Frequent detection of Merkel cell polyomavirus DNA in tissues from 10 consecutive autopsies.

Merkel cell polyomavirus (MCPyV) has been identified in samples of Merkel cell carcinoma (MCC), an aggressive skin cancer. Seroepidemiologic studies indicated a high frequency of MCPyV infection in humans, suggesting respiratory and faecal-oral routes, or transmission by skin contact. Since MCC is more frequent in immunocompromised patients, a reactivation of MCPyV latently infecting target cells has been proposed. However, neither definite ways of transmission nor specific target organs have been identified with certainty. Ten autopsies with an extensive organ sampling for a total of 121 specimens (tissue and blood samples) were collected. All tissue specimens were fixed in formalin and embedded in paraffin. Real-time PCR was performed to quantify the copy number of the large T antigen (LT) gene and the capsid VP1 gene of MCPyV. MCPyV LT and/or VP genes were detected in all of the collected specimens. A high prevalence of MCPyV was found in the blood (six cases) and lung (five cases); the brain was positive in three cases. The highest viral copy number was detected in blood from two autopsies (21 610 570.09 copies per 105 cells and 380 413.25 copies per 105 cells), whereas the viral copy number in the other organs was low. Our data confirm the high frequency of MCPyV infection in the general population, which seems to indicate that the respiratory tract is a possible route for viral transmission and viral persistence in the brain. The frequent detection of MCPyV DNA in blood suggests that circulating leukocytes could be one of the reservoirs of MCPyV, whereas the high viral copy number also seems to indicate the possibility of viral reactivation in immunocompetent adults.

A new clinical cut-off of cytokeratin 19 mRNA copy number in sentinel lymph node better identifies patients eligible for axillary lymph node dissection in breast cancer.

AIMS: Cytokeratin 19 (CK19) mRNA copy number predicts the probability of tumour load in axillary lymph nodes (ALN) and can help in decision-making regarding the axillary dissection. The purpose of this study was to define a new cut-off of CK19 mRNA copy number using the one-step nucleic acid amplification (OSNA) assay on metastatic sentinel lymph nodes (SLN) in order to identify cases at risk of having one or more positive ALN. METHODS: 1296 SLN from 1080 patients were analysed with the OSNA assay. 194 patients with positive SLN underwent ALN dissection and the mean value of CK19 copy number (320 000) of their SLN was set as initial cut-off. Receiver operative characteristics curve identify a best cut-off of 7700 (sensitivity 78%, specificity 57%). A comparison between our and the traditional cut-off (5000) was performed. RESULTS: The cut-off of 7700 successfully identifies patients with positive ALN (p=0.001, false- negative cases: 17%). In the range between 5000 and 7700, one patient with positive ALN would not undergo axillary dissection, whereas eight patients with negative ALN would be correctly identified. CONCLUSIONS: We suggest that the level of CK19 mRNA copy number could be the only parameter to consider in the intraoperative management of the axilla.

Mutation analysis of KRAS in primary colorectal cancer and matched metastases by means of highly sensitivity molecular assay.

Mutation analysis of KRAS is needed before starting treatment with anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer (CRC). In most of the cases, testing is performed on primary tumors, assuming that KRAS mutation status does not change in metastasis although correlation studies gave conflicting results. We evaluated the KRAS status concordance rate between primary tumors and related metastasis using a highly sensitive molecular assay. Forty-five primary tumors and related metastases from patients with CRC (28/45 male-62.2% and 17/45 female-37.8%; mean age 66.4 years) were analyzed by using TheraScreen: KRAS mutational kit. Metastatic samples were collected from lymph nodes (8/45-17.8%) and visceral sites (37/45-82.2%); 23 were synchronous (49%) and 22 were metachronous (51%), obtained after a mean of 30.8 months after the first diagnosis of CRC. Twenty-eight patients had KRAS mutations in both primary CRC and related metastases (62.2%). No differences in type and frequency of mutations were identified, despite different metastatic sites and time of onset of metastatic disease. Our results indicate that the mutation status of KRAS is the same in primary CRC and metastasis, suggesting that in clinical practice, KRAS testing can be performed on both tumor tissues when using a highly sensitive molecular assay.

Epidermal growth factor receptor gene analysis with a highly sensitive molecular assay in routine cytologic specimens of lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) gene mutational analysis is critical for guiding the treatment of lung adenocarcinoma. In everyday clinical practice, EGFR testing is frequently centralized in referral laboratories that may receive paucicellular cytologic specimens, often fixed in various ways. We conducted a search for EGFR mutations in 108 cytologic samples of lung adenocarcinoma from different hospitals using the TheraScreen EGFR29 kit. These samples included 80 (74.1%) fine-needle aspirations, 13 (12%) pleural/ascitic fluids, 13 (12%) bronchial washings, and 2 bronchial brushings. The samples were fixed in ethanol (n = 79), Duboscq-Brasil (n = 18) or formalin (n = 10); 1 was unfixed. Ninety-two (85.2%) were amplified, 16 (14.8%) were not. Mutations were detected in 22 (23.9%) of 92 amplified samples, 9 containing less than 200 cancer cells, and 4 with less than 50% cancer cells. DNA was amplified in 12 of 18 Duboscq-Brasil-fixed samples. These findings indicate that cytologic specimens are adequate for EGFR testing when a highly sensitive assay is used, even if they are paucicellular or not optimally fixed.