Molecular and Histological Changes in Post-Treatment Biopsies of Non-Squamous Non-Small Cell Lung Cancer: A Retrospective Study.

BACKGROUND: Recently, in advanced non-small cell lung cancer (NSCLC), standard chemotherapy was flanked by biological agents directed against genomic abnormalities, including EGFR and ALK alterations, that significantly improved patient outcome. Despite these achievements, tumour progression almost always occurs and a reassessment of the tumour genetic profile may contribute to modulating the therapeutic regimen. Resampling may provide tissue for additional tests to detect acquired resistance and/or new genetic alterations, but the currently available information is limited. PATIENTS AND METHODS: Histological and genetic reassessments of biopsy or surgical tissue samples from 50 non-squamous NSCLC patients before and after at least one systemic treatment were performed. EGFR, KRAS, BRAF, PIK3CA and HER2 mutations were sequenced, p.T790M was identified with real-time PCR, and ALK and MET genomic alterations by fluorescence in situ hybridization. RESULTS: Overall in baseline biopsies, 37/50 (74 %) tumours had genetic alterations, either single (52 %) or multiple (22 %). Among them, 16 were EGFR mutations and 6 ALK rearrangements. In the second tissue sampling, 54 % of cases had additional genomic changes, including newly acquired alterations (81 %) or losses (18 %). The commonest changes were MET amplification and p.T790M mutation. One case had a histological shift from adenocarcinoma to small cell carcinoma. CONCLUSIONS: The remarkable number of molecular changes following systemic therapy and the genetic complexity of some cases underline the value of histological and molecular re-evaluation of lung cancer to tailor the most appropriate therapy during disease progression.

Characterization of thyroid 'follicular neoplasms' in fine-needle aspiration cytological specimens using a panel of immunohistochemical markers: a proposal for clinical application.

The distinction of benign from malignant follicular thyroid neoplasms remains a difficult task in diagnostic fine-needle aspiration cytology, and some discrepant results have been reported for the individual immunocytochemical markers of malignancy proposed so far. The aim of this study was to test if the combined use of a panel of markers could improve the diagnostic accuracy in the preoperative cytological evaluation of 'follicular neoplasms' in an attempt to reduce the number of thyroidectomies performed for benign lesions. The immunocytochemical expression of galectin-3, HBME-1, thyroperoxidase, cytokeratin-19 and keratan-sulfate was retrospectively analyzed in 125 consecutive fine-needle aspiration samples (cell blocks) of indeterminate diagnoses of 'follicular thyroid neoplasm', and compared with their corresponding surgical specimens, including 33 follicular carcinomas, 42 papillary carcinomas and 50 follicular adenomas. Statistical analysis on each marker confirmed that galectin-3 and HBME-1 were the most sensitive (92% and 80% respectively) and specific (94% and 96% respectively) molecules. The use of these two markers sequentially in non-oncocytic lesions (testing HBME-1 as a second marker whenever galectin-3 proved negative) increased the sensitivity and specificity up to 97% and 95% respectively. In oncocytic lesions, HBME-1 proved to be less sensitive, and the sequential combination of galectin-3 and cytokeratin-19 reached 100% of both specificity and sensitivity. Our data showed that, as compared with the use of single markers, the sequential combination of two markers represents the most accurate immunohistochemical panel in managing patients with a fine-needle aspiration biopsy diagnosis of 'follicular neoplasms', especially in otherwise controversial categories such as oncocytic tumours. The combination of three or more markers did not substantially improve the diagnostic accuracy of the test.

Management of neuroendocrine differentiated breast carcinoma.

In neuroendocrine differentiated breast cancer, the coexistence of both neuroendocrine and exocrine components may raise some uncertainty about the best clinical approach to adopt. We describe the case of a patient with neuroendocrine differentiated breast carcinoma with lung metastases, who experienced a partial response after epirubicin chemotherapy. During subsequent maintenance hormone therapy with letrozole, plasma chromogranin A was consistently elevated even though CT showed disease stabilization. The patient was scheduled for surgery and radical resection was performed. She is still alive and disease free after over 37 months. Anthracyclines are effective in the treatment of neuroendocrine differentiated breast carcinoma. Surgical resection of metastatic lesions can lead to a durable disease-free status. Serial evaluation of circulating chromogranin A is useful in the follow-up of these patients.

Galectin-3: presurgical marker of thyroid follicular epithelial cell-derived carcinomas.

Preoperative follicular lesion characterisation represents an unsolved diagnostic problem in thyroid nodular disease. Although fine-needle aspiration biopsy is the most reliable preoperative diagnostic procedure, it shows inherent limitations in differentiating adenoma from follicular carcinoma and, sometimes, follicular variants of papillary carcinoma. Galectin-3 cytoplasmic neoexpression has been proposed as a peculiar feature of thyroid malignant cells, easily detectable in cytological and histological samples. The aim of this study was to re-evaluate the galectin-3 expression in a large sample of thyroid lesions using an immunohistocytochemical biotin-free detection system and a specific anti-human-galectin-3 monoclonal antibody in order to avoid the interference of technical factors, a cause of conflicting results recently reported by some authors. We analysed galectin-3 expression of 39 follicular carcinomas, 26 papillary carcinomas, and 105 adenomas in both cell-block samples and their histological counterparts. All cell-block and histological papillary carcinoma samples showed high levels of galectin-3 immunoreactivity. Thirty-four follicular carcinomas were positive, whereas 5 were negative in cell-blocks but positive in their histological counterparts. Twelve out of 105 adenomas expressed galectin-3 in cell-blocks and histological samples. The diagnostic accuracy of preoperative galectin-3 evaluation in adenomas vs follicular carcinomas was 90.0%. Galectin-3 expression was also investigated in 22 minimally-invasive follicular carcinomas. All of them showed galectin-3 immunoreactivity in both cytological and histological specimens with the exception of two cases, where galectin-3 positivity was observed only in the surgical material. The routine correct use of galectin-3, by increasing the diagnostic accuracy of conventional cytology, improves the management of thyroid nodules and can lead to a sensitive reduction of useless thyroid surgeries.

Interleukin-6 producing pheochromocytoma presenting with acute inflammatory syndrome.

Pheochromocytomas are tumors able to produce catecholamines and a variety of biologically active neuropeptides. We report the case of a 36-yr-old female patient with pheochromocytoma exhibiting headache, intermittent fever, thrombocytosis, and marked inflammatory signs. Nonsteroidal anti-inflammatory drugs were ineffective in lowering the body temperature, while a corticosteroid agent obtained excellent results. IL-6 was found elevated (20 pg/ml); it fell to 4.5 pg/ml 3 weeks after the adrenalectomy, in parallel to normalization of other laboratory data. The interleukin-6 (IL-6) over-production can either be ascribed directly to the tumor (as confirmed by immunohistochemistry) or indirectly accounted for by tumoral production, as a consequence of the high levels of circulating norepinephrine. To our knowledge, our paper represents the 6th case report of IL-6 secreting pheochromocytoma associated with clinical markers of inflammatory response.

Galectin-3 as a presurgical immunocytodiagnostic marker of minimally invasive follicular thyroid carcinoma.

Thyroid nodules are a common occurrence in the general population, but only a small number of them are eventually diagnosed as cancers. Fine-needle aspiration biopsy (FNAB) is the most accurate and cost-effective method for the presurgical management of thyroid nodules, but it misses the differential diagnosis between thyroid follicular adenomas and follicular carcinomas. Among them, minimally invasive follicular carcinoma (MIC), also defined as encapsulated tumor, only differs from follicular adenoma for the exhibition of minimal, but entire thickness, infiltration of the capsule and/or vascular invasion. This feature cannot be assessed in FNAB and can occasionally be hard to recognize in surgical specimens. As reported in several studies, galectin-3 is a reliable marker of thyroid malignancy, but no data are available on MICs. We analyzed the immunohistochemical expression of galectin-3 in 17 MICs and 52 follicular adenomas in both preoperative paraffin-embedded cytological human thyroid sediments (cell blocks) obtained by FNAB and in the corresponding surgical specimens. Among the MICs, all surgical samples showed galectin-3 immunoreactivity in the cytoplasm, whereas 16 of 17 corresponding FNAB cell blocks were positive. No evidence of cytoplasmic galectin-3 expression was observed in 48 of 52 adenomas in both cell blocks and histological tissues. These findings indicate that galectin-3 is a reliable presurgical molecular marker of MIC, improving the accuracy of conventional FNAB. It also proves to be useful in the histopathological assessment of resected tumors having suspected malignant features.

p53 expression in cultured blood human monocytes infected with mycobacterial strains.

BACKGROUND: An upregulation of the cell-cycle associated proteins p53 and p21/Waf1/Cip1 induced by mycobacteria was previously reported. We aimed to evaluate the expression of such proteins in peripheral blood human monocyte cultures infected with strains of different mycobacterial pathogens. METHODS: The study relied on the immunocytochemical determination of p53, p21/Waf1/Cipl, bcl-2 and on the Tunel detection of apoptosis in monocytes populations cultured on four-welled chamber slides (10(6) cells/well) infected with Mycobacterium tuberculosis, M. bovis and M. avium for four consecutive days (mycobacterium/monocyte ratio 10:1). The results were expressed as mean values and SD of the percentages of stainings recorded in five fields per slide. RESULTS: The statistical analysis with Fischer test demostrated that at most sampling times the p53 and p21/Waf1/Cip1 expression and the apoptosis index were significantly higher in M. tuberculosis infected cultures than in controls (p<0.05). The M. bovis related picture diverged from the previous one for a lower p53 expression (p<0.05) at all sampling times. The M. avium infected culture values did not diverge significantly from the controls. CONCLUSIONS: The p53 and p21/Wafl/Cipl upregulation is compatible with both host defense strategies and pathogen strategies (safeguard of intracellular sanctuaries). The discrepancies among different cultures suggest a direct relationship between p53 activation and mycobacterial ability to enter host cells.

p27(kip1) immunoreactivity correlates with long-term survival in pleural malignant mesothelioma.

BACKGROUND: Pleural malignant mesothelioma (PMM) is a rare and highly aggressive tumor, whose development is strictly related to occupational exposure to asbestos. The prognosis of PMM is generally poor (median survival, 4-12 months), but a few have a relatively long survival. The objective of this study was to evaluate the use of the cell cycle-related proteins p27(kip1) and MIB-1 as prognostic indicators of survival in PMMs. METHODS: Of 621 PMMs, the authors selected 27 cases with a relatively long-term survival (> 24 months) and a control group of 36 PMMs having a shorter (usual) survival (< 24 months). RESULTS: The expression of the p27(kip1) was significantly higher in the long-term survival group compared with the control (short survival) group (81.41% vs. 31.94%; P < 0.0001). The PMMs of epithelioid histotype had a significantly higher p27(kip1) immunoreactivity compared with those of biphasic type (59.24% vs. 38.94%; P = 0.02). In agreement with the data in the literature, the proliferative activity (as detected by MIB-1 immunoreactivity) was significantly higher in short than long survival PMMs (43.53% vs. 14.11%; P < 0.0001) and in the biphasic histotype than in the epithelioid type (43.19% vs. 26.02%; P = 0.006). CONCLUSIONS: The combined expression of high/low p27(kip1) and low/high Ki-67 values identified with 100% specificity and sensitivity long versus short survivors. p27(kip1) represents a reliable additional predictive factor for PMMs and a useful marker to identify patients having a more favorable prognosis.

Immunohistochemical assessment of Ki-67 in the differential diagnosis of adrenocortical tumors.

OBJECTIVES: To evaluate the utility of Ki-67 immunohistochemical analysis in the differential diagnosis between benign and malignant adrenocortical neoplasms. METHODS: Tissue specimens were obtained from 37 patients referred to our institute from 1990 to 1999. The indications for adrenalectomy were adrenal-dependent Cushing syndrome (n = 9), hyperandrogenism (n = 1), mineralocorticoid excess (n = 8), and nonfunctioning adrenal masses (n = 19). The histologic diagnosis was cortical adenoma in 26 of 37 patients and cortical carcinoma in the remainder. Normal adrenal glands were obtained from subjects who underwent radical nephrectomy because of initial renal carcinoma. Immunohistochemical analysis was performed using the monoclonal antibody anti-Ki-67 (clone MIB-1). The Ki-67 labeling index was expressed as the number of positive cells per 1000 cells.Results. The average Ki-67 expression was 2.0 per thousand +/- 1.2 per thousand (SD) in normal adrenal glands, 11.3 per thousand +/- 16.0 per thousand in adenomas, and 185.8 per thousand +/- 60.3 per thousand in carcinomas (P <0.0001). A threshold value of the Ki-67 labeling index between 70 per thousand and 90 per thousand reliably separated adenoma from carcinoma. A significant inverse correlation was found between Ki-67 expression and overall survival in patients with adrenal carcinoma (r = -0.74, P = 0.009). CONCLUSIONS: Immunohistochemical assessment of the nuclear antigen Ki-67 can be useful in the differential diagnosis between adrenocortical adenoma and carcinoma. High levels of Ki-67 seem to indicate patients with adrenocortical cancer with a worse prognosis.

Relationship between neuroendocrine features and prognostic parameters in human prostate adenocarcinoma.

PURPOSE: The biological behaviour of prostate cancer is highly variable and prediction by the commonly employed prognostic parameters is not sufficient. The concept of neuroendocrine (NE) differentiation in prostate adenocarcinoma has recently received increasing attention due to possible implications for prognosis and therapy. MATERIALS AND METHODS: Core needle biopsies from 142 newly diagnosed patients were immunohistochemically examined for the coexistence of NE differentiation using an antibody against chromogranin A (CgA). Circulating CgA was available in 106 of these patients. RESULTS: NE differentiation was found in 64 (45.1%) tumors. Among them 29 (20.4%) had CgA positive cells scattered or focally distributed in less than 5% per mm3 of tumor tissues, 26 (18.3%) between 5% and 10% and 9 (6.4%) more than 10%, respectively. There was a significant correlation between the extent of NE features and either Gleason score (P < 0.01) or stage of disease. Circulating CgA but not PSA correlated with immunohistochemical CgA (P < 0.03) particularly in metastatic cases. CONCLUSIONS: These data support the concept that NE differentiation in human prostate cancer has a negative prognostic significance. Circulating CgA levels reflect immunohistochemical findings.

Different immunohistochemical patterns of TGF-beta1 expression in benign and malignant adrenocortical tumours.

OBJECTIVE: Transforming growth-factor beta1 (TGF-beta1) influences a number of specific functions of adrenocortical cells in several animal species. The aim of our study was to evaluate by immunohistochemical analysis the presence and distribution of TGF-beta1 in normal adrenal tissue and in different adrenal tumours. PATIENTS: We analysed 8 functioning (5 adenomas and 3 carcinomas) and 15 non functioning (6 adenomas and 9 carcinomas) adrenal tumours and 6 normal adrenal glands. RESULTS: In normal adrenal glands, the glomerulosa and the reticularis zones displayed diffuse cytoplasmic staining, while the fasciculata zone was almost completely negative. Functioning adenomas displayed cytoplasmic staining restricted to compact cells while in nonfunctioning adenomas, prevalently composed by clear cells, no staining was observed. Overall, adrenal carcinomas were characterized by the lack of cytoplasmic positivity and by sporadic positive cells around vessels both in functioning and in nonfunctioning tumours. CONCLUSIONS: TGF-beta1 expression is associated with active steroid secretion in normal adrenal tissue, as well as in benign cortical adenomas, while this relationship is lost in primary adrenal malignancies. These data provide indirect evidence for a regulatory role played by TGF-beta1 on steroid secretory pathways.

Intravesical bacillus calmette-guerin (BCG) as inducer of tumor-suppressing proteins p53 and p21 Waf1-Cip1 during treatment of superficial bladder cancer.

PURPOSE: Previous in vitro investigations recorded an inhibition of cell proliferation by BCG when added to different cell cultures. The induction of apoptosis by BCG is controversial. Our study aimed to evaluate the influence of BCG on the expression of tumor suppressing proteins p53 and p21Waf1-Cip1 and apoptosis of the urothelial cells in vivo. MATERIALS AND METHODS: Twenty-one cases of superficial bladder cancer, treated with TUR and subsequent intravesical BCG, were studied retrospectively. The assays evaluated the expression of p53 and p21Waf1-Cip1 by immunochemistry (IHC), and the presence of apoptosis by TUNEL assay. The estimates were performed, in each case, on the following specimens: one tumor sample and one non-neoplastic sample collected during the TUR which preceded the administration of BCG; one non-neoplastic sample collected 3 months after the diagnosis; and one non-neoplastic sample collected in the first 2 weeks after the completion of the treatment. Samples of 6 cancer recurrences detected during BCG were examined too. RESULTS: As usual for non-neoplastic urothelium, the pre-BCG samples displayed poor p53 and p21Waf1-Cip1 immunoreactivity. By contrast, the samples collected during and in the aftermath of BCG showed an overall increase of the expression of both proteins. The rare occurrence of apoptosis proved to be chronologically unrelated to the BCG treatment. DISCUSSION: The relationship between changes of the IHC features and BCG suggests that BCG, at least under some circumstances, can induce the activation of wild type p53 and p21Waf1-Cip1 in the urothelium. The mechanism of the BCG-p53 status interaction and its role in the antitumor activity of BCG remain to be clarified.

Galectin-3 is a presurgical marker of human thyroid carcinoma.

Galectin-3 is a carbohydrate-binding protein endowed with an affinity for beta-galactosides. It has been shown to play an important role in cell-cell and cell-matrix interactions and in pre-mRNA splicing. Furthermore, it is involved in the control of cell growth, neoplastic transformation, and metastasis. Interestingly, high levels of galectin-3 expression have been recently described in malignant thyroid neoplasias, but not in adenomas or in normal thyroid tissue. We investigated galectin-3 expression in human presurgical specimens obtained by fine-needle aspiration biopsy. We analyzed galectin-3 expression by immunoperoxidase staining in both paraffin-embedded cytological thyroid sediments (cell blocks) obtained by fine-needle aspiration biopsy and their histological counterparts. A total of 64 samples were examined: 17 follicular carcinomas; 18 papillary carcinomas; and 29 follicular adenomas. All cell blocks and histological samples of papillary carcinomas expressed high levels of galectin-3 at either the cytoplasmic or nuclear level. Among follicular carcinomas, all histological samples expressed galectin-3, whereas 14 of 17 corresponding cell blocks were positive in the cytoplasm. No evidence of cytoplasmic galectin-3 expression was observed in 26 of 29 follicular adenomas. Hence, cytoplasmic galectin-3 staining seems to be a reliable, easy, and cheap marker for presurgical diagnosis of follicular carcinomas and an even more suitable one for papillary carcinomas.

Molecular heterogeneity of B-lineage diffuse large cell lymphoma.

B-lineage diffuse large cell lymphoma (B-DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B-DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B-DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B-DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B-DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TPS3 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TPS3. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B-DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity.

Evaluation of c-ras oncogene product (p21) in superficial bladder cancer.

OBJECTIVES: The aim of our study is the evaluation of the prognostic importance of p21 protein in superficial bladder cancer. METHODS: One hundred and fourteen patients with an initial diagnosis of monofocal bladder cancer (stage Ta-T1) following TUR were investigated. On the tissue removed by TUR, besides the usual pathological evaluation, an immuno-histochemical investigation was carried out in order to ascertain the presence of c-ras oncogene product (protein p21). The actuarial curves concerning the time free from the first recurrence were computed, comparing different subgroups in regard to protein p21 presence, grade and stage of the tumour. RESULTS: The analysis of the results shows the importance of tumour stage as a predictor of recurrence, as well as that of the presence of c-ras products. This last factor increases the risk of recurrence almost 2-fold, in the same time lag, for c-ras-positive patients (p < 0.001). The prognostic significance of c-ras is independent of stage. CONCLUSION: Our data underline the possibility of acquiring important information on the prognosis of superficial bladder cancer patients, pointing out the significance of c-ras oncogene product.

High frequency of p53 expression in colo-rectal adenomatous polyps.

Previous studies on p53 protein expression in colonic adenomas showed controversial results. The present study evaluates the p53 expression in colonic adenomas, at different dysplasia degrees, by immunohistochemical analysis, using a newly introduced monoclonal anti-p53 antibody. Paraffin embedded sections of 48 colorectal adenomas, 5 colonic carcinomas and 11 normal colonic biopsies were studied by immunohistochemical analysis using a monoclonal mouse anti-p53 antibody (clone DO-1). Normal colonic mucosa specimens and 5/48 adenomas were found negative for p53 staining. p53-positive nuclei were less than 10% in 22/48 and between 10 and 40% in 15/48 adenomas. In 6/48 adenomas and in 4/5 carcinomas we found a high percentage of p53-positive nuclei (> 40%). Immunohistochemical p53-positivity is a common event in colonic adenomas, not dependent on dysplasia degree. It might be the result of p53 wild-type increase, due to the typical genomic instability of colonic adenomas.

Immunohistochemical evaluation of P-glycoprotein in human malignancies by monoclonal antibody MC57.

P-glycoprotein expression was analyzed on 137 formalin-fixed, paraffin-embedded human tumours by monoclonal antibody (MAb) MC57. This MAb recognizes an extracellular human specific P-glycoprotein epitope and defines their multidrug resistance (MDR) phenotype by its binding on cells. Immunohistochemistry indicated that this MAb reacted in human cells and tissues in the same pattern as that found with other MAbs to P-glycoprotein. However, the present extensive study demonstrated that MAb MC57 is a highly specific reagent for the evaluation of an extracellular P-glycoprotein epitope preserved after fixation procedures and that this MAb is available to assess P-glycoprotein expression in routinely processed human tumour specimens.

Prognostic significance of Ki67 labelling in resected non small cell lung cancer.

One hundred and eleven tissue samples of primary non small cell lung cancer obtained from patients undergoing radical surgery for resectable disease were investigated for the presence and distribution of Ki67 related antigen using an immunohistochemical technique, as a marker of the proliferative activity of the tumour. No correlation was seen between Ki67 expression and clinico-pathological variables (sex, age, histology, grading and pTNM stage) but disease-free survival was significantly lower in patients with higher Ki67 score (> 25% positive cells) at diagnosis (P < 0.03). Growth fraction evaluated by Ki67 labelling may provide a complementary prognostic parameter in non small cell lung cancer.

Detection of multidrug resistance associated P-170 glycoprotein in previously untreated non small cell lung cancer.

Samples of Non Small Cell Lung Cancer (NSCLC) and normal bronchial tissue obtained in patients submitted to radical surgery and without previous exposure to cytotoxic drugs were investigated for the expression of P-170 glycoprotein using C-219 monoclonal antibody and an immunohistochemistry technique. In normal bronchial tissue the immunostaining was confined to the lumenal surface of the epithelium. Fifteen out of 86 NSCLC had more than 1/4 of examined cells positive for P-170 glycoprotein, but the heterogeneity of the expression ranged from rare scattered cells to a positive pattern for nearly all cells considered, without any relationship with pathologic and clinical prognostic variables.