Luca Molinaro

Thymidylate Synthase But Not Excision Repair Cross-Complementation Group 1 Tumor Expression Predicts Outcome in Patients With Malignant Pleural Mesothelioma Treated With Pemetrexed-Based Chemotherapy

PURPOSE The relationship between thymidylate synthase (TS) expression and outcome in patients with malignant pleural mesothelioma (MPM) treated with pemetrexed (P) was retrospectively evaluated. PATIENTS AND METHODS Sixty histologically confirmed patients with MPM previously treated with P and platinum (45 of 60) or as single agent (15 of 60) were retrospectively considered. Eighty-one control patients with MPM not P-treated were also evaluated. TS and excision repair cross-complementation group 1 (ERCC1) gene expression levels were evaluated by real-time polymerase chain reaction and by immunohistochemistry using the H-score. RESULTS Median TS H-score value was 90 (range, 5 to 240). A significant correlation between low TS protein expression and longer time to progression (TTP; 17.9 v 7.9 months; hazard ratio [HR], 2.05, 95% CI, 1.19 to 3.77; P = .02) or overall survival (OS; 30 v 16.7 months; HR, 2.38; 95% CI, 1.15 to 4.91; P = .019) was found when patients were divided according to median H-score. Conversely, TS mRNA levels were not significantly correlated with outcome. In platinum-treated patients (n = 45), no correlation was found with survival according to ERCC1 median H-score, but patients in the lower tertile had a significantly shorter survival (HR, 3.06; 95% CI, 1.08 to 8.69; P = .035). In control MPMs, TS had no prognostic role. At multivariate analysis, TS protein levels were the only independent prognostic factor for both TTP (HR, 2.71; 95% CI, 1.13 to 6.49; P = .02) and OS (HR, 6.91; 95% CI, 1.90 to 25.07; P = .003). CONCLUSION In patients with MPM treated with P-based chemotherapy, low TS protein levels are predictive of improved TTP and OS. The role of TS assessment is worth of prospective validation in future studies on MPM.

MammaPrint Molecular Diagnostics on Formalin-Fixed, Paraffin-Embedded Tissue

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Accuracy of CT-guided transthoracic needle biopsy of lung lesions: Factors affecting diagnostic yield

PurposeThis study was performed to analyse the variables affecting the diagnostic accuracy of computed tomography (CT)-guided transthoracic needle biopsy of pulmonary lesions.Materials and methodsA retrospective study of 612 consecutive procedures with confirmed final diagnoses was undertaken. Benign and malignant needle biopsy results were compared with final outcomes to determine diagnostic accuracy. A statistical analysis of factors related to patient characteristics, lung lesions and biopsy technique was performed to determine possible influences on diagnostic yield. A p value less than 0.05 was interpreted as statistically significant.ResultsThere were 508 (83%) malignant and 104 (17%) benign lesions. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for a diagnosis of malignancy were 90.2%, 99.0%, 99.8%, 67.3% and 91.7%, respectively. Overall diagnostic accuracy was 83.3%. Variables affecting diagnostic accuracy were the final diagnosis (benign 67%, malignant 92%; p<0.001) and lesion size (lesions<1.5 cm 68%, lesions 1.5–5.0 cm 87%, lesions>5 cm 78%; p<0.05).ConclusionsIn CT-guided transthoracic needle biopsy, the final diagnosis and lesion size affect diagnostic accuracy: benign lung lesions and lesions smaller than 1.5 cm or larger than 5.0 cm in diameter provide lower diagnostic yield.RiassuntoObiettivoValutare i fattori che influenzano l’accuratezza diagnostica dell’agobiopsia transtoracica percutanea TC-guidata di lesioni polmonari.Materiali e metodiSono state considerate retrospettivamente 612 procedure bioptiche consecutive per le quali è risultata disponibile la diagnosi definitiva. I reperti bioptici benigni e maligni sono stati confrontati con la diagnosi finale, ricavando l’accuratezza diagnostica della metodica. È stata quindi condotta un’analisi statistica relativamente a variabili proprie del paziente, della lesione e della tecnica bioptica per determinare possibili influenze sui valori di accuratezza. I dati ottenuti sono stati considerati statisticamente significativi per valori di p<0,05.RisultatiCinquecentootto lesioni (83%) sono risultate, alla diagnosi finale, maligne, mentre 104 (17%) benigne. I valori di sensibilità, specificità, valore predittivo positivo, valore predittivo negativo ed accuratezza, riferiti ad una diagnosi di malignità, sono risultati rispettivamente del 90,2%, 99,0%, 99,8%, 67,3% e 91,7%. L’accuratezza diagnostica globale della procedura è stata dell’83,3%. Le variabili che hanno influenzato significativamente i livelli di accuratezza sono risultate la diagnosi finale (espansi benigni 67%, maligni 92%; p<0,001) ed il diametro medio della lesione (lesioni<1,5 cm 68%, tra 1,5 e 5,0 cm 87%, >5 cm 78%; p<0,005).ConclusioniLa diagnosi finale (benignità versus malignità) e le dimensioni della lesione influenzano l’accuratezza diagnostica della metodica: addensamenti polmonari di natura benigna e lesioni con diametro <1,5 cm o >5 cm sono caratterizzati da livelli minori di accuratezza diagnostica.

Prognostic role of osteopontin expression in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) represents highly aggressive neoplasms with a mean survival of approximately 10 months. Osteopontin, a glycoprotein involved in cell-matrix interactions correlated with invasion and metastatic spread in several tumors, has recently been proposed as a serum marker of MPM in asbestos-exposed subjects. The aim of this study was to define the prognostic role of osteopontin in MPM. For the study, 32 long-term survivors (>24 months) and a random sample of 69 short-term survivors (<or=24 months) were matched according to the main clinicopathologic features. Immunohistochemical osteopontin expression in tissue specimens was quantified through the HScore (histologic scoring) method and correlated with clinicopathologic parameters and survival. Osteopontin expression was significantly lower in long-term compared with short-term survivors (P< .0001), and overall survival analysis showed that low osteopontin expression was associated with longer survival; multivariate analysis confirmed the value of osteopontin expression as an independent prognostic factor (P< .0001).

Comparative diagnostic and prognostic performances of the hematoxylin-eosin and phospho-histone H3 mitotic count and Ki-67 index in adrenocortical carcinoma

Mitotic count on hematoxylin and eosin slides is a fundamental morphological criterion in the diagnosis and grading of adrenocortical carcinoma in any scoring system employed. Moreover, it is the unique term strongly associated with patient’s prognosis. Phospho-histone H3 is a mitosis-specific antibody, which was already proven to facilitate mitotic count in melanoma and other tumors. Therefore, a study was designed to assess the diagnostic and prognostic role of phospho-histone H3 in 52 adrenocortical carcinomas, comparing manual and computerized count to standard manual hematoxylin- and eosin-based method and Ki-67 index. Manual hematoxylin and eosin and phospho-histone H3 mitotic counts were highly correlated (r=0.9077, P<0.0001), better than computer-assisted phospho-histone H3 evaluations, and had an excellent inter-observer reproducibility at Bland-Altman analysis. Three of 15 cases having <5 mitotic figures per 50 high-power fields by standard count on hematoxylin and eosin gained the mitotic figure point of Weiss Score after a manual count on phospho-histone H3 slides. Traditional mitotic count confirmed to be a strong predictor of overall survival (P=0.0043), better than phospho-histone H3-based evaluation (P=0.051), but not as strong as the Ki-67 index (P<0.0001). The latter further segregated adrenocortical carcinomas into three prognostic groups, stratifying cases by low (<20%), intermediate (20–50%), and high (>50%) Ki-67 values. We conclude that (a) phospho-histone H3 staining is a useful diagnostic complementary tool to standard hematoxylin and eosin mitotic count, enabling optimal mitotic figure evaluation (including atypical mitotic figures) even in adrenocortical carcinomas with a low mitotic index and with a very high reproducibility; (b) Ki-67 proved to be the best prognostic indicator of overall survival, being superior to the mitotic index, irrespective of the method (standard on hematoxylin and eosin or phospho-histone H3-based) used to count mitotic figures.

Minimum biopsy set for HER2 evaluation in gastric and gastro-esophageal junction cancer

Background and study aims: The HER2 status of small endoscopic biopsies is important for predicting the eligibility of patients with metastatic HER2-positive gastric cancer or gastro-esophageal junction (GEJ) cancer for anti-HER2 therapy approved by the U.S. Food and Drug Administration. The aim of this study was to identify the minimum biopsy set required to evaluate the HER2 status with confidence. Patients and methods: A total of 103 consecutive patients with resected gastric cancer or GEJ cancer were retrospectively selected; 2 formalin-fixed, paraffin-embedded samples of each surgical specimen and all paired endoscopic biopsies were analyzed for HER2 status with both immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. A total of 10 virtual biopsies were constructed by selecting areas 2.6 mm in diameter on the luminal side of digitalized slides obtained from the surgical specimens. The results of evaluating HER2 status in virtual biopsies, slides containing complete surgical specimens, and endoscopic biopsies were compared. The resulting minimum biopsy set was applied to the endoscopic biopsy series for validation. Results: A biopsy set containing a minimum of 5 samples was identified as the most accurate in predicting HER2 status (sensitivity, 92 %; specificity, 97 %). In only 3 of the 103 cases (2.9 %) did a comparison of the HER2 evaluation of virtual biopsies and that of entire slides show inconsistent results. Overall agreement between the endoscopic biopsies and surgical samples for HER2 IHC status increased from 78.4 % to 92.3 % when biopsy sets containing 4 or fewer samples were compared with biopsy sets containing 5 or more samples. Conclusions: Although the recommendations suggest that 8 to 10 biopsies are necessary, the results show that a minimum set of 5 biopsies may be sufficient for reliable HER2 assessment in gastric cancer and GEJ cancer. However, endoscopists should be aware that a smaller sample size may be less accurate in selecting patients eligible for anti-HER2 therapy.

Approaching heterogeneity of human epidermal growth factor receptor 2 in surgical specimens of gastric cancer.

Gastric cancer shows intratumoral heterogeneity for human epidermal growth factor receptor 2 expression. We evaluated whether the number of tissue blocks analyzed or the antibodies used may influence the immunohistochemical results in gastrectomy specimens. Clinicopathologic data from 148 patients receiving gastric surgery for cancer were collected. One tissue block for each of 88 primary tumors and 60 paired primary tumors and metastases was examined for human epidermal growth factor receptor 2 status by immunohistochemistry using 3 different antibodies (HercepTest, CB11, and 4B5) and by fluorescent in situ hybridization. Two additional tissue blocks of the primary tumor were tested by immunohistochemistry if the results were negative on the first tissue block. The concordance among the 3 antibodies was 94.5% (testing 1 tissue block). Two cases showed a clinically significant discrepancy between primary tumor (score 0) and lymph nodes metastases (score 3+). Additional block analysis increased both the sensitivity (from 63% to 83%) and the accuracy (from 91% to 94%) of immunohistochemistry as compared with fluorescent in situ hybridization. The multiblock approach could potentially identify a greater number of human epidermal growth factor receptor 2-positive gastric cancers, particularly those with higher levels of intratumor heterogeneity. In turn, human epidermal growth factor receptor 2 positivity correlated with a worse prognosis (P=.011) and was an independent variable in multivariate analysis (hazard ratio, 1.57). In conclusion, testing more than 1 tissue block of cancer from specimens of gastric resection provides a more reliable human epidermal growth factor receptor 2 assessment regardless of the antibody used.

Comparison of Anti-Estrogen Receptor Antibodies SP1, 6F11, and 1D5 in Breast Cancer Lower 1D5 Sensitivity but Questionable Clinical Implications

We compared the anti-estrogen receptors (ER) SP1, 6F11, and 1D5 antibodies in breast carcinoma cases with different ranges of positive cells to evaluate whether this could generate different therapies for patients. We selected 66 cases of breast cancer, each of which was immunostained with the 3 antibodies. 1D5 was less sensitive than SP1 and 6F11, as seen in 26, 20, and 21 negative cases, respectively. Nine cases showed differences in endocrine-therapy indications, of which 8 1D5-negative cases showed low positivity for SP1 and/or 6F11. However, these cases were prevalently G3, progesterone receptor-negative or low-positive, with high Ki-67 and positive HER-2 findings, all biological features associated with endocrine resistance. Finally ER values obtained with these 3 antibodies had no implications for chemotherapy.

Spontaneous and pronase-induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines†

A subgroup of HER2‐overexpressing breast tumours co‐expresses p95

Caveolin 1 expression independently predicts shorter survival in oligodendrogliomas

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