Giancarlo Troncone

Cyclin dependent kinase inhibitor p27Kip1 expression in normal and neoplastic cervical epithelium.

AIM: To investigate whether there is loss of the p27Kip1 protein in developing cervical cancer and whether p27Kip1 immunoreactivity has any relation to the proliferative indicator Ki-67. METHODS: The expression of p27Kip1 and Ki-67 was assessed by immunohistochemistry in serial sections from normal epithelium (13), low grade (27) and high grade (19) squamous intraepithelial lesions (LSIL, HSIL), and invasive cervical cancer (23). In the SIL cases the presence of human papillomavirus (HPV) genomic sequences was assessed by in situ hybridisation. The results were evaluated by image analysis, and reported as mean score of the percentage of p27Kip1 and of Ki-67 positive cells in each histological group. RESULTS: In general, p27Kip1 immunostaining was related to squamous differentation, and was intense in normal epithelium (47%), while it was reduced in SIL lesions as an effect of the decreased number of differentiating cells. However, decrease in the p27Kip1 expression was more evident in LSIL (36%) than in HSIL (39%); in the latter, p27Kip1 had a different intraepithelial distribution in that the staining extended to the basal cells. The average levels of p27Kip1 were similar in SIL lesions associated to low, intermediate, and high risk HPV types. Compared with normal epithelium and dysplasia, invasive cancer showed significantly lower p27Kip1 levels (23%). There was no relation between p27Kip1 and Ki-67 labelling indices in any of the histological groups examined. CONCLUSIONS: A reduction in p27Kip1 protein occurs in cervical cancer independently of the proliferative status. The changes in p27Kip1 expression may be related to the unregulated kinetics of developing cervical cancer.

KRAS mutation detection by high-resolution melting analysis significantly predicts clinical benefit of cetuximab in metastatic colorectal cancer

Background:Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are restricted to KRAS wild-type (WT) metastatic colorectal cancers (mCRCs), usually identified by direct sequencing, that may yield false negative results because of genetic heterogeneity within the tumour. We evaluated the efficiency of high-resolution melting analysis (HRMA) in identifying KRAS-mutant (MUT) tumours.Methods:We considered 50 mCRC patients scored as KRAS-WT by direct sequencing and treated with cetuximab-containing chemotherapy, and tested the correlations between HRMA findings and response rate (RR), progression-free (PFS) and overall survival (OS).Results:Aberrant melting curves were detected in four (8%) cases; gene cloning confirmed these mutations. Response rate (RR) of HRMA KRAS-WT patients was 28.3%. There was no response in HRMA KRAS-MUT patients. Disease control rate (responsive plus stable disease) was 58.7% in HRMA KRAS-WT patients and 25% in HRMA KRAS-MUT patients. There was no correlation between HRMA KRAS status and RR (P=0.287) or disease control (P=0.219). Median PFS (4.8 vs 2.3 months; hazard ratio (HR)=0.29, P=0.02) and OS (11.0 vs 2.7 months; HR=0.11, P=0.03) were significantly longer for the HRMA KRAS-WT than for HRMA KRAS-MUT patients.Conclusions:High-resolution melting analysis identified 8% more KRAS-MUT patients not responding to cetuximab-containing regimens, suggesting that HRMA may be more effective than direct sequencing in selecting patients for anti-EGFR antibodies.

PAX8 is expressed in anaplastic thyroid carcinoma diagnosed by fine-needle aspiration: a study of three cases with histological correlates.

OBJECTIVEnIt is difficult to diagnose anaplastic thyroid carcinoma (ATC) in a fine-needle aspiration (FNA) sample because, given the loss of morphological and immunophenotypical follicular thyroid features, its cytology resembles that of other undifferentiated neoplasms. Recent studies have shown that immunostaining for paired box gene 8 (PAX8), a transcription factor expressed in normal thyroid, is effective for diagnosing ATCs on histology. The aim of this study was to evaluate whether PAX8 could be used to identify ATCs on cytology also.nnnDESIGN AND METHODSnWe selected three PAX8-immunostained undifferentiated FNA samples previously diagnosed as suspected ATCs, whose cell block had been negative for the expression of TGB and thyroid transcription factor-1. Matched histological samples, available in two cases, were also processed for PAX8 immunohistochemistry.nnnRESULTSnAll three FNA samples were PAX8 positive. Two samples that had an epithelioid pattern showed a diffuse, intense nuclear signal. The third sample, which had a spindle-cell pattern, showed less intense and more patchy staining. Matched histology yielded overlapping results.nnnCONCLUSIONSnPAX8 immunocytochemistry can help cytopathologists to diagnose ATCs.

Toll-like receptor 9 agonist IMO cooperates with everolimus in renal cell carcinoma by interfering with tumour growth and angiogenesis

Background:Targeting the mammalian target of rapamycin by everolimus is a successful approach for renal cell carcinoma (RCC) therapy. The Toll-like receptor 9 agonist immune modulatory oligonucleotide (IMO) exhibits direct antitumour and antiangiogenic activity and cooperates with both epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors.Methods:We tested the combination of IMO and everolimus on models of human RCC with different Von-Hippel Lindau (VHL) gene status, both in vitro and in nude mice. We studied their direct antiangiogenic effects on human umbilical vein endothelial cells.Results:Both IMO and everolimus inhibited in vitro growth and survival of RCC cell lines, and their combination produced a synergistic inhibitory effect. Moreover, everolimus plus IMO interfered with EGFR-dependent signaling and reduced VEGF secretion in both VHL wild-type and mutant cells. In RCC tumour xenografts, IMO plus everolimus caused a potent and long-lasting cooperative antitumour activity, with reduction of tumour growth, prolongation of mice survival and inhibition of signal transduction. Furthermore, IMO and everolimus impaired the main endothelial cell functions.Conclusion:A combined treatment with everolimus and IMO is effective in VHL wild-type and mutant models of RCC by interfering with tumour growth and angiogenesis, thus representing a potentially effective, rationale-based combination to be translated in the clinical setting.

p27Kip1 protein expression in Hashimoto’s thyroiditis

Aims: Hashimoto’s thyroiditis (HT) is an autoimmune disease in which both proliferation and apoptosis are enhanced. p27Kip1 protein protects tissues from disease mechanisms that involve excessive cell proliferation and apoptosis. This study investigated whether there is loss of p27Kip1 expression in HT and whether p27Kip1 immunoreactivity has any relation to the proliferative indicator Ki-67. Because p27Kip1 is regulated through either degradation, mediated by the S phase kinase associated protein 2 (Skp2), or sequestration, via D3 cyclin, the expression of these proteins was also investigated. Methods: Immunohistochemistry was used to assess p27Kip1, Ki-67, Skp2, and cyclin D3 expression in 19 cases of HT and in 10 normal thyroids. The results were evaluated by image analysis and reported as labelling indices (LIs) in both groups. Results: The p27Kip1 LI was lower in HT than in normal thyroid (28% v 75%; p < 0.001), whereas Ki-67 (1.13% v 0.13%), Skp2 (0.74% v 0.15%), and cyclin D3 (1.56% v 0.00%) LIs were higher in HT than in normal thyroids (p < 0.001). There was no correlation between p27Kip1 and the expression of Ki-67, Skp2, and cyclin D3. Conclusions: p27Kip1 downregulation is not exclusive to tumours but occurs also in HT, independently of the proliferative status and of changes in Skp2 and cyclin D3 expression. Further investigation is required to understand the mechanisms leading to p27 deregulation because these observations suggest that the regulation of p27Kip1 expression in epithelial thyroid cells may play a role in HT pathogenesis.

MASC is indistinguishable from acinic cell carcinoma, papillary‐cystic variant on salivary gland FNA cytomorphology: case report with histological and immunohistochemical correlates

1. Barroeta JE, Farkas T. Merkel cell carcinoma and chronic lymphocytic leukemia (collision tumor) of the arm: a diagnosis by fine-needle aspiration biopsy. Diagn Cytopathol 2007;35:293–5. 2. Soares FA, Potenciano O, Saldanha JC, Laemmel A. Fine needle aspiration of squamous cell carcinoma of the skin metastatic to the site of leukemic lymphadenopathy. A case report. Acta Cytol 1992;36:407–9. 3. McElroy C, Velilla R, Chaudhary H, Al-Abbadi MA. Fine-needle aspiration diagnosis of squamous cell carcinoma in a lymph node involved with small lymphocytic lymphoma: case report and review of the literature. Diagn Cytopathol 2009;37:48–50. 4. Flezar MS, Prevodnik VK, Kirbis IS, Strojan P. Cutaneous squamous cell carcinoma metastatic to chronic lymphocytic leukaemia: diagnostic potential of fine needle aspiration cytology. Cytopathology 2006;17:288–94. 5. Caraway NP, Wojcik EM, Saboorian HM, Katz RL. Concominant lymphoma and metastatic carcinoma in a lymph node: diagnosis by fine-needle aspiration biopsy in two cases. Diagn Cytopathol 1997;17:287–90. 6. Engels EA, Pfeiffer RM, Fraumeni JF Jr et al. Spectrum of cancer risk among US solid organ transplant recipients. JAMA 2011;306:1891–901.

Ultimobranchial Body Remnants (Solid Cell Nests) as a Pitfall in Thyroid Pathology

We report the history of a pitfall in thyroid histopathology of a 47-yr-old man with a euthyroid nodular goiter involving the right lobe. The ultrasound-guided fineneedle aspiration of the dominant (3 cm) nodule, showing benign-appearing follicular cells, colloid, and scattered Hurthle cells, was consistent with a benign hyperplastic nodule (1). Because the left lobe had not shown evidence of nodular disease, the patient was submitted to a right lobectomy. The pathologist described a heavy lymphoplasmacyticbackgroundwithoccasionalgerminalcenter formation, as observed in Hashimoto thyroiditis, and a microscopic ( 1cm) follicularproliferationofepithelial thyroidcellswhose nucleidisplayedmembrane irregularityandchromatinclearing with occasional grooves. Thus, a histological report of a follicular variant of papillary microcarcinoma was issued. The patient underwent a completion thyroidectomy at the Federico II UniversityofNaples.At thatoccasion, thecompletesetofslides from both surgeries was reviewed by expert pathologists in the field of thyroid disease.

Performance of EGFR mutant-specific antibodies in different cytological preparations: a validation study.

Molecular testing for epidermal growth factor receptor (EGFR) mutations is required to select the most appropriate treatment for advanced‐stage non‐small cell lung cancer (NSCLC). In routine practice, cytological samples are often the only specimens available for testing. When the number of neoplastic cells is large, DNA‐based assays are the gold standard. When cytological samples contain only a few neoplastic cells, immunocytochemistry (ICC) using anti‐EGFR mutant‐specific antibodies may be more effective. We aim to assess the specificity and sensitivity of IHC staining in cytological specimens using mutated cell lines subjected to different cytopreparations and staining methods.

A case of anaplastic thyroid carcinoma with exceptional growth

A 73-year-old man was admitted to our department because of a sudden and rapid enlarging neck mass. The patient reported the start of the enlargement of the neck 40 days prior. He was a farmer and lived in a rural village. He had smoked 20 cigarettes per day for 20 years. His past medical history was unremarkable. Physical examination revealed a very large painless neck mass, associated with swelling of the face, upper extremities and trunk. Other clinical findings included dullness on percussion and absence of vesicular sounds at the left lung base. He complained of hoarseness and dysphagia. There were no other symptoms such as fever, weight loss, cough or asthenia and his vital signs were normal. Laboratory results were within normal ranges, except for a neutrophil leukocytosis (white blood cells: 18,540/mmc, neutrophils: 15,230/mmc), increased a1 and a2 and a C-reactive protein of 2.64 mg/dl (limit value 0.5 mg/dl). Thyroid function tests and tumor markers were normal, but serum thyroglobulin was 1077 ng/ml (reference range 1.4–78 ng/ml). An X-ray study of the chest documented a right lung base opacity and a small left-sided pleural effusion. The analyses with 12-lead electrocardiogram (ECG), and abdominal and pelvic ultrasonography showed no clinically significant findings. A pericardial effusion was found with echocardiography. Thyroid ultrasound imaging showed several small laterocervical lymph nodes and a diffuse thyroid enlargement. The thyroid was inhomogeneous in echotexture because of many nodules. The biggest one (greater diameter 10 cm) was characterized by a mixed echotexture and extended to the mediastinum, causing tracheal compression and deviation. An ultrasound-guided needle aspiration of the thyroid lesion was carried out. Moreover, a diagnostic thoracentesis revealed the presence of tinged pleural fluid. A contrast-enhanced CT scan of the neck and the chest confirmed the presence of a substernal multinodular goiter (transverse diameter 15 cm and anterior–posterior 10 cm) causing right deviation of the trachea (Fig. 1). The CT scan also showed a bilateral pleural thickening and effusion with multiple pleural and pulmonary solid nodules associated with the presence of several enlarged mediastinal lymph nodes. The patient’s clinical presentation suggested the presence of an aggressive tumor. The following diagnostic hypotheses were formulated: thyroid tumor, multinodular goiter with lung cancer, lymphoma, thymoma and mesothelioma with metastases. To better clarify the nature of the lesion, we performed a biopsy of the mediastinal mass. The Papanicolaou stained smears showed anaplastic epithelioid dissociated cells displaying dense and ‘‘glassy’’ cytoplasm. The matched core biopsy confirmed the anaplastic thyroid carcinoma diagnosis. The cells showed dense cytoplasmic inclusions eccentrically displacing the nucleus as observed in the rhabdoid variant. These cells were dishesive, entrapping the residual normal follicles and infiltrating several striated muscle bundles. Necrotic areas were also recognized. During the hospitalization, the patient’s clinical conditions worsened: the patient complained of severe dysphagia and dyspnea. To improve the compressive symptoms, we C. Altruda M. Venafro A. Pagano F. C. Sasso (&) Department of Internal and Clinical Medicine ‘‘MagrassiLanzara’’, Second University of Naples, Naples, Italy e-mail: ferdinando.sasso@unina2.it

CD10 is useful to identify gastrointestinal contamination in endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) cytology from pancreatic ductal adenocarcinoma

Endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) cytology is an effective tool to diagnose pancreatic ductal adenocarcinoma (PDA). Standard morphological criteria are usually reliable. When contaminating gastrointestinal (GI) epithelial cells are prevalent among neoplastic cells, these can be highlighted by carcinoembryonic antigen (CEA) staining. CD10 is a cell‐surface metallopeptidase normally expressed by the GI epithelial apical border, whose expression is decreased or lost in PDA. We included CD10 in a panel, together with CEA, to discriminate the GI contaminant cells from PDA cells on cell blocks.