Elisa Capizzi

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples.

Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

Blood and Tissue Biomarkers in Prostate Cancer: State of the Art

The prevalence of prostate cancer (PCa) is high and increases with age. PCa is the most common cutaneous cancer in American men. Prostate-specific antigen (PSA) screening has impacted the detection of PCa and is directly responsible for a dramatic decrease in stage at diagnosis. Gleason score and stage at the time of diagnosis remain the mainstay to predict prognosis, in the absence of more accurate and reliable tissue or blood biomarkers. Despite extensive research efforts, very few biomarkers of PCa have been introduced to date in clinical practice. Even screening with PSA has recently been questioned. A thorough analysis of all tissue and serum biomarkers in prostate cancer research cannot be easily synthesized, and goes beyond the scope of the present article. Therefore the authors focus here on the most recently reported tissue and circulating biomarkers for PCa whose application in clinical practice is either current or expected in the near future.

Studies on immunoproteasome in human liver. Part I: Absence in fetuses, presence in normal subjects, and increased levels in chronic active hepatitis and cirrhosis

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.

Real time RT-PCR approach for the evaluation of ERBB2 overexpression in breast cancer archival samples: a comparative study with FISH, SISH, and immunohistochemistry.

We tested the reliability of real time reverse transcription polymerase chain reactions as an alternative method for the assessment of ERBB2 status in paraffin-embedded tissues of 83 patients with breast cancer and 20 non-neoplastic controls. PCR was also compared with the immunohistochemical (IHC) HercepTest score and with fluorescence (FISH) and silver (SISH) in-situ hybridization, in 42 selected cases. ERBB2 mRNA was overexpressed in 26/83 (31%) breast cancer samples, using a cutoff calculated as the mean value of the controls plus 3 SD or with the receiver operating curve. The PCR test showed a 96% sensitivity and a 100% specificity when compared with FISH, with an area under the receiver operating curve of 98.4%. Overexpression of ERBB2 at PCR was also significantly correlated with amplification in FISH (P<0.001, Mann-Whitney test) and in SISH (P<0.001, Mann-Whitney test), and with the IHC HercepTest scores 2 or 3 (P<0.001, Spearman rank correlation). FISH, SISH, and IHC were also compared with each other. ERBB2 amplification in FISH significantly correlated with that in SISH (P=0.002, χ2 test with a concordance of the 87%), but not with IHC HercepTest scores (P=0.214, χ2 test). Real time PCR is a reliable and cost-effective method for the assessment of ERBB2 status in archival breast cancer samples, compared with FISH. Its introduction in routine diagnostic pathology practice is feasible even if it requires amendments to the current clinical oncology protocols.

Blood Monitoring of Granzyme B and Perforin Expression After Intestinal Transplantation : Considerations on Clinical Relevance

Background. The use of biomarkers for rejection monitoring represents a major goal in intestinal transplantation. We analyzed the blood expression of Granzyme B (GB) and Perforin (PF) in the following pathological conditions after intestinal transplantation: acute rejection (AR), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection, and posttransplant lymphoproliferative disease (PTLD). The diagnostic accuracy and the clinical utility of these tests are finally discussed. Methods. GB and PF levels were measured by real time polymerase chain reaction on peripheral blood samples from 32 intestinal recipients. Blood samples (n=494) after comparison of clinical, histological, and microbiological data were assigned to the following groups: normal (n=307), AR (n=30), EBV infection (n=107), CMV infection (n=25), and PTLD (n=25). Results. Mean levels of GB and PF in the AR (GB=279.7; PF=256.7), PTLD (GB=199; PF=185.9), EBV (GB=133.2; PF=143.7), and CMV (GB=151.3; PF=144) groups were significantly higher than in the normal group (GB=100.1; PF=101.1) (all P<0.05, except for PF in CMV infection). The best accuracy was obtained for the diagnosis of AR with sensitivity and specificity of 80% and 79% for GB and 70% and 79% for PF, respectively. The area under the receiver-operator characteristics curve was 0.87 for GB and 0.82 for PF. Conclusions. GB and PF are diagnostic molecular markers of AR. GB and PF blood levels are also increased in case of viral infections or PTLD. Serial blood testing for GB and PF might be predictive of early intestinal graft dysfunction and should be interpreted in the context of the histological and virological analyses.

OATP 1B1/1B3 expression in hepatocellular carcinomas treated with orthotopic liver transplantation

The organic anion transporter peptides (OATP) 1B1 and 1B3 are hepatocytic-specific transporters determinant for the uptake of the contrast media Gd-EOB-DTPA during magnetic resonance, but variably lost in hepatocellular carcinoma (HCC). Here, we studied a series of HCCs from livers that underwent liver transplantation (OLT) and correlated the expression of OATP 1B1/1B3 with HCC morphological features and the expression of the biliary-type keratins K7 and K19, the latter previously correlated with a worse prognosis after OLT. Seventy-five HCCs from 69 OLT patients were evaluated by histology and immunohistochemistry with monoclonal antibodies against OATP 1B1/1B3, K7, and K19. Histopathological and immunohistochemical features were therefore compared to recipient follow-up data. Thirty-four (45%) HCCs were completely OATP−, and 18 (24%) showed positivity for K7 and/or K19. We observed a significant inverse correlation between OATP and K7/19 expression (P < 0.001): all OATP+ cases were K7/19−, while all K7+ and/or K19+ cases were OATP−. Sixteen cases were negative for all antibodies. No correlation was found between histopathological features and immunohistochemistry. Twenty-five recipients experienced HCC recurrence, and ten died from neoplastic recurrence. Neither OATP nor keratin expressions were correlated with HCC recurrence, while OATP negativity significantly correlated with HCC-related death after recurrence (P = 0.036). In conclusion, HCCs show a progressive loss in OATP immunoreactivity that correlates with the gain of a biliary phenotype. Although further studies are required to define these findings better, our results support the idea that OATP could be used together with K7/19 to identify a phenotypical “spectrum” in HCC progression.

Oestrogen receptor 1 mRNA is a prognostic factor in ovarian cancer patients treated with neo-adjuvant chemotherapy: Determination by array and kinetic PCR in fresh tissue biopsies

Oestrogen receptors (ESRs) regulate the growth and differentiation of normal ovarian epithelia. However, to date their role as biomarkers in the clinical setting of ovarian cancer remains unclear. In view of potential endocrine treatment options, we tested the role of ESR1 mRNA expression in ovarian cancer in the context of a neo-adjuvant chemotherapy trial. Study participants had epithelial ovarian or peritoneal carcinoma unsuitable for optimal upfront surgery and were treated with neo-adjuvant platinum-based chemotherapy before surgery. RNA was isolated from frozen tumour biopsies before treatment. RNA expression of ESR1 was determined by microarray and reverse transcriptase kinetic PCR technologies. The prognostic value of ESR1 was tested using univariate and multivariate Cox proportional hazards models, Kaplan-Meier survival statistics and the log-rank test. ESR1 positively correlates with proliferation markers and histopathological grading. ESR1 was a significant predictor of survival as a continuous variable in the univariate Cox regression analysis. In multivariate analysis, elevated baseline ESR1 mRNA levels predicted prolonged progression-free survival (P=0.041) and overall survival (P=0.01) after neo-adjuvant chemotherapy, independently of pathological grade and age. We conclude that pretreatment ESR1 mRNA is associated with tumour growth and is a strong prognostic factor in ovarian cancer, independent of the strongest clinical parameters used in clinical routine. We suggest that ESR1 mRNA status should be considered in order to minimize possible confounding effects in ovarian cancer clinical trials, and that early treatment with anti-hormonal agents based on reliable hormone receptor status determination is worth investigating.

Quantification of free plasma DNA before and after chemotherapy in patients with advanced epithelial ovarian cancer

Objectives A nonrandomized trial was planned to investigate the role of free plasma DNA (FPDNA) in patients with epithelial ovarian cancer before and after chemotherapy. Twenty-two patients with advanced stage ovarian cancer not suitable for debulking were treated with a neoadjuvant platinum/taxanes chemotherapy. Patients with clinical complete or partial response underwent radical hystero-oophorectomy, omentectomy, and lymphadenectomy and were followed up every 3 to 6 months. Methods Blood samples were obtained from each patient before chemotherapy, before each cycle, before and after surgery. FPDNA was quantified by real-time quantitative polymerase chain reaction using the Quantifiler Human Quantification Kit and expressed in ng/mL. Fifty female healthy blood donor volunteers were used as controls. Results Median FPDNA quantities discriminated between patients before chemotherapy (29.6±22.7 ng/mL) and controls (6.4±4.0 ng/mL) using a 14.5 ng/mL cutoff with 77% sensitivity and 96% specificity (P<0.001). Mean FPDNA concentrations significantly decreased after chemotherapy (17.9±14.5 ng/mL, P=0.001). A peak of FPDNA levels (66.2±45.2 ng/mL) was observed in association with surgery (P<0.001). Median follow-up and median progression-free survival time were 13.4±5.1 and 11.7±5.6 months, respectively. Eight patients with FPDNA values above the cutoff after chemotherapy showed disease progression or died, whereas 7 patients with FPDNA below the cutoff were free from disease. Patients with FPDNA levels above and below the cutoff showed significantly separated progression-free survival curves (P=0.007, log-rank test). Conclusions FPDNA quantification significantly discriminates between cancer patients and controls and correlates with response to chemotherapy. Although performed in a limited series, we demonstrated a correlation between FPDNA values and clinical behavior of ovarian cancer patients.

Spitzoid tumors in children and adults: a comparative clinical, pathological, and cytogenetic analysis.

Spitzoid neoplasms may represent a difficult diagnosis in the practice of dermatopathology. We evaluated the concordance of the fluorescence in-situ hybridization (FISH) assay, histopathology, and dermoscopy in a group of adults and in a group of children with spitzoid neoplasms. The FISH assay, designed to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes and of centromere 6 (Cep 6), was performed in a group of children and in a group of adults with a histopathologic diagnosis of spitzoid neoplasms. FISH data were compared with dermoscopy and histopathology. Fifteen spitzoid neoplasms were collected from 13 patients (five children and eight adults): nine lesions were histologically diagnosed as typical Spitz nevi; three lesions were melanomas and three were atypical Spitz nevi. The conventional FISH criteria were concordant with the clinical and histopathologic diagnosis of Spitz nevi in four adults and in three children. FISH criteria of the other neoplasms showed a concordance with the histopathologic diagnosis in three cases. Discordant results were obtained in five cases (two children, three adults). The FISH melanoma assay proved more reliable in spitzoid lesions found in adults than in children. This assay should be interpreted carefully in pediatric patients with Spitz nevi in the context of histological features as melanomas in the pediatric population may show distinct chromosomal aberrations.

Nodal Occult Metastases in Intermediate- and High-Risk Prostate Cancer Patients Detected Using Serial Section, Immunohistochemistry, and Real-Time Reverse Transcriptase Polymerase Chain Reaction: Prospective Evaluation With Matched-Pair Analysis

BACKGROUND The purpose of the study was to prospectively evaluate the incidence of nodal OCM assessed using SS, IHC, and RT-PCR in prostate cancer patients compared with the standard pathological evaluation (SPE), and to evaluate short-term oncological outcomes of patients with OCM. PATIENTS AND METHODS Fifty-four consecutive patients with intermediate- or high-risk prostate cancer treated with radical prostatectomy and extended pelvic LN dissection comprised the study population (StP). The central sections with the largest diameter of each LN of the StP and a matched-pair population (MpP) with identical characteristics as the StP were used to assess the improved detection rate of OCM. Pathological characteristics and biochemical recurrence-free survival were assessed according to the presence of macroscopic or OCM. RESULTS A total of 1064 LNs were processed in the 54 patients of the StP, with 11 (20.4%) patients with evident metastases at SPE and 7 with OCM (13.0% additional patients); the percentage of positive patients improved from 16.6% (18 of 108) of the MpP to 33.3% (18 of 54) of the StP (16% additional patients). The mean diameter of the 10 additional LNs with OCM found at SS only and of the 6 additional LNs found at IHC only was significantly lower than the mean diameter of the 28 metastases found at routine pathologic examination (RPE) (P < .0001). Patients with OCM showed risk of biochemical recurrence similar to patients with no LN metastases (P = .008). CONCLUSION SS, IHC, and RT-PCR can detect a not negligible percentage of OCM missed at RPE. Patients with OCM showed short-term oncological outcomes more similar to those with macroscopic metastases. Longer follow-up studies considering cancer-specific survival are needed.