Francesca Maletta

Expression of p63 is the sole independent marker of aggressiveness in localised (stage I–II) Merkel cell carcinomas

Merkel cell carcinoma of the skin is a malignant neuroendocrine tumour, whose prognostic criteria are a matter of dispute. Specifically, no predictor is presently available in stage I–II tumours. We collected clinical and follow-up data from 70 Merkel cell carcinomas of the skin. The same cases were studied for p63 expression by immunohistochemistry, by reverse-transcription PCR (RT-PCR) and TP63 gene status by FISH and for presence of Merkel cell polyomavirus by PCR. Stage emerged as a significant prognostic parameter (P=0.008). p63 expression, detected in 61% (43/70) of cases by immunohistochemistry, was associated with both decreased overall survival (P<0.0001) and disease-free survival (P<0.0001). Variable expression patterns of the different p63 isoforms were found only in cases immunoreactive for p63. In these latter lesions, at least one of the N-terminal p63 isoforms was detected and TAp63α was the most frequently expressed isoform. TP63 gene amplification was observed by FISH in only one case. Presence of Merkel cell polyomavirus DNA sequences was detected in 86% (60/70) of Merkel cell carcinomas and did not emerge as a significant prognostic parameter. Merkel cell carcinoma cases at low stage (stage I-II) represented over half (40/70 cases, 57%) of cases, and the clinical course was uneventful in 25 of 40 cases while 15 cases died of tumour (10/40 cases) within 34 months or were alive with disease (5/40 cases) within 20 months. Interestingly, a very strict correlation was found between evolution and p63 expression (P<0.0001). The present data indicate that p63 expression is associated with a worse prognosis in patients with Merkel cell carcinoma, and in localised tumours it represents the single independent predictor of clinical evolution.

Gene Status in HER2 Equivocal Breast Carcinomas: Impact of Distinct Recommendations and Contribution of a Polymerase Chain Reaction-Based Method

BACKGROUND The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Cytological detection of papillary thyroid carcinomas by nuclear membrane decoration with emerin staining

Decoration of the nuclear membrane by emerin staining enhances detection of nuclear irregularities typical of papillary thyroid carcinoma (PTC). Our study aims to verify whether staining with emerin is a helpful diagnostic marker in fine-needle aspiration (FNA) cytology. We first designed a prospective study on smears, Thin Prep, and cell block specimens to prove the feasibility of the procedure; subsequently, we designed a retrospective study of 78 FNA cell blocks from cases that, after surgery, turned out to be either benign (34 cases) or malignant lesions (44, of which 31 PTC). From each sample, we obtained two slides, one stained with hematoxylin and eosin (H&E) and the other with emerin. In cases classified as Thy3, HBME-1 and galectin 3 (Gal3) stains had also been performed. Two blinded observers made the judgment concerning Thy categories (as proposed by the British Thyroid Association), first on H&E, then on emerin, HBME-1, and Gal3 stained slides. On cytological preparation, emerin staining represents an effective tool for the detection of nuclear irregularities, allowing for the identification of cases of PTC. In Thy3 cases, emerin stainings sensitivity and specificity (64% and 96%) proved higher than HBME-1s (60% and 88%) and Gal3s (61% and 68%). In conclusion, the immunohistochemical definition of the nuclear membrane, as determined by emerin stain, is a useful tool in the cytological diagnosis of thyroid lesions and can help to solve inconclusive cases by highlighting nuclear irregularities typical of PTC.

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.

MALAT1 Long Non-coding RNA Expression in Thyroid Tissues: Analysis by In Situ Hybridization and Real-Time PCR

Long non-coding RNAs (lncRNAs) are important for transcription and for epigenetic or posttranscriptional regulation of gene expression and may contribute to carcinogenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNA involved in the regulation of the cell cycle, cell proliferation, and cell migration, is known to be deregulated in multiple cancers. Here, we analyzed the expression of MALAT1 on 195 cases of benign and malignant thyroid neoplasms by using tissue microarrays for RNA in situ hybridization (ISH) and real-time PCR. MALAT1 is highly expressed in normal thyroid (NT) tissues and thyroid tumors, with increased expression during progression from NT to papillary thyroid carcinomas (PTCs) but is downregulated in poorly differentiated thyroid cancers (PDCs) and anaplastic thyroid carcinomas (ATCs) compared to NT. Induction of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta in a PTC cell line (TPC1) led to increased MALAT1 expression, supporting a role for MALAT1 in EMT in thyroid tumors. This is the first ISH study of MALAT1 expression in thyroid tissues. It also provides the first piece of evidence suggesting MALAT1 downregulation in certain thyroid malignancies. Our findings support the notion that ATCs may be molecularly distinct from low-grade thyroid malignancies and suggest that MALAT1 may function both as an oncogene and as a tumor suppressor in different types of thyroid tumors.

Interpretation of p16INK4a/Ki‐67 dual immunostaining for the triage of human papillomavirus‐positive women by experts and nonexperts in cervical cytology

The triage of human papillomavirus (HPV)‐positive women is needed to avoid overreferral to colposcopy. p16INK4a immunostaining is an efficient triage method. p16INK4a/Ki‐67 dual staining was introduced mainly to increase reproducibility and specificity compared with stand‐alone p16INK4a staining.

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

Introduction Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). Methods One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. Results RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). Conclusion RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

The Pre-analytical Phase in Surgical Pathology

Several sequential passages are involved in the pre-analytical handling of surgical specimens from resection in the surgical theater to paraffin-embedding and storage. Each passage is highly critical and can significantly affect the preservation of morphology, antigens, and nucleic acids. Some key points in this process are still undefined and are subject to high variability among hospitals. High quality and standardization are demanded and pathologists should therefore work to comply with all novel clinical requests (such as genomic and antigenic testing for targeted molecular therapies). Under-vacuum sealing of surgical pieces can be a safe and reliable alternative to storage in large formalin-filled boxes; it prevents dehydration and favors cooling by removing air. Moreover, it implements tissue banking and preservation of nucleic acids. After transport of specimens to pathological anatomy laboratories, the next passage, fixation, has been the object of several attempt to find alternatives to formalin. However, none of the substitutes proved successful, and formalin fixation is still considered the gold standard for preservation of morphology and antigens. RNA has instead been found to be heavily affected by degradation and fragmentation in formalin-fixed tissues. Based on the hypothesis that RNA degradation would be inhibited by maintaining a low temperature, a protocol based on processing tissues with formalin at low temperature (cold fixation) was evaluated and proved useful in obtaining a reduction in RNA fragmentation. Finally, the problem of storage is discussed, in order to find ways to guarantee feasibility of molecular analyses even years after the original diagnosis.

Somatic mutation profiling of hobnail variant of papillary thyroid carcinoma

Hobnail variant of papillary thyroid carcinoma (HPTC) represents a recently described, aggressive and rare group of thyroid tumors with poorly understood pathogenesis. Molecular data about this group of cancers are few, and a more detailed molecular characterization of these tumors is needed. The main objective of the study is to define a comprehensive molecular typing of HPTC. Eighteen patients affected by HPTC, including eighteen primary tumors and four lymph node metastases, were screened for NRAS, KRAS, HRAS, BRAF, TP53, PIK3CA, hTERT, PTEN, CDKN2A, EGFR, AKT1, CTNNB1 and NOTCH1 gene mutations. Sequencing is conducted on the MiSEQ system, and molecular data are compared with clinical-pathologic data and follow-up. The patients include 14 women and 4 men. Ages range from 23 to 87 years. All 18 primary tumors of HPTC showed ≥30% hobnail features. BRAF and TP53 mutations are by far the most common genetic alterations in primary HPTC (72.2% and 55.6%, respectively), followed by hTERT (44.4%), PIK3CA (27.8%), CTNNB1 (16.7%), EGFR (11.1%), AKT1 (5.5%) and NOTCH1 (5.5%). The mutational pattern in primary tumors and metastasis was usually maintained. Univariate Cox regression analyses with bootstrap procedure indicated a significantly increased mortality risk in patients harboring BRAF mutation and BRAF mutation associated with TP53 and/or PIK3CA mutations. The detection of these multiple mutations appears to allow the identification of a subset of more aggressive tumors within the group and to bear information that should be useful for prognostic stratification of these patients including the planning of adjuvant therapy.

Leiomyomata of the genitourinary tract: A case series from the “rare urological neoplasm” registry

Abstract Objective. Leiomyomata are benign neoplasms that are rarely encountered in the genitourinary tract outside the uterus. Pathological confirmation is always needed for definitive diagnosis, in order to rule out malignancies such as leiomyosarcoma. In cases of small leiomyomata, a surveillance strategy can be suggested, although the preferred approach is complete resection. Prognosis seems to be excellent, but available data are based on few, inconsistent series, as is often the case with rare neoplasms. This article presents long-term follow-up data from the most heterogeneous series, in terms of anatomical location, currently available. Material and methods. The present series of 33 genitourinary leiomyomata was retrieved after joining the “rare urogenital neoplasm” registry of two Italian regions. Two pathologists jointly reviewed all pathological slides and confirmed the diagnosis of leiomyoma. Results. Sixteen cases were localized to the scrotum, eight to the bladder, five to the kidney, two to the prostate, one to the urethra and one to the penis. Mean patient age was 58.4 (range 32–80) years. Mean follow-up was 15.5 (range 2–20) years. Conclusions. In this highly heterogeneous series, the disease showed excellent long-term recurrence- and progression-free interval. The accuracy of pathological diagnosis, along with the indolent long-term course, make the role of active surveillance amenable for asymptomatic cases, particularly where surgery may result in overtreatment.