Piera Balzarini

BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations

The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1-associated protein 1 (BAP1) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1 protein is frequently lost in mesothelioma, especially of epithelioid/biphasic subtype and is commonly associated with homozygous BAP1 deletion. BAP1 immunostain represents an excellent biomarker with an unprecedented specificity (100%) in the distinction between benign and malignant mesothelial proliferations. Finding BAP1 loss in mesothelial cells should prompt to immediately reevaluate the patient; moreover, it might be useful in mapping tumor extent and planning surgical resection.

Sinonasal mucosal melanoma: Molecular profile and therapeutic implications from a series of 32 cases

Primary sinonasal mucosal melanomas are aggressive tumors with a poor clinical control by current treatments, raising the urgent need of novel strategies.

Application of alternative fixatives to formalin in diagnostic pathology.

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

Chromosome changes in sarcomatoid renal carcinomas are different from those in renal cell carcinomas

Sarcomatoid renal carcinoma (SRC) is an uncommon and highly malignant renal tumor with sarcomatous morphology or with both sarcomatous and carcinomatous components. The tumors are generally referred to as dedifferentiated or transformed renal cell carcinomas (RCC). Information on their genomic changes is scarce. Data from a cytogenetic study of four cases of SRC are presented in this article. Combined with a few other cases from the literature, it appears that genomic changes in most cases of SRC have little in common with those characterizing conventional RCC. Sarcomatoid transformation thus may occur occasionally in RCC without the characteristic chromosome changes but also, and perhaps more frequently, in a subgroup of RCC without the characteristic chromosome changes.

Cytogenetic features, clinical significance and prognostic impact of type 1 and type 2 papillary renal cell carcinoma

The purpose of this paper is to evaluate the clinical, pathologic, and cytogenetic features, as well as the disease-free survival in patients with papillary renal cell carcinoma (PRCC) subdivided into types 1 and 2, according to the definition given by Delahunt and Eble. The clinical, surgical, and follow-up data for the PRCC cases treated since 1995 were taken from an institutional database. The samples were revised by an experienced pathologist, who subdivided them into types 1 and 2. The data from the cases in which the tumor karyotype was available were analyzed. Out of 1,150 patients surgically treated for renal cancer, 132 cases of PRCC were detected (prevalence 11.5%), 57 with type 1 and 75 with type 2, followed for a mean period of 50 months. Tumor diameter, peri-renal tissues, as well as venous invasion, lymphnodal, and distant metastasis were highlighted to be distributed with a significant difference between the two groups, which indicated higher aggressiveness in type 2 cases. Survival analysis has showed a significantly higher-progression risk and a shorter disease-free survival in type 2 cases. An evaluable tumoral karyotype was obtained in 26 cases. An overlapping distribution was detected in chromosomes 7, 17, 12, 16, and 20, while some alterations in chromosomes 10, 5, 6, 11, 15, 18, 22, and 8 appeared as typical of type 2 cases. In conclusion, types 1 and 2 PRCC have different pathologic and cytogenetic features and a radically different biologic behavior - indolent in type 1 and aggressive in type 2.

Diagnostic usefulness of fluorescent cytogenetics in differentiating chromophobe renal cell carcinoma from renal oncocytoma: a validation study combining metaphase and interphase analyses.

We investigated the usefulness of interphase fluorescence in situ hybridization (FISH) analysis to differentiate between 11 chromophobe renal carcinomas and 12 renal oncocytomas, showing different clinical outcomes, when compared with conventional metaphase cytogenetics by karyotyping. Karyotypically, 3 chromophobe renal cell carcinomas showed losses of chromosomes, 3 were polyploid, 1 was normal, and 4 failed to grow. Of 12 oncocytomas, 5 showed a normal numeric karyotype and 6 additional structural rearrangements. FISH on chromophobe renal cell carcinomas showed a high percentage of cases (10/11 [91%]) with multiple numeric losses among chromosomes 1, 2, 6, 10, and 17; this interphase pattern was observed irrespective of the 3 different metaphase karyotypes. Of 12 oncocytomas, 11 (92%) revealed a normal numeric chromosomal status showing at least 2 chromosomes without aneusomy by interphase FISH. The study demonstrates that indeed FISH performed on formalin-fixed, paraffin-embedded tissue can provide clinically useful information more reliably than karyotyping of most of these tumors.

Mucinous cystic carcinoma of the pancreas: a unique cell line and xenograft model of a preinvasive lesion

Pancreatic mucinous cystic tumors (MCT) are proliferations of mucin-producing epithelia supported by an ovarian-like stroma. They are classified into adenomas (MCA), borderline (MCB) and noninvasive or invasive carcinomas (MCC). The molecular mechanisms underlying their clinical behavior are poorly understood, partly due to the lack of cellular models. We report the establishment of MCC1, the first cell line from a pancreatic MCT, deriving from the highly dysplastic cell component of a noninvasive MCC. MCC1 has mutations in codon 12 of K-RAS (GGT>GAT), codon 58 of P16 (CGA>TGA) and codon 132 of P53 (AAG>AGG). The FHIT and DPC4 genes are unaltered. Immunohistochemistry shows abnormal expression of MUC1 and p53, loss of p16 and retention of Fhit and Dpc4 in both the cell line and the highly dysplastic cells of the primary lesion. The morphological and molecular features of MCC1 and its corresponding primary tumor are consistent with a model for non-invasive MCC, where K-RAS, P16, P53 and MUC1 alterations are pre-invasive changes associated with progression of malignancy of MCT from adenoma to carcinoma. MCC1 is sensitive to 5-fluorouracil, representing the first assessment of drug sensitivity for MCC. Finally, MCC1 is a suitable model for preclinical studies, as it grows in immunodeficient mice.

ALK Testing in Lung Adenocarcinoma Technical Aspects to Improve FISH Evaluation in Daily Practice

Introduction: Anaplastic lymphoma kinase (ALK) gene rearrangement characterizes a subgroup of patients with lung adenocarcinoma who may benefit from ALK inhibitors. Fluorescence in situ hybridization (FISH) with a break-apart/split-signal strategy is the gold standard to investigate ALK. The cutoff to define ALK positivity has been settled at 15% or greater. A subset of patients has ALK borderline status, showing 15% ± 5% positive cells. Several aspects, both biological and technical, might influence signals evaluation, making FISH interpretation a challenging task. To improve ALK evaluation, we classified the different FISH patterns on the basis of the type of the split signals, namely short, long, far away, and deleted. Methods: We investigated ALK gene status by FISH in 244 lung adenocarcinomas and in a series of ALK negative cell lines samples, collected in three Institutions. Results: ALK positive profile was found in 12% of patients; long, deleted, and far-away splits were the primary patterns observed. ALK borderline profile characterized 10% of samples; long and deleted splits were significantly more frequent in those borderline finally classified as ALK positive, whereas short split were mostly detected in those borderline patients finally classified as ALK negative (p = 3.4 × 10−3). In the ALK negative control series, short split was the predominant pattern. Concordance was observed among different operators and probes for both samples and controls. Conclusions: Difficulties in ALK FISH signal interpretation might be bypassed using this detailed scoring system, which is highly reproducible, helps clarify borderline samples (according to split type), and provides experimental evidence that 15% is a reasonable cutoff to overcome the assay-dependent background noise.

HER-2/neu in breast cancer: a comparative study between histology, immunohistochemistry, and molecular technique (FISH).

HER-2/neu is a protooncogene frequently overexpressed in breast cancer. Fluorescence in situ hybridization (FISH) is a technique targeting the gene amplification, while immunohisto-chemistry detects the protein expression. Usually both are applied to paraffin-embedded tissue. The authors studied HER-2 by FISH and immunohistochemistry (HercepTest) in 81 breast carcinomas. The results showed an overall concordance (correlation coefficient 0.64). In all cases with HercepTest score 0 and 1+, nonamplification of the gene was observed. Gene amplification was found in 20% of cases with a 2+ score and in 77.78% of cases with a 3+ score. Data described in literature for 3+ carcinomas showed a 3% to 10% discrepancy between protein expression and gene amplification, while in this study this difference was up to 22.22%. As a consequence, even if it is usually considered important to analyze only 2+ cases by FISH, 3+ scores nonamplified for HER-2/neu may be a new, interesting subset. Furthermore, the authors investigated the two-variables correlation between chromosome 17 copy number, protein over-expression, gene amplification, and presence of metastatic lymph nodes. Interesting results came from the correlation between the HercepTest score and the HER-2/neu gene amplification evaluation, HercepTest and chromosome 17 aneusomy, and gene amplification and lymph nodes status. In conclusion, the FISH technique can be an important and useful diagnostic tool to integrate the results of the HercepTest and to select patients for immunotherapy.

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-β1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-β1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-β1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-β1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-β1 with anti-TGF-β1 antibodies or with Ly-364947 (a specific inhibitor TGF-β1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-β1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.