A Real-World Application of Liquid Biopsy in Metastatic Colorectal Cancer: The Poseidon Study.

BACKGROUND: First-line decision making is the key to the successful care of mCRC patients and RAS/BRAF status is crucial to select the best targeted agent. In hub centers, a relevant proportion of patients referred from small volume centers may not have standard tissue-based (STB) molecular results available at the time of the first visit (T0). Liquid biopsy (LB) may help circumvent these hurdles. METHODS: A monoinstitutional prospective head-to-head comparison of LB versus (vs.) STB testing was performed in a real-world setting. Selection criteria included: mCRC diagnosis with unknown RAS/BRAF status at T0, tumoral tissue archived in external centers, no previous treatment with anti-EGFR. At T0, patients underwent plasma sampling for LB testing and procedure for tissue recovery. RAS/BRAF genotyping was carried out by droplet digital PCR on circulating-tumoral (ct) DNA. The primary endpoint was the comparison of time to LB (T1) vs. STB (T2) results using the Mann-Whitney U test. Secondary endpoints were the concordance between LB and STB defined as overall percent agreement and the accuracy of LB in terms of specificity, sensitivity, positive and negative predictive value. We also performed an exploratory analysis on urinary (u) ctDNA. RESULTS: A total of 33 mCRC patients were included. Mean T1 and T2 was 7 and 22 days (d), respectively (p < 0.00001). T2 included a mean time for archival tissue recovery of 17 d. The overall percent agreement between LB and STB analysis was 83%. Compared to STB testing, LB specificity and sensitivity were 90% and 80%, respectively, with a positive predictive value of 94% and negative one of 69%. In detail, at STB and LB testing, RAS mutation was found in 45% and 42% of patients, respectively; BRAF mutation in 15%. LB results included one false positive and four false negative. False negative cases showed a significantly lower tumor burden at basal CT scan. Concordance between STB and uctDNA testing was 89%. CONCLUSIONS: Faster turnaround time, high concordance and accuracy are three key points supporting the adoption of LB in routinary mCRC care, in particular when decision on first-line therapy is urgent and tissue recovery from external centers may require a long time. Results should be interpreted with caution in LB wild-type cases with low tumor burden.

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases.

PURPOSE: More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD). PATIENTS AND METHODS: We analyzed a cohort of 112 patients with mCRC who had undergone metastatic resection with curative intent as part of the PREDATOR clinical trial. The study evaluated the prognostic value of ctDNA, correlating MRD status postsurgery with clinical outcomes by using a personalized and tumor-informed ctDNA assay (bespoke multiple PCR, next-generation sequencing assay). Postresection, systemic therapy was given to 39.2% of the patients at the discretion of the treating physician. RESULTS: Postsurgical, MRD positivity was observed in 54.4% (61 of 112) of patients, of which 96.7% (59 of 61) progressed at the time of data cutoff (hazard ratio [HR]: 5.8; 95% CI, 3.5 to 9.7; P < .001). MRD-positive status was also associated with an inferior overall survival: HR: 16.0; 95% CI, 3.9 to 68.0; P < .001. At the time of analyses, 96% (49 of 51) of patients were alive in the MRD-negative arm compared with 52.4% (32 of 61) in the MRD-positive arm. Patients who did not receive systemic therapy and were MRD-negative in the combined ctDNA analysis at two time points had an overall survival of 100%. In the multivariate analysis, ctDNA-based MRD status was the most significant prognostic factor associated with disease-free survival (HR: 5.78; 95% CI, 3.34 to 10.0; P < .001). CONCLUSION: This study confirms that in mCRC undergoing resection of metastases, postoperative MRD analysis is a strong prognostic biomarker. It holds promises for being implemented in clinical decision making, informing clinical trial design, and further translational research.

Synaptophysin expression in V600EBRAF-mutated advanced colorectal cancers identifies a new subgroup of tumours with worse prognosis.

BACKGROUND: Neuroendocrine differentiation has been extensively associated with worse prognosis and to mechanisms of therapy resistance in several epithelial cancers. A high prevalence of neuroendocrine differentiation was recently described in V600EBRAF-mutated (BRAFmt) metastatic colorectal cancers (mCRCs) but no data are available about its prognostic impact in this setting. METHODS: We assessed synaptophysin immunohistochemical expression in a multi-institutional series of 159 BRAFmt mCRCs with matched clinical and pathological information. Tumours were dichotomized as synaptophysin high and low. Overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier and log-rank tests. RESULTS: Thirty-five tumours (22.0%) showed any level of positivity for synaptophysin, and 18 (11.3%) were characterized by positivity in at least 20% of tumour cells. Four cases resulted 100% synaptophysin positive. The histotype of synaptophysin-positive tumours (i.e. ≥20%) was not otherwise specified in 11 cases (61.1%) and mucinous adenocarcinoma in 4 cases (22.2%). Four cases were DNA mismatch repair deficient (22.2%) and 7 (38.9%) were characterized by a high number of tumour-infiltrating lymphocytes. At multivariate analysis, high synaptophysin expression was a negative independent prognostic factor for both PFS (HR = 2.00, 95% confidence interval [CI] 1.21-3.33, p = 0.006) and OS (HR = 2.27, 95% CI 1.35-3.85, p = 0.001). CONCLUSIONS: Among BRAFmt mCRCs, synaptophysin-positive tumours are characterized by worse PFS and OS. Further studies should investigate the molecular mechanisms involved in the acquisition of the neuroendocrine phenotype to identify novel-targeted treatment strategies.

The heterogeneous clinical and pathological landscapes of metastatic Braf-mutated colorectal cancer.

Colorectal cancer (CRC) is a complex and molecularly heterogeneous disease representing one of the most frequent causes of cancer-related death worldwide. About 8-15% of CRCs harbor a mutation in BRAF gene, a proto-oncogene involved in cell proliferation, differentiation and survival through the MAPK signaling cascade. The acquisition of BRAF mutation is an early event in the "serrated" CRC carcinogenetic pathway and is associated with specific and aggressive clinico-pathological and molecular features. Despite that the presence of BRAF mutation is a well-recognized negative prognostic biomarker in metastatic CRC (mCRC), a great heterogeneity in survival outcome characterizes these patients, due to the complex, and still not completely fully elucidated, interactions between the clinical, genetic and epigenetic landscape of BRAF mutations. Because of the great aggressiveness of BRAF-mutated mCRCs, only 60% of patients can receive a second-line chemotherapy; so intensive combined and tailored first-line approach could be a potentially effective strategy, but to minimize the selective pressure of resistant clones and to reduce side effects, a better stratification of patients bearing BRAF mutations is needed.

Prediction of Benefit from Checkpoint Inhibitors in Mismatch Repair Deficient Metastatic Colorectal Cancer: Role of Tumor Infiltrating Lymphocytes.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is highly effective in microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC); however, specific predictive biomarkers are lacking. PATIENTS AND METHODS: Data and samples from 85 patients with MSI-H mCRC treated with ICIs were gathered. Tumor infiltrating lymphocytes (TILs) and tumor mutational burden (TMB) were analyzed in an exploratory cohort of "super" responders and "clearly" refractory patients; TILs were then evaluated in the whole cohort of patients. Primary objectives were the correlation between the number of TILs and TMB and their role as biomarkers of ICI efficacy. Main endpoints included response rate (RR), progression-free survival (PFS), and overall survival (OS). RESULTS: In the exploratory cohort, an increasing number of TILs correlated to higher TMB (Pearson's test, p = .0429). In the whole cohort, median number of TILs was 3.6 in responders compared with 1.8 in nonresponders (Mann-Whitney test, p = .0448). RR was 70.6% in patients with high number of TILs (TILs-H) compared with 42.9% in patients with low number of TILs (odds ratio = 3.20, p = .0291). Survival outcomes differed significantly in favor of TILs-H (PFS: hazard ratio [HR] = 0.42, p = .0278; OS: HR = 0.41, p = .0463). CONCLUSION: A significant correlation between higher TMB and increased number of TILs was shown. A significantly higher activity and better PFS and OS with ICI in MSI-H mCRC were reported in cases with high number of TILs, thus supporting further studies of TIL count as predictive biomarker of ICI efficacy. IMPLICATIONS FOR PRACTICE: Microsatellite instability is the result of mismatch repair protein deficiency, caused by germline mutations or somatic modifications in mismatch repair genes. In metastatic colorectal cancer (mCRC), immunotherapy (with immune checkpoint inhibitors [ICIs]) demonstrated remarkable clinical benefit in microsatellite instability-high (MSI-H) patients. ICI primary resistance has been observed in approximately 25% of patients with MSI-H mCRC, underlining the need for predictive biomarkers. In this study, tumor mutational burden (TMB) and tumor infiltrating lymphocyte (TIL) analyses were performed in an exploratory cohort of patients with MSI-H mCRC treated with ICIs, demonstrating a significant correlation between higher TMB and increased number of TILs. Results also demonstrated a significant correlation between high number of TILs and clinical responses and survival benefit in a large data set of patients with MSI-H mCRC treated with ICI. TMB and TILs could represent predictive biomarkers of ICI efficacy in MSI-H mCRC and should be incorporated in future trials testing checkpoint inhibitors in colorectal cancer.

Prognostic Role of a New Index (RAPID Index) in Advanced Hepatocellular Carcinoma Patients Receiving Sorafenib: Training and Validation Cohort.

BACKGROUND AND AIMS: The aim of the present study is to evaluate a new index influenced by the balance between the immune system, α-fetoprotein (AFP), and lactate dehydrogenase (LDH) (RAPID index) as a prognostic factor in patients treated with sorafenib. METHODS: This study was conducted on a training cohort of 159 hepatocellular carcinoma (HCC) patients and a validation cohort of 68 HCC patients treated with sorafenib. The RAPID index was calculated as neutrophil/lymphocyte count × LDH × AFP. RESULTS: In the training cohort, the median overall survival (OS) was 23.2 months (95% CI 11-25) and 12.1 months (95% CI 9-15) for patients with a low (≤3,226) and high (>3,226) RAPID index, respectively (ref. <3,226, HR = 0.56, 95% CI 0.35-0.88, p = 0.017). Following adjustment for clinical covariates, multivariate analysis confirmed the RAPID index ≤3,226 versus >3,226 (HR = 0.37, 95% CI 0.18-0.74, p = 0.0054) as an independent prognostic factor for OS. In the validation cohort, the median OS was 26.9 months (95% CI 17.6-26.9) and 7.0 months (95% CI 6.2-9.2) for patients with a low (≤ 3,226) and high (>3,226) RAPID index, respectively (ref. <3,226, HR = 0.19, 95% CI 0.10-0.36, p < 0.0001). Performing the same multivariate analysis of the training cohort (AFP, Eastern Cooperative Oncology Group, aspartate aminotransferase, neutrophil, platelet, systemic inflammatory index and RAPID index), the RAPID index <3,226 versus >3,226 (HR = 3.86, 95% CI 1.45-10.29, p = 0.007) was found to be an independent prognostic factor for predicting OS. CONCLUSION: The low cost, easy assessment, and reproducibility of a full blood count make the RAPID index a promising tool for assessing HCC prognosis in future clinical practice.

CK7 and consensus molecular subtypes as major prognosticators in V600EBRAF mutated metastatic colorectal cancer.

BACKGROUND: V600EBRAF mutated metastatic colorectal cancer (mCRC) is a subtype (10%) with overall poor prognosis, but the clinical experience suggests a great heterogeneity in survival. It is still unexplored the real distribution of traditional and innovative biomarkers among V600EBRAF mutated mCRC and which is their role in the improvement of clinical prediction of survival outcomes. METHODS: Data and tissue specimens from 155 V600EBRAF mutated mCRC patients treated at eight Italian Units of Oncology were collected. Specimens were analysed by means of immunohistochemistry profiling performed on tissue microarrays. Primary endpoint was overall survival (OS). RESULTS: CDX2 loss conferred worse OS (HR = 1.72, 95%CI 1.03-2.86, p = 0.036), as well as high CK7 expression (HR = 2.17, 95%CI 1.10-4.29, p = 0.026). According to Consensus Molecular Subtypes (CMS), CMS1 patients had better OS compared to CMS2-3/CMS4 (HR = 0.37, 95%CI 0.19-0.71, p = 0.003). Samples showing less TILs had worse OS (HR = 1.72, 95%CI 1.16-2.56, p = 0.007). Progression-free survival analyses led to similar results. At multivariate analysis, CK7 and CMS subgrouping retained their significant correlation with OS. CONCLUSION: The present study provides new evidence on how several well-established biomarkers perform in a homogenousV600EBRAF mutated mCRC population, with important and independent information added to standard clinical prognosticators. These data could be useful to inform further translational research, for patients' stratification in clinical trials and in routine clinical practice to better estimate patients' prognosis.

A validated prognostic classifier for V600EBRAF-mutated metastatic colorectal cancer: the 'BRAF BeCool' study.

BACKGROUND: Despite the well-known negative prognostic value of the V600EBRAF mutation in patients with metastatic colorectal cancer (mCRC), its outcome is quite heterogeneous, and the basis for this prognostic heterogeneity should be better defined. METHODS: Two large retrospective series of V600EBRAF-mutated mCRC from 22 institutions served as an exploratory and validation set to develop a prognostic score. The model was internally and externally validated. RESULTS: A total of 395 V600EBRAF-mutated mCRCs were included in the exploratory set. Performance status, CA19.9, lactate dehydrogenase, neutrophil/lymphocyte ratio, grading and liver, lung and nodal involvement emerged as independent prognostic factors for overall survival (OS). Two different scoring systems were built: a 'complete' score (0-16) including all significant covariates and a 'simplified' score (0-9), based only on clinicopathological covariates, and excluding laboratory values. Adopting the complete score, proportions of patients with a low (0-4), intermediate (5-8) and high (9-16) score were 44.7%, 42.6% and 12.6%, respectively. The median OS was 29.6, 15.5 (hazard ratio [HR] for intermediate vs low risk: 2.16, 95% confidence interval [CI]: 1.44-3.22, p < .001) and 6.6 months (HR for high vs low risk: 4.72, 95% CI: 2.72-8.20, p < .001). Similar results were observed also after adjusting for the type of first-line treatment and adopting the simplified score. The simplified prognostic score derived from the exploratory set was then applied to the validation set for external confirmation. CONCLUSIONS: These scoring systems are based on easy-to-collect data and defined specific subgroups with relevant differences in their life expectancy. These tools could be useful in clinical practice, would allow better stratification of patients in clinical trials and may be adopted for proper adjustments in exploratory translational analyses.

Class 1, 2, and 3 BRAF-Mutated Metastatic Colorectal Cancer: A Detailed Clinical, Pathologic, and Molecular Characterization.

PURPOSE: BRAF mutations are grouped in activating RAS-independent signaling as monomers (class 1-V600E) or as dimers (class 2-codons 597/601), and RAS-dependent with impaired kinase activity (class 3-codons 594/596). Although clinical, pathologic, and molecular features of V600EBRAF-mutated metastatic colorectal cancer (mCRC) are well known, limited data are available from the two other classes. EXPERIMENTAL DESIGN: Data from 117 patients with BRAF (92 class 1, 12 class 2, and 13 class 3)-mutated mCRC were collected. A total of 540 BRAF wt mCRCs were included as control. IHC profiling was performed to determine the consensus molecular subtypes (CMS), cytokeratin 7/20 profiles, tumor-infiltrating lymphocyte infiltration, and BM1/BM2 categorization. Overall survival (OS) and progression-free survival were evaluated by Kaplan-Meier and log-rank test. RESULTS: Class 3 BRAF-mutated mCRC was more frequently left sided (P = 0.0028), pN0 (P = 0.0159), and without peritoneal metastases (P = 0.0176) compared with class 1, whereas class 2 cases were similar to class 1. Hazard ratio for OS, as compared with BRAF wt, was 2.38 [95% confidence interval (CI), 1.61-3.54] for class 1, 1.90 (95% CI, 0.85-4.26) for class 2, and 0.93 (95% CI, 0.51-1.69) for class 3 (P < 0.0001). Class 2 and 3 tumors were all assigned to CMS2-3. A higher median CD3/CD8-positive lymphocyte infiltration was observed in BRAF-mutated class 2 (P = 0.033) compared with class 3 cases. CONCLUSIONS: For the first time, different clinical and pathologic features and outcome data were reported according to the three BRAF mutation classes in mCRC. Specific targeted treatment strategies should be identified in the near future for such patients.

A New Genetic Risk Score to Predict the Outcome of Locally Advanced or Metastatic Breast Cancer Patients Treated With First-Line Exemestane: Results From a Prospective Study.

INTRODUCTION: Approximately 50% of locally advanced or metastatic breast cancer (MBC) patients treated with first-line exemestane do not show objective response and currently there are no reliable biomarkers to predict the outcome of patients using this therapy. The constitutive genetic background might be responsible for differences in the outcome of exemestane-treated patients. We designed a prospective study to investigate the role of germ line polymorphisms as biomarkers of survival. PATIENTS AND METHODS: Three hundred two locally advanced or MBC patients treated with first-line exemestane were genotyped for 74 germ line polymorphisms in 39 candidate genes involved in drug activity, hormone balance, DNA replication and repair, and cell signaling pathways. Associations with progression-free survival (PFS) and overall survival (OS) were tested with multivariate Cox regression. Bootstrap resampling was used as an internal assessment of results reproducibility. RESULTS: Cytochrome P450 19A1-rs10046TC/CC, solute carrier organic anion transporter 1B1-rs4149056TT, adenosine triphosphate binding cassette subfamily G member 2-rs2046134GG, fibroblast growth factor receptor-4-rs351855TT, and X-ray repair cross complementing 3-rs861539TT were significantly associated with PFS and then combined into a risk score (0-1, 2, 3, or 4-6 risk points). Patients with the highest risk score (4-6 risk points) compared with ones with the lowest score (0-1 risk points) had a median PFS of 10 months versus 26.3 months (adjusted hazard ratio [AdjHR], 3.12 [95% confidence interval (CI), 2.18-4.48]; P < .001) and a median OS of 38.9 months versus 63.0 months (AdjHR, 2.41 [95% CI, 1.22-4.79], P = .012), respectively. CONCLUSION: In this study we defined a score including 5 polymorphisms to stratify patients for PFS and OS. This score, if validated, might be translated to personalize locally advanced or MBC patient treatment and management.

Candidate germline polymorphisms of genes belonging to the pathways of four drugs used in osteosarcoma standard chemotherapy associated with risk, survival and toxicity in non-metastatic high-grade osteosarcoma.

This study aimed to identify associations between germline polymorphisms and risk of high-grade osteosarcoma (HGOS) development, event-free survival (EFS) and toxicity in HGOS patients treated with neo-adjuvant chemotherapy and surgery.Germline polymorphisms of 31 genes known to be relevant for transport or metabolism of all four drugs used in HGOS chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) were genotyped in 196 patients with HGOS and in 470 healthy age and gender-matched controls. Of these 196 HGOS patients, a homogeneously treated group of 126 patients was considered for survival analyses (survival cohort). For 57 of these, treatment-related toxicity data were available (toxicity cohort).Eleven polymorphisms were associated with increased risk of developing HGOS (p < 0.05). The distribution of polymorphisms in patients was characterized by a higher Shannon entropy. In the survival cohort (n = 126, median follow-up = 126 months), genotypes of ABCC2_1249A/G, GGH_452T/C, TP53_IVS2+38G/C and CYP2B6*6 were associated with EFS (p < 0.05). In the toxicity cohort (n = 57), genotypes of ABCB1_1236T/C, ABCC2_1249A/G, ABCC2_3972A/G, ERCC1_8092T/G, XPD_23591A/G, XRCC3_18067T/C, MTHFR_1298A/C and GGH_16T/C were associated with elevated risk for toxicity development (p < 0.05).The results obtained in this retrospective study indicate that the aforementioned germline polymorphisms significantly impact on the risk of HGOS development, EFS and the occurrence of chemotherapy-related toxicity. These findings should be prospectively validated with the aim of optimizing and tailoring HGOS treatment in the near future.

Polymorphic thymidylate synthase gene impacts on overall survival of patients with epithelial ovarian cancer after platinum-based chemotherapy.

AIM: High levels of TS have been associated with a worse clinical outcome in several cancers including epithelial ovarian cancer (EOC). The TS gene (TYMS) is highly polymorphic and has an effect on mRNA/protein expression. MATERIALS & METHODS: Six TYMS polymorphisms were investigated for overall survival (OS) in 216 EOC patients: TYMS 1494ins/del, TSER (variable number of tandem repeats of 28 bp), TSER G>C, TYMS 1053C>T, TYMS IVS6-68C>T and TYMS 1122A>G. RESULTS: In a multivariate analysis, TYMS 1494 del/del genotype was associated with a significant increased OS compared with the ins/ins genotype (hazard ratio: 0.36; 95% CI: 0.16-0.82, p = 0.01). Similar results were obtained for the mutant genotypes TYMS 1053TT and TYMS IVS6-68TT. The event-free survival was significantly higher in TYMS 1053TT patients compared with wild-type patients (p = 0.05). CONCLUSION: TYMS 1494ins/del, 1053C>T and IVS6-68C>T polymorphisms can be prognostic markers for OS in patients with EOC, independently from stage at diagnosis, median age and tumor histotype.

miRNA pharmacogenomics: the new frontier for personalized medicine in cancer?

In recent years pharmacogenomic research has highlighted several genetic biomarkers of treatment toxicity and efficacy, dealing with drug metabolism, transport and mechanism of action. More recently, polymorphisms in miRNA encoding genes, their targets or factors involved in their maturation are rising as new pharmacogenomic markers in cancer. miRNAs are brief ncRNAs involved in DNA translational control, with an effect on mRNA and protein-expression levels. The study of genetic polymorphisms in genes involved in miRNA translational control machinery could give innovative insights in pharmacogenomics. This review summarizes the most recent and promising results in the field and gives an overview of the future perspective of personalized cancer therapy.

Drug interactions among the epidermal growth factor receptor inhibitors, other biologics and cytotoxic agents.

The epidermal growth factor receptor (EGFR) signalling pathway is a key element in the growth of several epithelial malignancies. Small molecules tyrosine kinase inhibitors (TKIs) and anti-EGFR monoclonal antibodies (mAbs) prevent the phosphorylation of the receptor, leading to cell cycle arrest at G(1) phase, apoptosis, inhibition of angiogenesis and metastasis. To increase the antitumoral effects of EGFR inhibitors (EGFRIs), a number of combinatory regimens have been evaluated and planned with standard cytotoxic drugs and/or inhibitors of EGFR complementary pathways such as mTOR, VEGF and Ras/Raf/ERK. Compared to EGFRI monotherapy, the combination approach is a promising strategy to improve tumor response and survival. However, pharmacokinetic (absorption, distribution, metabolism and excretion) and pharmacodynamic drug interactions can occur, affecting the outcome. Pharmacokinetics of TKIs can be affected by drugs used in combination: conversely, pharmacokinetic interactions have not been reported for EGFR mAbs. Potential pharmacokinetic interactions occur between EGFRIs and other factors such as food and hydrocarbons in tobacco smoke were also considered. EGFRIs are characterized by a number of pharmacodynamic interactions that must be taken into consideration to avoid adverse events, to increase antitumoral activity, and define potential new strategies for developing efficient combination regimens. In this context, treatment schedule and drug sequence appear to be particularly relevant for combination regimens with EGFRIs. Improved molecular characterisation of the EGFR pathway and its complementary pathways in tumor cells is required to better define predictive pharmacokinetic and pharmacodynamic biomarkers for optimum treatment outcome.

Effect of TP53 Arg72Pro and MDM2 SNP309 polymorphisms on the risk of high-grade osteosarcoma development and survival.

PURPOSE: The germ-line polymorphisms TP53 Arg72Pro and MDM2 SNP309 T>G are risk factors for tumor development and affect response to chemotherapy and survival in several cancers, but their prognostic and predictive value in patients with high-grade osteosarcomas is not yet defined. The purpose of this study was to investigate the effect of the TP53 Arg72Pro and the MDM2 SNP309 on the risk of osteosarcoma development and survival. EXPERIMENTAL DESIGN: The relative risk to develop osteosarcomas and the overall survival associated to TP53 Arg72Pro and MDM2 SNP309 polymorphisms were investigated in 201 patients. Correlations with event-free survival (EFS) were analyzed in a homogeneous subgroup of 130 patients with high-grade osteosarcomas of the limbs, nonmetastatic at diagnosis, which underwent neoadjuvant chemotherapy. RESULTS: Multivariate analysis showed that the MDM2 polymorphism T309G was associated with an increased risk of developing osteosarcomas [GG versus TT; odds ratio, 2.09; 95% confidence interval (95% CI), 1.15-3.78]. A case/control gender approach evidenced a significant increased risk only for female osteosarcoma patients (GG versus TT; odds ratio, 4.26; 95% CI, 1.61-11.25). Subjects carrying the TP53 Arg72Pro polymorphism were found to have a significantly increased death risk (Pro/Pro versus Arg/Arg; hazard ratio, 2.90; 95% CI, 1.28-6.66). In the subgroup of 130 high-grade osteosarcomas, the TP53 Arg72Pro was an independent marker of EFS (Pro/Pro versus Arg/Arg; hazard ratio, 2.67; 95% CI, 1.17-6.11). CONCLUSION: The study provides evidence supporting the association of MDM2 SNP309 with high-grade osteosarcoma risk in females and shows that TP53 Arg72Pro has a prognostic value for overall survival and EFS in osteosarcoma patients.

Predictive role of the UGT1A1, UGT1A7, and UGT1A9 genetic variants and their haplotypes on the outcome of metastatic colorectal cancer patients treated with fluorouracil, leucovorin, and irinotecan.

PURPOSE: UGT1A1*28 is considered the main pharmacogenetic predictor of the toxicity outcome of irinotecan-treated patients. We evaluated the effect of other UGT1A variants and haplotypes involved in 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation on severe toxicity and efficacy of fluorouracil, leucovorin, and irinotecan (FOLFIRI). PATIENTS AND METHODS: In addition to UGT1A1*28, UGT1A1*60, UGT1A1*93, UGT1A7*3, and UGT1A9*22 were genotyped in 250 metastatic colorectal cancer patients, and associations with severe hematologic and nonhematologic toxicity, objective response, time to progression (TTP), and overall survival were evaluated. In a subset of 71 patients, pharmacokinetic data were also available. RESULTS: UGT1A7*3 was the only marker of severe hematologic toxicity after the first cycle (odds ratio [OR], 3.94; 95% CI, 1.05 to 14.82; P = .04) in a multivariate analysis. It was also associated with glucuronidation ratio (SN-38G area under the curve [AUC]/SN-38 AUC) and biliary index (irinotecan AUC) x (SN-38 AUC/SN-38G AUC). Haplotype I (all the reference sequence alleles but UGT1A9*22) was a predictor of severe hematologic toxicity during the entire course of therapy (OR, 0.39; 95% CI, 0.19 to 0.82; P = .01), together with sex (OR, 2.08; 95% CI, 1.01 to 4.28; P = .05). In addition to UGT1A1*28, haplotype II (all the variant alleles but UGT1A9*22) was associated with a response rate (OR, 8.61; 95% CI, 1.75 to 42.38; P = .01). UGT1A1*28 was the only marker associated with TTP. CONCLUSION: We propose that UGT1A variants additional to UGT1A1*28 might improve the prediction of the outcome of colorectal cancer patients treated with FOLFIRI. A UGT1A haplotype-based approach might be an efficacious strategy to achieve treatment individualization of FOLFIRI.

The role of UGT1A1*28 polymorphism in the pharmacodynamics and pharmacokinetics of irinotecan in patients with metastatic colorectal cancer.

PURPOSE: UGT1A1*28 polymorphism has been associated with decreased glucuronidation of SN38, the active metabolite of irinotecan. This could increase toxicity with this agent. PATIENTS AND METHODS: In a prospective study, 250 metastatic colorectal cancer patients were treated with irinotecan, fluorouracil, and leucovorin as first-line treatment. UGT1A1*28 polymorphism was investigated with respect to the distribution of hematologic and nonhematologic toxicity, objective response rate, and survival. Pharmacokinetics was investigated in a subgroup of patients (71 of 250) who had been analyzed with respect to toxicity and efficacy. RESULTS: UGT1A1*28 polymorphism was associated with a higher risk of grade 3 to 4 hematologic toxicity (odds ratio [OR], 8.63; 95% CI, 1.31 to 56.55), which was only relevant for the first cycle, and was not seen throughout the whole treatment period for patients with both variant alleles TA7/TA7 compared with wild-type TA6/TA6. The response rate was also higher in TA7/TA7 patients (OR, 0.32; 95% CI, 0.12 to 0.86) compared with TA6/TA6. A nonsignificant survival advantage was observed for TA7/TA7 when compared with TA6/TA6 patients (hazard ratio, 0.81; 95% CI, 0.45 to 1.44). Higher response rates were explained by a different pharmacokinetics with higher biliary index [irinotecan area under the curve (AUC)x(SN38 AUC/SN38G AUC)] and lower glucuronidation ratio (SN38G AUC/SN38 AUC) associated with the TA7/TA7 genotype and a higher response rate, indicating that the polymorphism is functionally relevant. CONCLUSION: The results indicate that UGT1A1*28 polymorphism is of some relevance to toxicity; however, it is less important than discussed in previous smaller trials. In particular, the possibility of a dose reduction for irinotecan in patients with a UGT1A1*28 polymorphism is not supported by the result of this analysis.

Carboxylesterase isoform 2 mRNA expression in peripheral blood mononuclear cells is a predictive marker of the irinotecan to SN38 activation step in colorectal cancer patients.

PURPOSE: Irinotecan (CPT11) is a prodrug activated in humans mainly by carboxylesterase 2 (CES2) generating the SN38 metabolite responsible for the drug efficacy and toxicity. The interpatients variability in CPT11 activation step could cause unpredictable toxicity. To identify a predictive molecular marker for CPT11 activation in cancer patients, we investigated the CES2 mRNA expression in peripheral blood mononuclear cells (PBMC) and correlated it to CPT11 activation rate, toxic effects, and response. EXPERIMENTAL DESIGN: Forty-five colorectal cancer patients were treated with a CPT11-including regimen (FOLFIRI). CES2 mRNA expression in PBMC was quantified by reverse transcription-PCR in real time. Plasma concentrations of CPT11, SN38, and SN38-glucuronide were determined by high-performance liquid chromatography and the pharmacokinetic variables calculated adopting the noncompartmental model. Toxicity was evaluated by the National Cancer Institute Common Toxicity Criteria scale and response by the WHO criteria. RESULTS: A high interindividual variability in CES2 mRNA relative expression was observed (median, 1.45; range, 0.01-28.21). CES2 mRNA expression level was significantly associated with CPT11 activation ratio [(AUC(SN38) + AUC(SN38G))/AUC(CPT11)]. Patients with CES2 mRNA expression above the median cutoff value presented an activation ratio higher (median, 0.25; range, 0.15-0.42) than those with CES2 mRNA below the median (median, 0.20; range, 0.10-0.40; P = 0.013). This was associated with a nonsignificant trend of 1.34-fold increase of SN38 AUC in the group of patients with high CES2 mRNA expression (mean, 1.03 +/- 0.62 versus 0.77 +/- 0.32 micromol/L hour). Eight of 23 high CES2 mRNA-expressing patients (34.8%) developed grade 3 to 4 neutropenia or diarrhea compared with 2 of 22 (9.1%) in the low CES2-expressing group (P = 0.071). CONCLUSION: Our data support a predictive power of CES2 mRNA expression in PBMC for the activation rate of CPT11.

Sarcomas and pharmacogenetics.

Sarcomas are a heterogeneous group of tumors, requiring different chemotherapeutic approaches. Recently, several regimens for metastatic tumors were evaluated with respect to the different responses to conventional chemotherapy of the various histologic subtypes of sarcomas. The impact of pharmacogenetics in the progress of chemotherapy appears to be crucial in defining the clinical response to many drugs, such as anthracycline or alkylating agents, that are widely used in treatment regimens for soft tissue sarcomas (STS) or sarcomas of the bone. Polymorphisms of metabolizing enzymes (e.g., cytochrome P450 and glutathione-S-transferase), transporter proteins (reduced folate carrier and P-glycoprotein) or target proteins (thymidylate synthase, methylenetetrahydrofolate reductase, dihydrofolate reductase, and c-KIT) may be responsible for an altered clinical outcome, in terms of both response and toxicity. The administration of new chemotherapeutic agents, such as imatinib for gastrointestinal tumors (GIST), requires the study of genetic polymorphisms possibly affecting the integrity of the target (c-KIT), which may provide valid information regarding possible developments of therapy. For STS and sarcoma of the bone, the genetic markers, which could be unambiguously predictive of the phenotypic profile of patients, are as yet undetermined.