Predictive genetic markers in neoadjuvant chemoradiotherapy for locally advanced esophageal cancer: a long way to go. Review of the literature.

The role of genetic molecular markers in neoadjuvant treatment for locally advanced esophageal cancer has been reviewed, focusing strictly on concurrent chemoradiation protocols followed by surgery. Eleven studies evaluated the role of mRNA expression profile; the end point was overall survival (OS) in two studies and different definitions of histological response in nine. Genes reported as significant were involved in cell cycle control (30), apoptosis (7), structural molecules (9), cell metabolism (6) and DNA repair (1). Seven studies reported about 15 microRNA (miRNA) molecules associated with OS (2) or histological response (13), however, defined with different classifications. Their target genes were prevalently involved in cell cycle control (4), apoptosis (1), cell adhesion (1), migration (1) and angiogenesis (1). Gene polymorphisms (single-nucleotide polymorphisms (SNPs)) have been evaluated in 8 studies reporting 10 variants associated with survival or pathological response. OS was the end point in six of these studies. SNPs reported as significant were involved in DNA repair system (4), detoxification (2), folate metabolism (6), drug efflux (2) and others (2). In a study, a panel including histology, pathological response and five SNPs discriminated two subsets of patients with 5-year survival rates of 79.3% and 26.3% (hazard ratio 6.25, P<0.0001). In another study, combination of stage, grade and 4 miRNAs improved prediction of pathological response (P=10-30). At present, given the great inconsistency of the data and the variability of the end points, definite conclusions are extremely difficult, if not impossible. More consistent data can derive only from analyses obtained from patients included in prospective randomized trials while panels combining genetic and clinical factors may improve prediction.

Reference miRNAs for colorectal cancer: analysis and verification of current data.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Genetic prediction of long-term survival after neoadjuvant chemoradiation in locally advanced esophageal cancer.

Candidate genes involved in DNA repair, 5-fluorouracil metabolism and drug detoxification were genotyped in 124 patients receiving neoadjuvant chemoradiation treatment for locally advanced esophageal cancer and their predictive role for long-term relapse-free survival (RFS) and cancer-specific survival (CSS) were evaluated. A panel including MTHFR 677TT, MDR1 2677GT, GSTP1 114CC, XPC 499CC and XPC 939AC+CC, defined as high-risk genotypes, discriminated subgroups with significantly different outcomes. When the panel was combined with histology, patients split into two subsets with 5-year RFS and CSS rates of 65% vs 27% (hazard ratio (HR) 3.0, P<0.0001) and 69% vs 31% (HR 2.9, P<0.0001), respectively. Combining the 5-single-nucleotide polymorphism (5-SNP) panel with pathological response defined two major informative risk classes with 5-year PFS and CSS rates of 79.4% vs 17.7% (HR 6.71, P<0.0001) and 79.3% vs 26.3% (HR 6.25, P<0.0001), respectively. This classification achieved a sensitivity of 79%, a specificity of 85.4% and an accuracy of 81.8%.

Predictive markers in elderly patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors: an array-based pharmacogenetic study.

So far, no reliable predictive clinicopathological markers of response to aromatase inhibitors (AIs) have been identified, and little is known regarding the role played by host genetics. To identify constitutive predictive markers, an array-based association study was performed in a cohort of 55 elderly hormone-dependent breast cancer (BC) patients treated with third-generation AIs. The array used in this study interrogates variants in 225 drug metabolism and disposition genes with documented functional significance. Six variants emerged as associated with response to AIs: three located in ABCG1, UGT2A1, SLCO3A1 with a good response, two in SLCO3A1 and one in ABCC4 with a poor response. Variants in the AI target CYP19A1 resulted associated with a favourable response only as haplotype; haplotypes with increased response association were also detected for ABCG1 and SLCO3A1. These results highlight the relevance of host genetics in the response to AIs and represent a first step toward precision medicine for elderly BC patients.

Genetic risk of subsequent esophageal cancer in lymphoma and breast cancer long-term survival patients: a pilot study.

The occurrence of a second primary esophageal carcinoma (EC) in long-term cancer survivors may represent a late effect of previous radio-chemotherapeutic treatment. To identify the genetic factors that could increase this risk, we analyzed nine variants within ERCC1, XPD, XRCC1 and XRCC3 DNA repair pathway genes, and GSTP1, TP53 and MDM2 genes in 61 patients who received radio-chemotherapy for a prior lymphoma or breast cancer; 29 of them had a second primary EC. This cohort consists of 22 esophageal squamous cell carcinoma (ESCC) and 7 esophageal adenocarcinoma (EADC) patients. A validation cohort of 154 patients with sporadic EC was also included. The XPD Asp312Asn (rs1799793) was found to be associated with the risk of developing second primary ESCC (P=0.015). The resultant variant was also involved in the onset of sporadic ESCC (P=0.0018). To know in advance who among long-term cancer survivors have an increased risk of EC could lead to a more appropriate follow-up strategy.

Epigenetic alteration: new insights moving from tissue to plasma - the example of PCDH10 promoter methylation in colorectal cancer.

BACKGROUND: Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features. METHODS: A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay. RESULTS: The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3-97.8%) and 5.9% (0-80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234). CONCLUSION: Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

Predictors of survival and toxicity in patients on adjuvant therapy with 5-fluorouracil for colorectal cancer.

The present study aimed at investigating whether the simultaneous evaluation of pharmacokinetic, pharmacogenetic and demographic factors could improve prediction on toxicity and survival in colorectal cancer patients treated with adjuvant 5-fluorouracil (5FU)/leucovorin therapy. One hundred and thirty consecutive, B2 and C Duke's stage colorectal cancer patients were prospectively enrolled. 5FU pharmacokinetics was evaluated at the first cycle. Thymidylate synthase (TYMS) 5'UTR and 3'UTR polymorphisms and methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms were assessed in peripheral leukocytes. Univariate and multivariate analyses were applied to evaluate which variables could predict chemotherapy-induced toxicity, disease-free survival (DFS) and overall survival (OS). Multivariate analysis showed that: (a) low 5FU clearance was an independent predictive factor for severe toxicity (OR=7.32; P<0.0001); (b) high-5FU clearance predicted poorer DFS (HR=1.96; P=0.041) and OS (HR=3.37; P=0.011); (c) advanced age was associated with shorter DFS (HR=3.34; P=0.0008) and OS (HR=2.66; P=0.024); (d) the C/C genotype of the MTHFR C677T polymorphism was protective against grade 3-4 toxicity (P=0.040); (e) none of the TYMS polymorphisms could explain 5FU toxicity or clinical outcome.

Predictive value of hematological and phenotypical parameters on postchemotherapy leukocyte recovery.

BACKGROUND: Grade IV chemotherapy toxicity is defined as absolute neutrophil count <500/microL. The nadir is considered as the lowest neutrophil number following chemotherapy, and generally is not expected before the 7th day from the start of chemotherapy. The usual prophylactic dose of rHu-G-CSF (Filgrastim) is 300 microg/day, starting 24-48 h after chemotherapy until hematological recovery. However, individual patient response is largely variable, so that rHu-G-CSF doses can be different. The aim of this study was to verify if peripheral blood automated flow cytochemistry and flow cytometry analysis may be helpful in predicting the individual response and saving rHu-G-CSF. METHODS: During Grade IV neutropenia, blood counts from 30 cancer patients were analyzed daily by ADVIA 120 automated flow cytochemistry analyzer and by Facscalibur flow cytometer till the nadir. "Large unstained cells" (LUCs), myeloperoxidase index (MPXI), blasts, and various cell subpopulations in the peripheral blood were studied. At nadir rHu-G-CSF was started and 81 chemotherapy cycles were analyzed. Cycles were stratified according to their number and to two dose-levels of rHuG-CSF needed to recovery (300-600 vs. 900-1200 microg) and analyzed in relation to mean values of MPXI and mean absolute number of LUCs in the nadir phase. The linear regressions of LUCs % over time in relation to two dose-levels of rHu-G-CSF and uni-multivariate analysis of lymphocyte subpopulations, CD34(+) cells, MPXI, and blasts were also performed. RESULTS: In the nadir phase, the increase of MPXI above the upper limit of normality (>10; median 27.7), characterized a slow hematological recovery. MPXI levels were directly related to the cycle number and inversely related to the absolute number of LUCs and CD34(+)/CD45(+) cells. A faster hematological recovery was associated with a higher LUC increase per day (0.56% vs. 0.25%), higher blast (median 36.7/microL vs. 19.5/microL) and CD34(+)/CD45(+) cell (median 2.2/microL vs. 0.82/microL) counts. CONCLUSIONS: Our study showed that some biological indicators such as MPXI, LUCs, blasts, and CD34(+)/CD45(+) cells may be of clinical relevance in predicting individual hematological response to rHu-G-CSF. Special attention should be paid when nadir MPXI exceeds the upper limit of normality because the hematological recovery may be delayed.

Pharmacokinetic and demographic markers of 5-fluorouracil toxicity in 181 patients on adjuvant therapy for colorectal cancer.

BACKGROUND: The relationship between 5-fluorouracil (5-FU) pharmacokinetics and toxicity following i.v. bolus administration has not been extensively studied. PATIENTS AND METHODS: One hundred and eighty-one patients on adjuvant therapy with 5-FU plus leucovorin for colorectal cancer were the study population. 5-FU pharmacokinetics was determined on day 2 of the first, third, and fifth cycles; type and the grade of adverse reactions were recorded on the next cycle. RESULTS: The 5-FU area under the curve (AUC) measured at the first cycle ranged between 146 and 1236 mg x min/l and was significantly correlated with drug dose, patients' body weight (BW) and gender, females having higher AUCs. These covariates explained only 23% of AUC variability. AUC and age were the only covariates which discriminated between toxic (grade > or =2) and nontoxic cycles (grade <2), with an optimal AUC cut-off value of 596 mg x min/l. Such a correlation was lost during the next cycles following dose reduction because of toxicity in 80 patients. CONCLUSIONS: A method for calculating the initial 5-FU dose is proposed which takes into account patient BW, gender and a target AUC of 596 mg x min/l. Nevertheless, it appears that a substantial part of 5-FU toxicity is not linked to pharmacokinetic factors and dose adjustments must still be on the basis of careful clinical surveillance.

A novel G/C single-nucleotide polymorphism in the double 28-bp repeat thymidylate synthase allele.

Thymidylate synthase (TYMS) is the main molecular target for fluoropyrimidine anticancer drugs, and its expression has been correlated with the number of repeats of a 28-bp sequence in the 5'-untranslated region of the TYMS gene and with the presence of a G --> C single-nucleotide polymorphism in the second repeat of 3R alleles. Based on this double polymorphism, three main TYMS alleles have so far been identified: TYMS 2R, TYMS 3RC and TYMS 3RG. During genetic analysis of TYMS polymorphisms in 100 colorectal cancer patients, three patients revealed an unexpected 113-bp band after electrophoresis of the restriction fragment length polymorphism analysis. Subsequent sequencing revealed two 28-bp repeats in the 5'-untranslated region and the presence in both repeats of cytosine instead of guanine at the 12th nucleotide. This allele variant (TYMS 2RC) has not been previously described in man. All three patients were heterozygotes for TYMS 2RC and experienced grade 2-3 chemotherapy-related toxicity.

Increased myeloperoxidase index and large unstained cell values can predict the neutropenia phase of cancer patients treated with standard dose chemotherapy.

OBJECTIVE: The objective of this study was to better understand neutropenia induced by standard dose chemotherapy and to verify if there are any hematological parameters for defining the phase and possibly the duration of neutropenia. METHODS: The kinetics of large unstained cells (LUCs) and lymphocytes was evaluated in 324 blood counts of 56 chemotherapy cycles through the use of a Technicon H2 or an ADVIA 120 hematology analyzer. Blood samples collected during the neutropenia phase were also studied by flow cytometry using a large panel of monoclonal antibodies. Parametric and nonparametric statistics were employed to compare the different variables analyzed. A linear regression between each variable before and after nadir and a simple linear correlation among the same variables in the neutropenic and recovery phase were performed. RESULTS: The percentage of LUCs reaches the higher value at nadir and the difference between the mean value of prenadir and nadir is statistically significant (P <.01). The number of LUCs increases during the pre and postnadir phase. Lymphocytes number appears stable in the prenadir phase. The MPXI index increases in the prenadir phase and falls at nadir and this difference is statistically significant(P <.01). LUCs are correlated with blasts and CD34+ cells in the pre and postnadir phase, with CD3+/CD4+ cells in the prenadir phase, and with CD2+/CD56+ in the postnadir phase. CONCLUSIONS: Our data have shown that the estimation of both percentage of LUCs and MPXI can predict the neutropenia phase and orient for its duration. The lymphocyte number may be regarded as a parameter of risk of fever after day 5 of chemotherapy and the number of blood CD34+ cells may be predicted by LUC count.

Altered tissue electric properties in lung cancer patients as detected by bioelectric impedance vector analysis.

Modifications of body composition are frequent in cancer patients. Bioelectric impedance analysis can specifically detect changes in tissue electric properties, which may be associated with outcome. We evaluated the distribution of the impedance vectors from 63 adult male patients with lung cancer, stages IIIB (33 patients) and IV (30 patients), in supportive therapy. Body weight change over the previous 6 m.o. was the same in both groups (stable/increased 36% and decreased in 62%). Patients were compared with 56 healthy subjects matched for gender, age, and body mass index (25 kg/m2). Impedance measurements (standard tetrapolar electrode placement on the hand and foot) were made with 50-kHz alternating currents. The resistance and reactance of the vector components were standardized by the height of the subjects and were plotted as resistance/reactance graphs. The impedance vector distribution was the same in patients with either stage IIIB or IV cancer. The mean vector position differed significantly between cancer patients and control subjects (Hotelling T2 test, P < 0.01) because of a reduced reactance component (i.e., a smaller phase angle) with preserved resistance component in both cancer groups. Patients with a phase angle smaller than 4.5 degrees had a significantly shorter, i.e., 18 m.o., survival. Body weight loss was not significantly associated with survival. In conclusion, impedance vectors from lung cancer patients were characterized by a reduced reactance component. The altered tissue electric properties were more predictive than weight loss of prognosis.

Quantitative analysis of ribonucleoside triphosphates in human lymphoid cells by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) was applied to develop an analytical method for quantitation of ribonucleoside triphosphates (rNTPs) in human lymphoid cells obtained from patients with B-chronic lymphocytic leukaemia (B-CLL) and cutaneous lymphomas. The results of this analysis showed a significant depression of intracellular rNTPs in patients with B-CLL, compared with rNTPs of healthy controls. These data are in agreement with other studies in which rNTP separations were performed with traditional high-performance liquid chromatography. MECC has proved to be a useful tool for intracellular rNTPs determination, revealing possible new applications in the study of the metabolic state of human cells. In addition, this method can be useful in monitoring the effect of many drugs (antiviral, antineoplastic) which interfere with nucleotide metabolism.

Effect of dipyridamole, 5'-(N-ethyl)-carboxamidoadenosine and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine on LOVO cell growth and morphology.

The effects of stable compounds acting on adenosine receptors, 5'-(N-ethyl)-carboxamidoadenosine (NECA: A2 and A1 adenosine receptor agonist) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX: selective A1 adenosine receptor antagonist) were evaluated in vitro on doxorubicin-resistant LoVo (LoVo-R) and doxorubicin-sensitive LoVo (LoVo-S) human metastatic cell lines by using the neutral red test for cell growth. The effect of dipyridamole, an adenosine uptake inhibitor, was also evaluated. The drugs had an inhibitory effect on LoVo cell growth. The association of the drugs with doxorubicin enhanced the inhibition of cell growth, particularly for NECA and PACPX on LoVo-R cells. Morphological observation with scanning electron microscopy indicated cytotoxicity of the tested compounds, alone or in association with doxorubicin both in LoVo-R or LoVo-S cells, supporting the hypothesis of inhibitory effect on tumor cell growth.