Lorenzo Daniele

Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms.

BACKGROUND Tumor-infiltrating lymphocytes (TIL) are considered important in anticancer immunosurveillance, although their role has not been clearly established yet. We examined prevalence, correlations, and prognostic significance of TIL among our patient population of resected lung neoplasms. METHODS From 1993 to 2006, the presence of TIL was retrospectively evaluated in 1,290 patients operated on for primary lung neoplasms. Tumor-infiltrating lymphocytes were defined as those intraepithelial lymphocytes located within the cancer cell nests. RESULTS Tumor-infiltrating lymphocytes were detected in 294 patients (23%). A significant difference was found between prevalence in non-small cell lung carcinomas versus neuroendocrine tumors (290 of 1,208, 24% versus 4 of 82, 5%; p = 0.0001). Prevalence was similar in adenocarcinomas, squamous-cell carcinomas, and large-cell anaplastic carcinomas. Logistic regression analysis indicates that TIL correlate with grading (odds ratio, 1.27; 95% confidence interval, 1.04 to 1.55; p = 0.02), tumor dimension (odds ratio, 0.86; 95% confidence interval, 0.79 to 0.94; p = 0.0008), and vascular invasion (odds ratio, 1.62; 95% confidence interval, 1.21 to 2.16; p = 0.0009). A not significantly better survival in the presence of TIL was observed overall (p = 0.20), becoming significant in squamous-cell carcinomas (p = 0.03). In patients with stage I disease, TIL is associated with a significant survival advantage in squamous-cell carcinomas (p = 0.03). The survival advantage increases with the duration of follow-up and is more evident after 4 to 6 years. CONCLUSIONS Tumor-infiltrating lymphocytes are observed in about one fourth of resected lung neoplasms: they are rare in neuroendocrine tumors. Tumor-infiltrating lymphocytes are more frequent in poorly differentiated tumors and in tumors with microscopic vascular invasion. The presence of TIL correlates with an improved survival in squamous cell carcinomas, particularly at early stage. The survival advantage increases with the duration of follow-up.

Epidermal Growth Factor Receptor Gene in Primary Tumor and Metastatic Sites from Non-small Cell Lung Cancer

Introduction: The majority of patients with non-small cell lung cancer (NSCLC) develop distant metastases. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are capable of reducing brain and adrenal metastases. However, the EGFR status may be discordant between primary NSCLC and the corresponding metastases. Methods: Using fluorescence in situ hybridization (FISH) analysis, the EGFR gene status was evaluated in a series of 38 cerebral or adrenal metastases collected from two institutions and in the corresponding primary tumors. Also, EGFR mutational analysis was performed using direct sequencing on the cerebral metastases. Results: EGFR FISH was positive in 28% of the primary tumors and in 45% of the metastases (p < 0.05). Among the seven cases FISH-positive at the metastatic site but negative in the primary tumor, six were brain metastases, and one was an adrenal metastasis; all were polysomic for chromosome 7, none were amplified. No EGFR mutations have been found in the cerebral metastases. Conclusion: Because the molecular asset of EGFR may change during the metastatic progression of NSCLC to brain (but not to adrenal), the selection of patients with brain metastasis for specific targeted therapies by EGFR FISH analysis should be performed on metastatic lesions rather than on their corresponding primary tumors.

Predicting gefitinib responsiveness in lung cancer by fluorescence in situ hybridization/chromogenic in situ hybridization analysis of EGFR and HER2 in biopsy and cytology specimens

In non–small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutational analysis is an excellent predictor of responsiveness to treatment with tyrosine kinase inhibitors, such as gefitinib. In up to 80% of NSCLCs, cytologic samples or endoscopic biopsies are the only specimens available for molecular analysis, but PCR amplification of DNA from small fixed and paraffin-embedded samples may create artifactual mutations. Fluorescence in situ hybridization (FISH) of EGFR and HER2 has been proposed as an alternative method of analysis. This project aimed to determine the optimal scoring method for FISH or chromogenic in situ hybridization (CISH) assays when analyzing small NSCLC samples to predict response. FISH or CISH analysis of EGFR and HER2 genes was done on 42 small samples derived from NSCLC patients treated with gefitinib. EGFR mutational analysis was done after quantity and quality controls of DNA. In seven of seven cases, a balanced increase in EGFR gene and chromosome 7 number was found to correlate with the presence of specific EGFR mutations. In addition, seven of seven cases with balanced EGFR/HER2 polysomy and two of three cases with balanced EGFR/HER2 trisomy responded to gefitinib (75% of responders). Instead, the EGFR mutations predicted only 7 of 12 (58%) of gefitinib-responsive patients. When only endoscopic biopsies or cytologic specimens are available, we propose using FISH/CISH for EGFR and HER2 as the test of choice for selecting patients for treatment with gefitinib and to consider as negative predictive factor the absence of EGFR/HER2 gene gain. [Mol Cancer Ther 2007;6(4):1223–9]

Columnar cell lesions associated with breast calcifications on vacuum-assisted core biopsies: clinical, radiographic, and histological correlations.

Columnar cell lesions of the breast are increasingly recognized at mammography for their tendency to calcify. We studied 392 vacuum-assisted core biopsies performed solely for calcifications to evaluate the frequency of columnar cell lesions, their relationship with radiological risk, appearance of calcifications, and clinical data. Management and follow-up of columnar cell lesions without and with atypia (flat epithelial atypia) was analyzed. Cases with architectural atypia (cribriform spaces and/or micropapillae) were excluded from flat epithelial atypia. Calcifications were within the lumen of acini affected by columnar cell lesions in 137 out of 156 biopsies diagnosed with some columnar cell lesions. These represented 37% of vacuum-assisted core biopsies and 62% of low radiological risk (BI-RADS3) calcifications. High-risk (BI-RADS5) calcifications were never associated with columnar cell lesions. Age and menopausal status were comparable in columnar and in not-columnar cell lesions. Atypia was associated with long-term hormone replacement therapy in both lesions. Surgical biopsy was recommended for all cases with atypia. Flat epithelial atypia, as the only histological findings on vacuum-assisted core biopsies, was never associated with malignancy at surgery. In conclusion, we suggest that surgical excision is not mandatory when flat epithelial atypia is found as the most advanced lesion on vacuum-assisted core biopsy performed for low radiological risk calcifications, and that women should be advised of the possible hormone dependency of this entity.

Caveolin‐1 expression in lung carcinoma varies according to tumour histotype and is acquired de novo in brain metastases

Aims:  To study caveolin‐1 (Cav‐1) expression in metastatic lung carcinomas.

Tumor staging but not grading is associated with adverse clinical outcome in neuroendocrine tumors of the appendix: a retrospective clinical pathologic analysis of 138 cases.

Appendiceal neuroendocrine neoplasms (NENs) are rare and usually incidentally discovered. Most cases are clinically indolent, although the rare aggressive ones are poorly predictable. The aim of this study was to test the applicability and prognostic significance of the new World Health Organization (WHO) classification and to test the several pathologic features and TNM staging systems (American Joint Committee on Cancer and European Neuroendocrine Tumor Society) in these tumors. A multi-institutional retrospective series of 138 appendiceal NENs was selected on the basis of the availability of both pathologic material and clinical information, including follow-up data. All cases were reviewed to record pathologic features and to apply year 2000 and 2010 WHO classifications, as well as European Neuroendocrine Tumor Society and American Joint Committee on Cancer TNM stages. Clinical and pathologic characteristics were compared with disease outcome by contingency, univariate, and multivariate survival analyses. Although up to one third of cases presented several malignancy-associated pathologic features, only 4 patients died of the disease. Adverse outcome was significantly associated with extramural extension (including mesoappendix), well-differentiated carcinoma diagnosis (2000 WHO classification), pT3-4 stage, older age, and presence of positive resection margins, but not with tumor size, mitotic or proliferative indexes, and, consequently, 2010 WHO grading. In the appendix, at variance with midgut/hindgut NENs, the 2000 WHO classification performs better than the grading-based 2010 WHO scheme and, together with tumor stage, is the most relevant parameter associated with clinical aggressiveness.

Gefitinib (ZD1839): therapy in selected patients with non-small cell lung cancer (NSCLC)?

PURPOSE To evaluate response rate, toxicity and epidermal growth factor (EGFR) mutations and gene copy number as outcome predictive factors in Italian patients with non-small cell lung cancer (NSCLC) treated with gefitinib (Iressa) in an expanded access program (EAP). PATIENTS AND METHODS A total of 137 patients with advanced NSCLC received gefitinib as first line treatment or after failure of chemotherapy. In 43 cases, tissue specimens were available for EGFR status evaluation: immunohistochemical (IHC) for EGFR, fluorescence in situ hybridisation (FISH) or Chromogenic in situ hybridisation (CISH)-(ISH) analysis for EGFR and HER2 gene copy number, and PCR-DNA sequencing for mutational analysis of EGFR were performed. RESULTS In the study population, response rate (PR) was 13%; disease stabilization (DS) 26%; overall disease control rate 39%; median survival 6.3 months and time to progression 2.7 months. Toxicity was mild (G3 skin toxicity in 3% and G3 liver toxicity in 4% of patients). An EGFR-mutation was detected in 9/43 patients: Eight deletions in exon 19 and 1 missense mutation in exon 21. Increased gene copy number for EGFR and/or HER2 was detected in 17/43 patients. Response rate was significantly higher in women, non-smokers, in mutation carriers than in wild type carriers, in EGFR-trisomy/polysomy carriers and HER2-trisomy/polysomy carriers. CONCLUSIONS In this study, response rate and toxicity to gefitinib treatment were consistent with previously reported data for whites. Female gender, absence of smoking history, EGFR-mutations, EGFR and HER2-polysomy were significantly associated with response to gefitinib therapy in NSCLC patients.

A randomized double-blind placebo controlled phase I-II study on clinical and molecular effects of dietary supplements in men with precancerous prostatic lesions. Chemoprevention or "chemopromotion"?

Antioxidants effectiveness in prostate cancer (PCa) chemoprevention has been severely questioned, especially after the recent results of the Selenium and Vitamin E Cancer Prevention Trial. We present the results of a double‐blind randomized controlled trial (dbRCT) on the pharmacokinetic, clinical, and molecular activity of dietary supplements containing lycopene, selenium, and green tea catechins (GTCs) in men with multifocal high grade prostatic intraepithelial neoplasia (mHGPIN) and/or atypical small acinar proliferation (ASAP).

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT‐PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step‐sectioning protocol at 100 micron‐intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT‐PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT‐PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT‐PCR negative cases, metastases were detected only on sections distant from those analysed by RT‐PCR. CEA and/or CK19 were positive by RT‐PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.

Imatinib inhibits in vitro proliferation of cells derived from a pleural solitary fibrous tumor expressing platelet-derived growth factor receptor-beta.

We examined the in vitro effects of imatinib (Novartis Pharma AG, Basel, Switzerland) as a possible inhibitor of PDGFR pathway on cells derived from a recurrence of a pleural malignant solitary fibrous tumor (SFT). Primary cell culture was characterised by immunofluorescence. SFT-derived cells were treated with imatinib at different time points. Western blotting for PDGFR-beta, phospho-PDGFR-beta or smooth muscle actin (SMA) was performed before and after 96 h of treatment with imatinib. SFT-derived cells treated with imatinib for 96 h showed a dose dependent decrease of Ki67 expression. Results were confirmed by growth curve. Western blotting showed that PDGFR-beta was highly expressed and phosphorylated in SFT-derived cells and imatinib treatment reduced PDGFR-beta phosphorylation and SMA expression. With the limit of experimental findings, our results support a possible future application of imatinib as a candidate molecule in the target therapy of malignant SFTs over-expressing wild-type PDGFR.