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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.

Correlative immunohistochemical and reverse transcriptase polymerase chain reaction analysis of somatostatin receptor type 2 in neuroendocrine tumors of the lung.

Somatostatin receptors type 2 (sst2) have been frequently detected in neuroendocrine tumors and bind somatostatin analogues, such as octreotide, with high affinity. Receptor autoradiography, specific mRNA detection and, more recently, antisst2 polyclonal antibodies are currently employed to reveal sst2. The aim of the present study was to investigate by three different techniques the presence of sst2 in a series of 26 neuroendocrine tumors of the lung in which fresh frozen tissue and paraffin sections were available. It was possible, therefore, to compare, in individual cases, RNA analysis studied by reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH), and immunohistochemistry. A series of 20 nonneuroendocrine lung carcinoma samples served as controls. RT-PCR was positive for sst2 in 22 of 26 samples, including 15 of 15 typical carcinoids, 5 of 6 atypical carcinoids, and 2 of 5 small-cell carcinomas. The sst2 mRNA signal obtained by RT-PCR was strong in the majority (87%) of typical carcinoids and of variable intensity in atypical carcinoids and small-cell carcinomas. A weakly positive signal was observed in 5 of 20 control samples. In immunohistochemistry, two different antibodies (anti-sst2) were employed, including a monoclonal antibody, generated in the Department of Pathology, University of Turin. In the majority of samples a good correlation between sst2 mRNA (as detected by RT-PCR) and sst2 protein expression (as detected by immunohistochemistry) was observed. However, one atypical carcinoid and one small-cell carcinoma had focal immunostaining but no RT-PCR signal. ISH performed in selected samples paralleled the results obtained with the other techniques. A low sst2 expression was associated with high grade neuroendocrine tumors and with aggressive behavior. It is concluded that 1) neuroendocrine tumors of the lung express sst2, and there is a correlation between the mRNA amount and the degree of differentiation; 2) immunohistochemistry and ISH are reliable tools to demonstrate sst2 in these tumors; and 3) sst2 identification in tissue sections may provide information on the diagnostic or therapeutic usefulness of somatostatin analogues in individual patients with neuroendocrine tumors.

Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure

We propose multicore tissue microarray (TMA) as an alternative to whole section for routine assessment of prognostic factors in breast cancer. Since 2004, we introduced the multicore TMA for testing estrogen (ER) and progesterone receptors (PR), proliferation activity by Ki67, and HER2 overexpression and amplification in routine work. At least four tumor foci were selected on the whole section, and a dedicated technician used a stereomicroscope for accurate sampling of the selected areas. To identify a specific case in the TMA, a separate file and a computerized reporting form with the TMA map were created. A preliminary pilot study comparing the TMA results with those obtained on whole sections showed the specificity of the procedure. Moreover, in everyday diagnosis, hormone receptors were repeated on full section when negative in TMA, without significant discrepancy. Retrospective analysis of the 237 breast carcinomas studied by TMA showed the expected correspondence of tumor-grade differentiation with the hormone receptor pattern, the proliferation activity, and HER2 immunohistochemical and FISH values. In conclusion, multicore TMA may be an efficient approach in the routine study of prognostic factors in breast cancer, significantly reducing costs, time, and burden of slides necessary to accomplish these mandatory tests.

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]

Reliability of whole sentinel lymph node analysis by one-step nucleic acid amplification for intraoperative diagnosis of breast cancer metastases.

Objective: To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. Background: Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. Methods: Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. Results: Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. Conclusions: Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.

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.

Adenosquamous lung carcinomas: A histologic subtype with poor prognosis

INTRODUCTION The aim of this study is to evaluate the prognostic factors and outcome of patients operated for adenosquamous (ADS) carcinoma of the lung, in comparison with adenocarcinoma (AD) and squamous cell carcinoma (SCC). METHODS a retrospective review of our thoracic cancer surgical database for patients operated for ADS, SCC and AD between January, 1995 and December, 2009 was done. RESULTS Forty-eight patients (39 males, 81.3%) had ADS; complete tumor resection and lymphadenectomy was accomplished in all patients. A higher stage at presentation was observed in ADS, as compared to AD or SCC (p=0.0001). Three and 5-year survival rates were 25% and 15%. ADS overall survival was worse than AD or SCC (p=0.0005). Three and 5-year survival rates of ADS Stage I were similar to those of Stage IIIA AD or SCC. More than half ADS patients developed distant metastases (MTS) or local recurrences. Brain MTS were the most frequent. Median survival for those patients was 8±2.3 months. Postoperative platinum-based chemotherapy statistically improved patients survival (p=0.02). In the multivariate analysis, the presence of MTS (p=0.001), the tumor perineural invasion (p=0.01) and the tumor stage (p=0.0005) were factors associated with poor prognosis. Adjuvant chemotherapy was a significant positive prognostic factor (p=0.00001). CONCLUSIONS ADS are uncommon and extremely aggressive lung tumors. Adjuvant chemotherapy should be administered even in Stage I radically resected tumors. A whole brain postoperative prophylactic radiotherapy could be proposed to reduce risk of developing brain MTS.

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.

Ultrastructural Features of Neuroendocrine Differentiated Carcinomas of the Breast

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.

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.