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Objective Cervical lymph node fine needle aspirates (FNAs) may represent the only specimens available for an initial characterisation of patients with lymphadenopathy. Morphology and human papillomavirus (HPV) DNA presence were evaluated in FNAs collected from patients with oropharyngeal squamous cell carcinoma (OPSCC) or cancer of unknown primary (CUP). FNA HPV results were compared with those of the respective formalin-fixed paraffin-embedded (FFPE) primary cancer. Methods Liquid-based cytology was performed on FNAs collected in PreservCyt. HPV-DNA was analysed by the INNO-LiPA HPV genotyping Extra II on both cytological and FFPE samples. The CINtec® Histology Kit was used to assess p16 expression in cancer tissues. Results Forty-seven FNAs were collected from OPSCC and 16 from CUP patients. Cancer cells were found in 35/47 cases (74.5%), while 11 (23.4%) showed only necrosis and one (2.1%) was negative for malignancy. HPV-DNA was detected in 30/47 FNAs (63.8%), mostly harbouring HPV16 (90.0%). An excellent agreement was observed between the FNA and corresponding FFPE HPV status (raw agreement: 97.5%; Cohen κ: 0.94). The HPV test result on the necrotic FNAs completely matched that of the respective primary cancer. FNA HPV testing correctly identified 26/27 HPV-driven OPSCCs (96.3%). HPV was detected in nine of 16 FNAs (56.2%) from CUP patients. Conclusions HPV status of metastatic cervical lymph node FNAs reflects that of the corresponding primary OPSCCs even when cell integrity in the FNA is not preserved and only necrotic debris are present. In patients with initial CUP, HPV-positivity on the FNA may guide the diagnostic workup and therapeutic management, since it suggests an oropharyngeal origin.

Whole exome sequencing (WES) was used to investigate two Italian siblings with wild-type RET genotype, who developed medullary thyroid cancers (MTCs) and, later, primary prostate and breast cancers, respectively. The proband's MTC harbored a p.Met918Thr RET mutation; his sister's MTC was RET/RAS wild-type. Both siblings had a germline mutation (p.Arg417Gln) in the extracellular Sema domain of the proto-oncogene MET. Experiments involving ectopic expression of MET p.Arg417Gln in MET-negative T47D breast cancer cells documented the mutant receptor's functionality and its ability to enhance cell migration and invasion. Our findings highlight a possible link between MET germline mutations and MTCs and suggest that MET p. Arg417Gln may promote an invasive malignant phenotype. The possibility that MTC can be driven/co-driven by a MET mutation has potential management implications, since the tyrosine-kinase inhibitor cabozantinib-approved for treating advanced MTCs-is a specific MET inhibitor.

Introduction Fine-needle aspiration cytology (FNAC) is a basic step in the diagnosis of salivary gland tumors that have a wide variety of histological types. The recent Milan system for reporting salivary gland cytopathology (MSRSGC) can correlate the risk of malignancy with precise cytological features. A revised version was recently proposed to improve the surgical relevance and facilitate uniform management. Material and methods A multicenter study retrospectively used the original and revised MSRSGC criteria to classify a series of patients who received surgery after FNAC. Results We enrolled 503 patients from three tertiary centers. The risk of malignancy for the MSRSGC resulted 19.5% in cat. I, 14.3% in cat. II, 17.6% in cat. III, 3.6% in cat. IVa, 24.6% in cat. IVb, 66.7% in cat. V, and 96.8% in cat. VI. The results from the revised MSRSGC were consistent with the original values. Conclusion The MSRSGC is a promising classification system. In our opinion, the revised version of the MSRSGC supplements FNAC with some crucial clinical information and can better identify the appropriate treatment in each category.

Background: Conventional transbronchial needle aspiration (c-TBNA) contributed to improve the bronchoscopic examination, allowing to sample lesions located even outside the tracheo-bronchial tree and in the hilo-mediastinal district, both for diagnostic and staging purposes. Methods: We have evaluated the sensitivity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the c-TBNA performed during the 2005–2015 period for suspicious lung neoplasia and/or hilar and mediastinal lymphadenopathy at the Thoracic endoscopy of the Thoracic Surgery Department of the Regina Elena National Cancer Institute, Rome. Data from 273 consecutive patients (205 males and 68 females) were analyzed. Results: Among 158 (58%) adequate specimens, 112 (41%) were neoplastic or contained atypical cells, 46 (17%) were negative or not diagnostic. We considered in the analysis first the overall period; then we compared the findings of the first [2005–2011] and second period [2012–2015] and, finally, only those of adequate specimens. During the overall period, sensibility and accuracy values were respectively of 53% and 63%, in the first period they reached 41% and 53% respectively; in the second period sensibility and accuracy reached 60% and 68%. Considering only the adequate specimens, sensibility and accuracy during the overall period were respectively of 80% and 82%; the values obtained for the first period were 68% and 72%. Finally, in the second period, sensibility reached 86% and accuracy 89%. Carcinoma-subtyping was possible in 112 cases, adenocarcinomas being diagnosed in 50 cases; further, in 30 cases molecular predictive data could be obtained. Conclusions: The c-TBNA proved to be an efficient method for the diagnosis/staging of lung neoplasms and for the diagnosis of mediastinal lymphadenopathy. Endoscopist’s skill and technical development, associated to thin-prep cytology and to a rapid on site examination (ROSE), were able to provide by c-TBNA a high diagnostic yield and molecular predictive data in advanced lung carcinomas.

Benevolo M, Terrenato I, Mottolese M, Marandino F, Muti P, Carosi M, Rollo F, Ronchetti L, Mariani L, Vocaturo G & Vocaturo A (2010) Histopathology57, 580–586 Comparative evaluation of nm23 and p16 expression as biomarkers of high-risk human papillomavirus infection and cervical intraepithelial neoplasia 2+ lesions of the uterine cervix Aims: To investigate the clinical role of nm23 expression in identifying both high-risk human papillomavirus (HR-HPV) and high-grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2+ (CIN2+)], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR-HPV infected cervical lesions. Methods and results: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low- and high-grade lesions and squamous carcinomas (SC). HR-HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico-vaginal samples of the same patients. In detecting CIN2+, p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P

The role of tumor cells in synthesizing pro-inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF-7 human mammary adenocarcinoma cell line induced activation of hypoxia-inducible factor 1alpha (HIF-1alpha) and nuclear factor-kappa B (NF-kappaB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up-regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute-phase protein pentraxin-3 (PTX3). Hypoxia also stimulated chemokine (C-X-C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal-derived factor-1alpha (SDF-1alpha) increased invasion and migration of hypoxic MCF-7 cells. Inhibition of HIF-1alpha by chetomin and NF-kappaB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF-7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser-capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF-1alpha and NF-kappaB and up-regulation of IR, CXCR4, estrogen receptor alpha (ERalpha), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF-7 cells acquire a pro-inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF-1alpha, NF-kappaB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells.

The differential diagnosis of osteolytic lesions is very difficult because it is to be distinguish among tumoral masses, infective lesions and metabolic diseases that might have a similar imaging. To allow a correct therapy is often necessary to perform biopsy, histopathology and infectious test. We report an unusual case of non-specific osteomyelitis where all clinical and radiological aspects suggested a diagnosis of osteosarcoma.

p16 INK4a is overexpressed in high-risk human papillomavirus (HR-HPV)–infected preneoplastic and neoplastic lesions of the uterine cervix. Our aim was to verify whether p16 is a diagnostic marker also in cervical liquid-based cytology. We performed p16 immunocytochemical analysis and the Hybrid Capture 2 (HC2) test (Digene, Gaithersburg, MD) for HR-HPV infection in 471 ThinPrep-processed (Cytyc, Boxborough, MA) cervicovaginal samples and correlated the results with histologic findings. A total of 32.3% of the specimens showed p16 immunoreactivity, whereas the HC2 test was positive in 41.2% of the cases (65.2% concordance rate). Correlating the cytologic, p16, and HPV results with histologic findings revealed HC2 as the most sensitive test for a diagnosis of cervical intraepithelial neoplasia 2 or worse, whereas cytologic examination was the most specific. The positive predictive value was significantly higher for cytologic examination than for p16 and HR-HPV testing. These data suggest that p16 evaluation in ThinPrep samples does not have better clinical effectiveness for identifying high-grade lesions than conventional morphologic examination and HPV testing.

Genital human papillomavirus (HPV) is the causative agent of cervical cancer and is the most common sexually transmitted infection. Only limited and controversial data are available regarding HPV transmission in male sexual partners of women with cervical intraepithelial neoplasia (CIN). The aim of this study was to investigate the prevalence and the genotype distribution of HPV in penile scrapings of a series of Italian men, who had no visible penile lesions and were partners of women who were affected, or had been affected previously by cervical intraepithelial neoplasia or who were infected with HPV. The concordance of the viral group in the infected partners was determined. A total of 77 penile scrapings were screened for HPV infection by the polymerase chain reaction, while 59 cervicovaginal brushings of their female partners were tested. 35% of evaluable male samples and 64% of female sexual partners were found to be HPV positive. In the 55 simultaneously evaluable couples, a concordance of 45% was found, 11 couples (20%) with both partners being HPV negative and 14 couples (25%) with both partners HPV positive (P=0.001). Six out of the 14 couples (43%), where both partners were HPV positive, harbored the same HPV genotype group. These data, although preliminary, could support further the hypothesis that male HPV infection is more frequent in sexual partners of HPV positive or women with cervical intraepithelial neoplasia indicating that men could represent an important source of HPV transmission between sex partners.

HPV is one of the most common sexually transmitted infections. However little is known about its prevalence in the male population and concordance with female partners.This cross-sectional study aimed to: (a) investigate HPV prevalence and genotype distribution among a series of stable male sexual partners of CIN/HPV positive women and (b) assess HPV infection and type-specific concordance between partners.378 stable and monogamous male partners of CIN/HPV positive women were selected. Of these, 238 cases were enrolled at the same time as their female partner. All the subjects were tested by the Linear Array HPV genotyping assay.Overall, 153/378 men (40.5%) and 122/238 women (51.3%) were positive for at least one of the 37 HPV types detectable by the assay used. Among the HPV-positive participants, 69 of the 378 men (18.2%) and 54 of the 238 women (22.7%) harboured multiple genotypes. 75 couples (31.5%) were concordantly HPV positive, while 102 couples (42.9%) were concordantly negative (Kappa value: 0.491, p0.0001). Among the couples in which both partners were HPV positive, 68% harboured at least one genotype in common. Results from a GEE model evidenced that when the male partner tested HPV positive for at least one genotype, this had a significant effect on the positivity of their relative female partner (p0.0001).We evidenced a high prevalence of HPV male infections and a moderate concordance between partners. However, we observed a significant HPV type-specific correlation between partners, which is unlikely to be coincidental.

To investigate the clinical role of nm23 expression in identifying both high-risk human papillomavirus (HR-HPV) and high-grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2(+) (CIN2(+) )], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR-HPV infected cervical lesions. Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low- and high-grade lesions and squamous carcinomas (SC). HR-HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico-vaginal samples of the same patients. In detecting CIN2(+) , p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P 0.0001). Concerning HR-HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% versus 47%, P0.0001), although the sensitivities were comparable (71% versus 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. In contrast, we observed a significant association between p16 overexpression and HPV16/18 genotypes. We confirm the diagnostic value of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV-E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2(+) or HR-HPV infection.

The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL−). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL−, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL− patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2− lesions (p

Background Adequate surgery still remains the only curative treatment of chordoma. Interesting clinical data on advanced disease with molecularly targeted therapies were reported. Methods We described the clinical outcome of a series of chordoma patients followed at Regina Elena National Cancer Centre of Rome from 2004 to 2008. Results Twenty-five consecutive patients with sacral (11 patients), spine (13 patients), and skull base (1 patient) chordoma went to our observation. Six patients (24%) had primary disease, 14(56%) a recurrent disease, and 5(20%) a metastatic spreading. Surgery was the primary option for treatment in 22 out of 25 patients. Surgical margins were wide in 5 (23%) and intralesional in 17(77%) patients; 3 out of 4 in-house treated patients obtained wide margins. After first surgery, radiotherapy (protons or high-energy photons) were delivered to 3 patients. One out of the 5 patients with wide margins is still without evidence of disease at 20 months from surgery; 2 patients died without evidence of disease after 3 and 36 months from surgery. Sixteen out of 17 (94%) patients with intralesional margins underwent local progression at a median time of 18 months with a 2-year local progression-free survival of 47%. The 5-year metastasis-free survival rate was 78.3%. Seventeen patients with locally advanced and/or metastatic disease expressing platelet-derived growth factor receptor (PDGFR) β were treated with imatinib mesylate. A RECIST stabilization of the disease was the best response observed in all treated cases. Pain relief with reduction in analgesics use was obtained in 6 out of 11 (54%) symptomatic patients. The 5- and 10-year survival rates of the entire series of patients were 76.7 and 59.7%, respectively. Conclusions Despite progress of surgical techniques and the results obtained with targeted therapy, more effort is needed for better disease control. Specific experience of the multidisciplinar therapeutic team is, however, essential to succeed in improving patients' outcome.

Although the majority of endometrial cancer (EC) patients can be cured by surgery, unexpected recurrent disease may also occur in early stage patients. In the present study, whether or not the analysis of multiple biopathological parameters might lead to more accurate predictions of the clinical outcome of EC patients with long-term follow-up (FU) was investigated.Estrogen and progesterone receptor (ER and PgR) positivity and HER2 overexpression by immunohistochemistry were evaluated. The peritoneal washings (PWs) were analyzed by cytology and immunocytochemistry employing AR-3 and B72.3 monoclonal antibodies.The patients with positive PW and HER2 positive tumors showed shorter overall survival compared to those bearing HER2 negative tumors (p =0.004). HER2 overexpression also influenced the patient outcome in the group with tumors lacking PgR (p = 0.004). At multivariate analysis PgR and HER2 overexpression emerged as independent prognostic factors.The combined analysis of these biopathological markers could provide useful information for the selection of patients to be enrolled in innovative therapeutic strategies.

Breast carcinoma remains the most common malignancy in women in western countries. The principal sites of metastases are the regional lymph nodes, liver, lung and bone; unusual sites of metastases are very rare. Infiltrating ductal carcinoma is the most common histological subtype (about 80%) as compared to lobular carcinoma (7-20%), which metastasizes more frequently to unusual sites. An 80-year-old female patient was submitted to resection of an infiltrating ductal carcinoma (stage I). After 4 years, the presence of a pelvic mass in the absence of local, distant or lymph node recurrence was found. Resection of the mass was performed. The pathological and immunohistochemical examination revealed a metastatic carcinoma compatible with a mammalian origin. After 36 months of follow-up the patient is alive and disease-free. Considering the age of the patient, the good performance status, the absence of other sites of relapse and the large size of the mass, surgery was performed. Surgery remains the only valid option when recurrence produces abdominal complications. In the absence of randomized prospective studies, however, we do not know whether this is always the correct therapeutic strategy.

Learning Objectives After completing this course, the reader will be able to: Explain the importance of an accurate evaluation of HER-2 status to select breast cancer patients for trastuzumab therapy.Describe the current methods that assess HER-2 status in breast cancer.Discuss the advantages and limits of the CISH method for the detection of HER-2/neu gene amplification in histology and ThinPrep®-processed breast cancer fine-needle aspirates. Access and take the CME test online and receive 1 AMA PRA Category 1 Credit™ at CME.TheOncologist.com The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTest™. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep®-processed FNAs.

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P

To determine whether phenotypic field changes occur in tissues adjacent to carcinoma, we assayed, by immunohistochemistry, the expression of HER-2, p53, Fas, and FasL in 72 breast cancers (BC) and multiple autologous peritumoral tissues (PTTs) sampled up to 5 cm distance and in 44 benign breast tumors (BBTs). About 5% and 3% of the PTTs and 4.5% and 6.8% of BBTs showed alterations in HER2 and p53 expression, respectively. Of interest, gene amplification was observed in 50% of HER2 positive PTTs, but not in any HER2 positive BBTs. Fas, highly expressed in BBTs and downregulated in BC, maintained its expression in PTTs, whereas FasL, usually negative in BBTs, was upregulated in BC as well as in the PTTs closest (1 cm) to the invasive lesion. Our data suggest that FasL could be a potential novel biomarker of transformation, which may identify, along with HER2 and p53, precursor lesions in a genetically altered breast tissue.

Metastatic epithelial malignant tumor involving the spermatic cord and epididymis is rare and the prognosis of these patients is poor. Usually gastrointestinal cancers show diffusion to liver, lung and bone. Several routes by which a colorectal cancer can metastasize to the testis have been reported in literature. Herein we report a case of an occult gastrointestinal cancer with an intrascrotal metastasis in an adult patient with possible spread through the spermatic veins due to primary intestinal carcinoma. In the case of a testicular mass or hydrocele evidence in a patient with an unusual age for primary testis tumor, a diagnosis of metastatic cancer should be considered.

The purpose of this study was to clarify the role and the predictive strength of the adhesion molecule CD44s (standard isoform) in colorectal carcinogenesis.CD44s immunohistochemical expression was evaluated in 100 patients with colon adenoma and 100 patients with colon adenocarcinoma and adjacent non-neoplastic mucosa (ANNM). The patients were followed-up for five years.CD44s immunoreactivity was expressed in low-moderate-high-grade dysplasia adenomas and associated with adenocarcinoma (p = 0.01), ANNM (p = 0.05) and pTNM stage (p = 0.00001). Univariate analysis revealed that CD44s expression was associated with overall survival (OS) in carcinomas (p = 0.01) and ANNM (p = 0.05). Bivariate analysis revealed that CD44s was associated with OS in stages I and II patients (p = 0.03). Multivariate analysis revealed that stage (p = 0.0001) and CD44s expression (p = 0.05) were independent predictors of OS.CD44s is involved in colon carcinogenesis and is associated with aggressive carcinomas. The immunohistochemical expression of CD44s may reveal cells that have lost their adhesion ability and therefore detect carcinomas with high metastatic power.

Summary Background Thyroid cancer is the most common endocrine malignant disease, but preoperative diagnosis remains a challenge. Fine-needle aspiration cytology has greatly improved the clinical management of thyroid nodules, but the preoperative characterisation of follicular lesions is very difficult. Many patients are thus referred to surgery more for diagnosis than for therapeutic necessity. We undertook an international multicentre study to assess the usefulness of immunohistocytochemical staining for two potential markers of malignant thyrocytes. Methods Expression of galectin-3 and CD44v6 was tested on 1009 thyroid lesions (tissue specimens and cytological cell-blocks) and 226 fresh cytological samples obtained preoperatively by ultrasound-guided fine-needle aspiration of thyroid nodules (prospective analysis). The test used monoclonal antibodies specific for CD44v6 and galectin-3, the indirect avidin-biotin complex immunoperoxidase method, and 3-amino-9-ethyl-carbazole as substrate. Findings The sensitivity, specificity, positive predictive value, and diagnostic accuracy of this test method (for coexpression of the two markers) in the prospective analysis were 88%, 98%, 91%, and 97%, respectively. The sensitivity and specificity of galectin-3 immunodetection alone in discriminating benign from malignant thyroid lesions were more than 99% and 98% respectively, and the positive predictive value and diagnostic accuracy were 92% and 99%. Interpretation The integration of galectin-3 immunostaining with conventional cytomorphological and clinical diagnostic procedures represents a sensitive and reliable diagnostic approach for preoperative identification of thyroid carcinomas. This test method improves the diagnostic accuracy of conventional cytology and provides the molecular basis for a new nosological assignation of the not yet classified thyroid neoplasms of indeterminate malignant behaviour.

1050 Background: Axillary lymph node dissection (ALND) may not be necessary in women with breast cancer (BC) who have micrometastasis in a sentinel lymph node (SLN), owing to the low risk of non-SLN (NSLN) involvement. In our Institute we validated and adopted the molecular diagnostic tool OSNA based on the quantitative measurement of Cytokeratin 19 (CK19) mRNA.The aims of our work in a subgroup of women with micrometastatic SLN, were: 1) to correlate the copy numbers of CK19 mRNA with the risk of additional positive NSLNs; 2) to assess the relationships between the molecular subtype classification and the probability of a positive ALND; 3) to verify whether a combination of the new above mentioned parameters is able to identify a subgroup of patients with a micrometastatic SLN and a negligible risk of positive NSLNs in whom ALND may be avoided. Methods: The SLN lysates from 709 patients were analyzed by OSNA assay. We considered only patients with a micrometastatic SLN (copy numbers between 250 and 5000/μL) and the probability of having a positive ALND was calculated by the logistic regression model. This series of BC patients were divided into four main subtypes taking in account the BC classification as defined by a combination of estrogen, progesteron receptors and HER2 status. Results: OSNA positivity for micrometastasis was reported in 91/709 cases (12,8%).The number of patients with positive ALND was 20 (22%). The statistical analyses showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers (>2000) of CK19 mRNA together with HER2 subtype. Otherwise none of the luminal A patients with a positive SLN but presenting a copy number

Background: The accuracy of the OSNA assay for metastases detection in sentinel lymph nodes (SLNs) has recently been validated in our Institute and adopted as an intra-operative test for breast cancer (BC) patient management. The aims of this study in a series of early BC patients, were: 1) to correlate the copy numbers of cytokeratin 19 (CK19) mRNA with the size of nodal metastases and with the risk of additional disease in non-sentinel lymph nodes (NSLNs); 2) to investigate the relationship between SLN status with OSNA method, and conventional bio-pathological factors taking into account the molecular BC classification: luminal A, luminal B, HER2 subtype, and triple-negative; 3) to identify a subgroup of patients with positive SLN with higher risk of NSLNs metastatic involvement. Material and Methods: 750 SLNs from 580 patients were clinically processed using both OSNA assay and post-operative histology. The results of these two methods were then compared. The correlation between the size of metastases and the copy numbers of CK19 mRNA was calculated using the Spearman correlation coefficient. Complementary axillary lymph node dissection (ALND) was performed concurrently in case of OSNA assay positivity and the probability of having a positive lymph node axillary dissection was calculated by the unconditional logistic regression model. This series of BC patients were divided into four main subtypes taking in account the BC classification based on the immunohistochemistry phenotypic patterns. Results: OSNA positivity was reported in 24.6% of the patients for a sensitivity of 94.2%. Considering the 145 patients with SLN positivity the size of metastatic foci was significantly correlated to the copy numbers of CK19 mRNA. The complex relationships among the bio-pathological variables analyzed by multiple correspondence analysis (MCA) showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers of CK19 mRNA and HER2 subtype tumors. OSNA specificity (95%) and negative predictive value (98.4%) clearly demonstrated its reliability to guide ALND decision. Conclusions: Our results showed that OSNA is an excellent method for the detection of metastases in SLN and its adoption in our clinical practice has determined a significant improvement in the diagnosis of metastatic BC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-01-09.

e11012 Background: The accuracy of the OSNA assay for metastases detection in sentinel lymph nodes (SLNs) has recently been validated in our Institute and adopted as an intra-operative test for bre...

609 Background: The aims of the study were 1) to assess the accuracy of a new intra-operative molecular diagnostic tool named OSNA, based on the measurement of cytokeratin 19 (CK19) mRNA, in the detection of axillary sentinel lymph node (SLN) metastases in patients with breast carcinoma 2) to determine the concordance of OSNA analysis with multilevel haematoxylin and eosin (H&E) and immunohistochemical (IHC) examination. Methods: A prospective series of 247 consecutive SLNs from 187 breast cancer patients was evaluated. The OSNA assay (Sysmex, Kobe - Japan) follows a short sample preparation step and subsequent rapid amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification. Each SLN was immediately divided into four slices. Two alternate slices were used for the intra-operative OSNA assay. The remaining two slices were investigated by six-level histopathology with 100 μm skip ribbons using routine H&E and CK19 IHC staining. The results of histology and OSNA were then compared. Results: Pts characteristics: infiltrating ductal/infiltrating lobular/mucinous/medullar/DCIS: 130/10/1/1/25. OSNA and histo-pathological methods identified 54 SLNs positive and 185 negative cases (2/3 contained isolated tumour cells). We found 8 discordant cases, 2 OSNA negative with micrometastasis found by IHC/H&E analysis, 5 OSNA positive for micrometastases but IHC/H&E negative and 1 case macrometastatic at OSNA, while negative at IHC/H&E analysis. The overall concordance of OSNA with histopathology was 96.7% with a specificity of 96.8% and sensitivity of 96.4%. These discordances could be due to sampling bias such that a micrometastasis was confined to the slices used for OSNA or the slices used for histology. Conclusions: The current study suggests that the performance provided by OSNA assay is comparable to intensive histopathological work-up even when using only half a lymph node. The method could be applied as a rapid and reliable intra-operative diagnostic tool thus preventing breast cancer patients from a diagnostic delay or second surgery due to a postoperatively diagnosed positive SLN. No significant financial relationships to disclose.

Abstract #1091 Focal adhesion kinase (FAK) is a protein tyrosine kinase which regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis and is a critical mediator of signalling events between cells and their extracellular matrix. FAK is highly expressed in invasive breast cancer (BC) where its upregulation has been implicated in the acquisition of an invasive tumor phenotype. Hierarchical cluster analysis identified subgroups of BC with separate gene expression profiles which detect four main tumor phenotypes, Luminal A (LA), Luminal B (LB), Basal-like (B), and HER2 subtype (HS) characterized by significant differences in prognosis. Up to now, there are no data concerning FAK distribution within the molecular BC subtypes. In this study FAK overexpression was evaluated, by immunohistochemistry (IHC), in a retrospective series of 193 invasive BC surgically treated at our Institution between 2002 and 2005. Associations with conventional pathological factors and with molecular subtypes were analyzed by Multiple Correspondence Analysis (MCA) and chi square test, aimed to determine the impact of FAK high positivity within BC molecular subtypes and to define a biologic profile associated to aggressive bio-pathologic variables such as tumor size (T), nodal status (N), grading (G), p53, Bcl2, Ki-67, p-AKT, p-MAPK . The anti-FAK 4.47 monoclonal antibody was used to detected FAK expression which was scored as high (2 or 3 intensity and ≥ 90% positive cells) or low (0 or 1 intensity and < 90% positive cells). FAK was overexpressed in 41.7% of LA, 70% of LB, in 48.0% of HER2 subtype and 52.2% of Basal-like BC. The relationships among the bio-pathological factors, analysed by MCA, showed that FAK+ is located in the quadrant containing more aggressive tumor phenotypes such as HER2 and Basal like subtypes together with T3-4, N+, G3 and p53+, high Ki67, Bcl2–, p-MAPK+, p-AKT+. Moreover, in LA tumors, FAK overexpression is significantly related to nodal status (p=0.05), grading (p=0.007), p53 nuclear accumulation (p=0.047) and p-AKT overexpression (p=0.001) and it appears to be a useful tool in identifying BC patients at higher risk of progression. The presence of FAK upregulation in LA subtype, may represent a useful tool in identifying those LA patients who would benefit from more aggressive adjuvant therapy. In this context, further investigations are necessary to better assess the prognostic role of FAK overexpression in low risk BC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1091.

Background Cervical cancer represents an important global public health problem. It is the 2nd most common cancer among women worldwide. Human Papillomavirus (HPV) infection is now well-established as a necessary cause of invasive cervical cancer (ICC) development. Only a few studies on HPV prevalence and type-specific distribution in ICC have been conducted in Italy. Aim To describe the prevalence of HPV and the HPV type-specific distribution in ICC cases identified in Rome, Italy. Methods 140 paraffin embedded tissue blocks of primary ICC diagnosed between 2001 and 2006 were identified at the Regina Elena Cancer Institute in Rome (Italy). HPV was detected through amplification of HPV DNA using SPF-10 HPV broad-spectrum primers followed by DEIA and then genotyping by LiPA25 (version 1). Results 134 cases were considered suitable for HPV DNA detection after histological evaluation; and overall, 90.3% (121/134) HPV prevalence was detected. 111 cases had a single HPV type, 4 cases had an uncharacterized type (HPVX) and 6 cases had multiple HPV infections. The five most common single HPV types among positive cases were: HPV16 (71/121; 58.7%), HPV18 (12/121; 9.9%), HPV31, HPV45 and HPV58 (5/121; 4.1% each). 2 (1.5%) of the single infections and 2 (1.5%) of the multiple infections contained low risk types. Statistically significant differences in the relative contribution of HPV18 were found when comparing squamous cell carcinomas with adenocarcinomas. Conclusions HPV16 and HPV18 accounted for almost 70% of all the HPV positive ICC cases. The study provides baseline information for further evaluation on the impact of recently introduced HPV vaccines in Italy.

Background The thymus arises in the ventral portion of the third and fourth pharyngeal pouch. It descends into the anterior mediastinum at 6th week of gestation. Any errors occurring during this process can cause dissemination of aberrant nodules that are responsible for most atypical thymomas. Case Presentation The authors report a unusual case of type-A thymoma located in D10 and D11 vertebral bodies. The histology showed a uniform growth of short, spindle shaped, mitotically inactive cells. A few small, normal lymphocytes were seen scattered or in small groups. The immunohistochemical investigation for neuroectodermal, neuroendocrine, vascular and muscular markers were negative. It also confirmed the presence of CD3+, CD5+ T lymphocytes and the absence of immature T-lymphocyte markers. Conclusions The case described shows a thymic hystogenesis for spindle cell tumours. To our knowledge no other cases of vertebral thymomas have been described in international literature.