatellite instability (MSI) subtype emerged as optimal candidates for immunotherapy. To date, immunohistochemistry (IHC) is the gold standard for MSI assessment in formalin-fixed paraffin-embedded (FFPE) specimens. However, IHC, although useful for diagnostic typing, cannot be used to analyze cell-free DNA (cfDNA) in liquid biopsy, a tool that could overcome tumor heterogeneity and enable longitudinal monitoring. In order to find an alternative diagnostic method to IHC, we analyzed 86 retrospective GEAs FFPE samples with multiplex PCR. Moreover, to verify the feasibility of MSI detection in liquid biopsy, cfDNA samples of five patients that resulted in having MSI in a prospective cohort of 35 patients were evaluated by multiplex PCR, real-time PCR and droplet digital PCR (ddPCR). Analysis of FFPE showed 100% concordance between multiplex PCR and IHC (Cohen's Kappa agreement = 1). On the contrary, only ddPCR was able to detect MSI in cfDNAs of T3/T4 GEA patients. In conclusion, data highlight the molecular analysis as an optimal alternative to IHC for the diagnostic typing and suggest that the ddPCR assay can be considered as the most reliable and promising molecular approach to detect MSI in the cfDNA of GEA patients.

Allelic Imbalance Analysis in Liquid Biopsy to Monitor Locally Advanced Esophageal Cancer Patients During Treatment.

Esophageal cancer (EC) is a highly aggressive tumor, and the current monitoring procedures are partially inadequate to evaluate treatment efficacy. The aim of this study was to investigate whether allelic imbalance analysis in liquid biopsy could be used as an additional tool to monitor tumor burden in EC patients. For this purpose, circulating cell-free DNA (cfDNA) from 52 patients with a locally advanced EC, which underwent neoadjuvant treatment and resection, was analyzed. Data from four representative longitudinally followed patients are also reported. Furthermore, 17 DNAs from formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed and compared to time-matched cfDNAs. To look for allelic imbalance, which is the main genetic alteration in both EC histotypes, we used a panel of five microsatellites (MSs) and three single-nucleotide polymorphisms (SNPs) near genes described as frequently altered. The Fisher exact and Mann-Whitney U tests were used to analyze categorical and continuous data, respectively. The correlation coefficient between cfDNA and FFPE-DNA was calculated with the Pearson's correlation test. We found that the selected tumor-related alterations are present in cfDNA of both adenocarcinoma (EADC) and squamous cell carcinoma (ESCC) with similar frequencies. The only exception were the MSs, one downstream and one upstream, of SMAD4 of which the loss was only observed in EADC (26 vs. 0%, P = 0.018). More interestingly, longitudinal studies disclosed that in patients with disease progression, tumor-related alterations were present in cfDNA before overt clinical or instrumental signs of relapse. In conclusion, our data indicate that the evaluation of tumor-related gene allelic imbalance in cfDNA might be a useful tool to complement the current monitoring procedures for EC patients and to guide their management.

Identification of host variants associated with overall survival of esophageal cancer patients receiving platinum-based therapy.

Aim: Clinical features of esophageal cancer (EC) patients have poor prognostic power. Thus, it is paramount to discover biomarkers that can allow a more accurate survival prediction. Methods: To detect genetic variants associated with survival, DNA from 120 patients treated with cisplatin-based neoadjuvant therapy were genotyped using drug metabolism enzymes and transporters array. Results: We identified two variants: the rs2038067 in PPARD (p = 0.0004) and the rs683369 (F160L) in SLC22A1 (p = 0.001). Their prognostic power was greater than that of clinical stage alone (p = 0.017) and comparable to that of response to neoadjuvant therapy (p = 0.71). Interestingly, the prognostic accuracy of response models increased significantly when genetic variables were included (p = 0.003). Conclusion: Our data, though preliminary, strengthen the potential utility of germline variants for a better-tailored management of EC patients.

Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients.

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett's esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

Detection of Loss of Heterozygosity in cfDNA of Advanced EGFR- or KRAS-Mutated Non-Small-Cell Lung Cancer Patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

Association Between ERCC1 rs3212986 and ERCC2/XPD rs1799793 and OS in Patients With Advanced Esophageal Cancer.

Esophageal cancer (EC) is a very aggressive tumor, and no reliable prognostic markers exist especially for resectable advanced neoplasia. The principal aim of this study was to investigate the association of germline polymorphisms in nucleotide excision repair (NER) pathway genes with the overall survival (OS) of patients with advanced EC. As a second aim, we also studied the association of NER gene variants with response to cisplatin-based chemotherapy. Among the EC patients referred to our Institution between 2004 and 2012, we selected a cohort of 180 patients diagnosed with a clinical tumor stage ranging from IIB and IVA. Patients were genotyped for four NER variants, two in the ERCC1 (rs11615 and rs3212986) and two in the ERCC2/XPD (rs1799793 and rs13181) genes. Kaplan-Meier analyses and Cox proportional hazards model were used to evaluate the associations of the selected variants with OS; association with response to neoadjuvant therapy was investigated using logistic regression. Results showed that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 were significantly associated with shorter OS. On the contrary, response association analysis displayed that, while rs11615 and rs3212986 in ERCC1 were associated with response, both ERCC2/XPD variants were not. By creating survival prediction models, we showed that the rs3212986 and the rs1799793 have a better predictability of the tumor stage alone. Furthermore, they were able to improve the power of the clinical model (AUC = 0.660 vs. AUC = 0.548, p = 0.004). In conclusion, our results indicate that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 could be used as surrogate markers for a better stratification of EC patients with advanced resectable tumor.

Detection of genetic alterations in cfDNA as a possible strategy to monitor the neoplastic progression of Barrett's esophagus.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. Despite the low absolute risk of neoplastic progression of BE, probability increases with the diagnosis of dysplasia. For this reason, BE patients undergo an endoscopy-based surveillance that is, however, burdensome for patients, subject to inter-observer subjectivity, and expensive for national health systems. Thus, less invasive and low-cost diagnostic tools are needed. This study is aimed at finding a simple and reliable method to detect in the circulating cell-free DNA (cfDNA) of BE patients evidence of the molecular instability that accompanies BE carcinogenesis. We chose the loss of heterozygosity analysis because chromosomal region gains or losses have been described in BE and esophageal adenocarcinoma. Furthermore, this analysis does not require an a priori knowledge of tumor specific mutations and/or rearrangements. Previous data showed a good consistency between tissue and cfDNA alterations. Here, we report that, in the cfDNA of dysplastic BE patients, the frequency of genetic alterations is statistically higher than that of metaplastic BE patients (P = 0.005). Interestingly, after endoscopic treatment, the alteration frequency dropped, suggesting that cfDNA can also be used to monitor curative effects. Among the used markers, those that map nearby TP53 gene were the most discriminant between metaplastic and dysplastic BE. Furthermore, longitudinal follow-up cases showed that genetic alterations can be found in cfDNA before the appearance of a detectable lesion. Altogether, our data suggest that the use of liquid biopsy could become a minimally invasive diagnostic tool to implement BE patient monitoring.

Fast method for skeletal tissue gene expression analysis.

Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.

Predictive role of host constitutive variants in neoadjuvant therapy of esophageal cancer.

Chemoradiotherapy followed by surgery is at present the standard therapeutic approach for esophageal cancer (EC) in patients with resectable tumor. However, response to neoadjuvant therapy is characterized by a strong interpatient variability, and the identification of markers predictive of outcome is mandatory. In this review, taking into account the currently available literature, we report the impact that host genetic variables can have on EC neoadjuvant therapy. We mainly focused on the gene variants involved in the pharmacokinetics and pharmacodynamics of the common chemotherapeutic drugs used to treat EC patients, commented on the weakness of the present knowledge, and discussed the future strategies to achieve a more personalized and effective EC treatment.

Liquid biopsy as a novel tool to monitor the carcinogenesis of Barrett's esophagus.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. For this reason, endoscopic-based surveillance protocols have been developed. This prevention program is, however, burdensome for the patients and expensive for the national health systems. Thus, diagnostic strategies with a low invasiveness and a reduced economic impact are required. This study investigated the power of plasma circulating free DNA (cfDNA) in predicting neoplastic transformation in the natural history of two BE patients who progressed to esophageal adenocarcinoma. Longitudinally collected DNAs from plasma and paired formalin fixed paraffin embedded samples were examined for both loss of heterozygosity (LOH) in areas proximal to TP53, FHIT and BRCA2 genes, and mutations in TP53 gene. Results showed that: (i) early BE molecular alterations are mainly localized proximal to, or within, TP53 gene; (ii) LOH events present in cfDNA not only retrace the time-matched biopsy profile but better represent the total alterations of the BE epithelium. In conclusion, our findings suggested that LOH analysis in plasma cfDNA could represent an additional, less invasive, diagnostic tool to monitor neoplastic progression of BE epithelium.

A germline predictive signature of response to platinum chemotherapy in esophageal cancer.

Platinum-based neoadjuvant therapy is the standard treatment for esophageal cancer (EC). At present, no reliable response markers exist, and patient therapeutic outcome is variable and very often unpredictable. The aim of this study was to understand the contribution of host constitutive DNA polymorphisms in discriminating between responder and nonresponder patients. DNA collected from 120 EC patients treated with platinum-based neoadjuvant chemotherapy was analyzed using drug metabolism enzymes and transporters (DMET) array platform that interrogates polymorphisms in 225 genes of drug metabolism and disposition. Four gene variants of DNA repair machinery, 2 in ERCC1 (rs11615; rs3212986), and 2 in XPD (rs1799793; rs13181) were also studied. Association analysis was performed with pTest software and corrected by permutation test. Predictive models of response were created using the receiver-operating characteristics curve approach and adjusted by the bootstrap procedure. Sixteen single nucleotide polymorphisms (SNPs) of the DMET array resulted significantly associated with either good or poor response; no association was found for the 4 variants mapping in DNA repair genes. The predictive power of 5 DMET SNPs mapping in ABCC2, ABCC3, CYP2A6, PPARG, and SLC7A8 genes was greater than that of clinical factors alone (area under the curve [AUC] = 0.74 vs 0.62). Interestingly, their combination with the clinical variables significantly increased the predictivity of the model (AUC = 0.78 vs 0.62, P = 0.0016). In conclusion, we identified a genetic signature of response to platinum-based neoadjuvant chemotherapy in EC patients. Our results also disclose the potential benefit of combining genetic and clinical variables for personalized EC management.

Genetic features of metachronous esophageal cancer developed in Hodgkin's lymphoma or breast cancer long-term survivors: an exploratory study.

BACKGROUND: Development of novel therapeutic drugs and regimens for cancer treatment has led to improvements in patient long-term survival. This success has, however, been accompanied by the increased occurrence of second primary cancers. Indeed, patients who received regional radiotherapy for Hodgkin's Lymphoma (HL) or breast cancer may develop, many years later, a solid metachronous tumor in the irradiated field. Despite extensive epidemiological studies, little information is available on the genetic changes involved in the pathogenesis of these solid therapy-related neoplasms. METHODS: Using microsatellite markers located in 7 chromosomal regions frequently deleted in sporadic esophageal cancer, we investigated loss of heterozygosity (LOH) and microsatellite instability (MSI) in 46 paired (normal and tumor) samples. Twenty samples were of esophageal carcinoma developed in HL or breast cancer long-term survivors: 14 squamous cell carcinomas (ESCC) and 6 adenocarcinomas (EADC), while 26 samples, used as control, were of sporadic esophageal cancer (15 ESCC and 11 EADC). RESULTS: We found that, though the overall LOH frequency at the studied chromosomal regions was similar among metachronous and sporadic tumors, the latter exhibited a statistically different higher LOH frequency at 17q21.31 (p = 0.018). By stratifying for tumor histotype we observed that LOH at 3p24.1, 5q11.2 and 9p21.3 were more frequent in ESCC than in EADC suggesting a different role of the genetic determinants located nearby these regions in the development of the two esophageal cancer histotypes. CONCLUSIONS: Altogether, our results strengthen the genetic diversity among ESCC and EADC whether they occurred spontaneously or after therapeutic treatments. The presence of histotype-specific alterations in esophageal carcinoma arisen in HL or breast cancer long-term survivors suggests that their transformation process, though the putative different etiological origin, may retrace sporadic ESCC and EADC carcinogenesis.

ERCC1 C8092A (rs3212986) polymorphism as a predictive marker in esophageal cancer patients treated with cisplatin/5-FU-based neoadjuvant therapy.

OBJECTIVE: At present, no consensus exists on the beneficial effect of preoperative cisplatin/5-fluorouracil (5-FU)-based chemotherapy versus primary surgery in the management of patients with esophageal cancer. The aim of this study was to evaluate the impact of some relevant genetic polymorphisms, within drug-related and DNA repair genes, on the clinical outcome of esophageal cancer patients subjected to cisplatin/5-FU-based neoadjuvant treatment. METHODS: DNA from 143 esophageal cancer patients, 63 receiving neoadjuvant therapy and 80 receiving primary surgery, was analyzed for the following polymorphisms: the GSTM1 null, GSTT1 null, and GSTP1 Ile105Val (rs16953) in glutathione S-transferase (GST) family, 2 in thymidylate synthase (TS) gene, and the ERCC1 Asn118Asn (rs11615), ERCC1 C8092A (rs3212986), XPD/ERCC2 Asp312Asn (rs1799793), and XPD/ERCC2 Lys751Gln (rs13181) of the nucleotide excision repair pathway. RESULTS: We found that the ERCC1 rs3212986, although not associated with therapeutic response, is an independent predictive marker of better outcome in a cisplatin/5-FU-based neoadjuvant setting (hazard ratio: 0.38, 95% confidence interval: 0.2-0.73, P=0.008). In contrast, no association with clinical outcome was observed for this polymorphism in the primary surgery group. CONCLUSION: Our study indicates the ERCC1 rs3212986 as a predictive marker in the cisplatin/5-FU-based neoadjuvant setting, and also suggests its use as a marker to select the appropriate therapeutic approach in esophageal cancer patients.

DMET™ (Drug-Metabolizing Enzymes and Transporters) microarray analysis of colorectal cancer patients with severe 5-fluorouracil-induced toxicity.

PURPOSE: 5-fluorouracil (5-FU) has been widely used since the 1980s, and it remains the backbone of many chemotherapeutic combination regimens. However, its use is often limited by the occurrence of severe toxicity. Although several reports have shown the detrimental effect of some dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS) gene polymorphisms in patients undergoing 5-FU-based treatment, they account for only a minority of toxicities. METHODS: Looking for new candidate genetic variants associated with 5-FU-induced toxicity, we used the innovative genotyping microarray Affymetrix Drug-Metabolizing Enzymes and Transporters (DMET)™ Plus GeneChip that interrogates 1,936 genetic variants distributed in 231 genes involved in drug metabolism, excretion, and transport. To reduce variability, we analyzed samples from colorectal cancer patients who underwent fairly homogenous treatments (i.e., Machover or Folfox) and experienced G3 or G4 toxicity; control patients were matched for therapy and selected from those who did not disclose toxicity (G0-G1). RESULTS: Pharmacogenetic genotyping showed no significant difference in DPYD and TYMS genetic variants distribution between cases and controls. However, other polymorphisms could account for 5-FU-induced toxicity, with the CHST1 rs9787901 and GSTM3 rs1799735 having the strongest association. CONCLUSIONS: Although exploratory, this study suggests that genetic polymorphisms not directly related to 5-FU pharmacokinetics and pharmacodynamics are involved in 5-FU-induced toxicity. Our data also indicates DMET™ microarray as a valid approach to discover new genetic determinants influencing chemotherapy-induced toxicity.