Peter Jebsen

Four DNA methylation biomarkers in biliary brush samples accurately identify the presence of cholangiocarcinoma

Early detection of the highly aggressive malignancy cholangiocarcinoma (CCA) remains a challenge but has the potential to render the tumor curable by surgical removal. This study evaluates a biomarker panel for the diagnosis of CCA by DNA methylation analyses of biliary brush samples. The methylation status of 13 candidate genes (CDO1, CNRIP1, DCLK1, FBN1, INA, MAL, SEPT9, SFRP1, SNCA, SPG20, TMEFF2, VIM, and ZSCAN18) was investigated in 93 tissue samples (39 CCAs and 54 nonmalignant controls) using quantitative methylation‐specific polymerase chain reaction. The 13 genes were further analyzed in a test series of biliary brush samples (15 CCAs and 20 nonmalignant primary sclerosing cholangitis controls), and the methylation status of the four best performing markers was validated (34 CCAs and 34 primary sclerosing cholangitis controls). Receiver operating characteristic curve analyses were used to evaluate the performance of individual biomarkers and the combination of biomarkers. The 13 candidate genes displayed a methylation frequency of 26%‐82% in tissue samples. The four best‐performing genes (CDO1, CNRIP1, SEPT9, and VIM) displayed individual methylation frequencies of 45%‐77% in biliary brushes from CCA patients. Across the test and validation biliary brush series, this four‐gene biomarker panel achieved a sensitivity of 85% and a specificity of 98%, with an area under the receiver operating characteristic curve of 0.944. Conclusion: We report a straightforward biomarker assay with high sensitivity and specificity for CCA, outperforming standard brush cytology, and suggest that the biomarker panel, potentially in combination with cytological evaluation, may improve CCA detection, particularly among primary sclerosing cholangitis patients. (Hepatology 2015;61:1651–1659)

Recurrent respiratory papillomatosis: HPV genotypes and risk of high-grade laryngeal neoplasia.

Patients with recurrent respiratory papillomatosis (RRP) in Norway treated between 1987 and 2009 were recruited to this cohort study. They were followed from disease onset and data recorded until January 2012. Here, we describe the distribution of human papillomavirus (HPV) genotypes, the prevalence of multiple HPV infections, and the risk of high-grade laryngeal neoplasia and respiratory tract invasive carcinoma in a large cohort of patients with RRP. We also examined whether HPV genotype, gender, age or clinical course are risk factors for this development. Clinical records and histological specimens were reviewed. Using formalin-fixed paraffin-embedded biopsies, HPV genotyping were performed by quantitative polymerase chain reaction assays identifying 15 HPV types. HPV-negative specimens were analyzed by metagenomic sequencing. Paraffin blocks were available in 224/238 patients. The DNA quality was approved in 221/224 cases. HPV DNA was detected in 207/221 patients and all were HPV 6 or HPV 11 positive, comprising HPV 6 in 133/207, HPV 11 in 40/207 cases and HPV 6/11 in 15/207 cases. Co-infection with one or two high-risk HPV types together with HPV 6 or HPV 11 was present in 19/207 patients. Metagenomic sequencing of 14 HPV-negative specimens revealed HPV 8 in one case. In total, 39/221 patients developed high-grade laryngeal neoplasia. 8/221 patients developed carcinoma of the respiratory tract (six patients with laryngeal carcinoma and two patients with lung carcinoma). High-grade laryngeal neoplasias were found more frequently in HPV-negative versus HPV-positive patients, (RR = 2.35, 95% CI 1.1, 4.99), as well as respiratory tract carcinomas (RR = 48, 95% CI 10.72, 214.91). In summary, the majority of RRP were associated with HPV 6 and/or 11. HPV-negative RRP biopsies occurred more frequently in adult-onset patients, and were associated with an increased risk of laryngeal neoplasia and carcinoma in the respiratory tract.

Vaginal involvement in genital erosive lichen planus

A specialized Vulva Clinic with dedicated gynecologists and dermatologists was established in Oslo, Norway, in 2003. Fifty‐eight women referred to the clinic in 2003–2009 were diagnosed with genital erosive lichen planus. All patients filled out a questionnaire. Gynecological examination, including vaginal inspection, was performed, if necessary in general anesthesia. Median age at symptom start was 51 years (range 17–78 years) with 15 women (26%) being younger than 40 years old. Sexual abstinence was reported by 36 women and dyspareunia by another 10. On examination, vaginal involvement was seen in 49 women, including vaginal synechiae in 29 and total obliteration of the vagina in 9. Of 56 women treated with topical corticosteroids for at least three months, two had complete response and 36 partial responses. Similarly, of 22 women treated with tacrolimus, three had complete and six partial response. We conclude that vaginal involvement is more common in genital erosive lichen planus than previously reported.

Carcinoma of the urachus

A 63-year-old man presented with haematuria and suprapubic pain. After transurethral resection of the prostate a tumour was seen in the bladder dome at cystoscopy. Biopsy revealed an adenocarcinoma. Based on intraoperative and histological examination of the partial cystectomy specimen, the tumour was judged to be an urachus carcinoma. Four years after diagnosis the patient has no evidence of tumour. While partial cystectomy is the most frequent treatment, a radical total cystectomy is advocated in poorly differentiated tumours.

Chronic Lymphocytic Leukemia Cells Express CD38 in Response to Th1 Cell–Derived IFN-γ by a T-bet–Dependent Mechanism

Chronic lymphocytic leukemia (CLL) is a B cell malignancy associated with increased levels of inflammatory cytokines. Similarly, expression of CD38 on CLL cells correlates with CLL cell survival and proliferation, but the mechanisms that regulate CD38 expression and inflammatory cytokines remain unclear. We have recently demonstrated that patients have CLL-specific Th cells that support CLL proliferation. In this article, we show that CLL cells attract such Th cells, thereby establishing an Ag-dependent collaboration. Blocking experiments performed in vitro as wells as in vivo, using a xenograft model, revealed that secretion of IFN-γ was a major mechanism by which CLL-specific Th cells increased CD38 on CLL cells. The expression of the transcription factor T-bet in peripheral blood CLL cells significantly correlated with CD38 expression, and transient transfection of CLL cells with T-bet resulted in T-bethiCD38hi cells. Finally, chromatin immunoprecipitation experiments revealed that T-bet can bind to regulatory regions of the CD38 gene. These data suggest that CLL cells attract CLL-specific Th cells and initiate a positive feedback loop with upregulation of T-bet, CD38, and type 1 chemokines allowing further recruitment of Th cells and increased type 1 cytokine secretion. This insight provides a cellular and molecular mechanism that links the inflammatory signature observed in CLL pathogenesis with CD38 expression and aggressive disease and suggests that targeting the IFN-γ/IFN-γR/JAK/STAT/T-bet/CD38 pathway could play a role in the therapy of CLL.

Ploidy Analysis by In Situ Hybridization of Interphase Cell Nuclei in Fine-Needle Aspirates From Breast Carcinomas: Correlation With Cytologic Grading

Fine‐needle aspirates from 54 breast cancer patients were investigated for numeric aberrations in chromosomes 6, 7, 12, and 17 by in situ hybridization (ISH) of interphase cell nuclei. Ploidy findings were compared with cytologic grading of tumors. Aneuploidy was found in 73% of cases. Chromosomes 6 and 7 showed numeric abnormalities in 63% and 62% of cases, respectively, whereas chromosome 17 retained a disome pattern in 2/3 of the tumors. Thirteen cancers (28% of 47 with four analyzed probes) had a normal signal number in all four chromosomes. In 17 (36%), all four had signal gain. Another 17 showed a mixed disome/aneusome pattern. They presented a continuum of increasing numeric abnormalities, 82% disomy for chromosome 17, and 13 of them were grade 2, indicating intermediate biologic properties. Correlation between grading and ploidy was good, with 10 of 11 grade 1 carcinomas showing diploidy, whereas 33 of 36 grade 2 and 3 tumors had numeric aberrations. Diagn. Cytopathol. 1997; 17:267–271.

Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patients

Purpose:  Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells.

nm23 protein expression in fine‐needle aspirates from breast carcinoma

nm23 has been recognized as a potential suppressor gene of metastasis. Reduced nm23 expression in breast carcinoma has been found to correlate with axillary lymph node metastases, high grade tumors, and shorter survival.

A Clinical Study of Cervical Dysplasia in Long-Term Survivors of Allogeneic Stem Cell Transplantation

This retrospective study examined the prevalence of and risk factors for cervical dysplasia and genital human papillomavirus (HPV) infection in 89 female recipients of allogeneic stem cell transplantation (allo-SCT) between 1985 and 2005 who survived for more than 5 years after transplantation. All patients underwent regular gynecologic examination and cervical cytological testing. The incidence rates of cervical cytological abnormalities and HPV infection were calculated. Various clinical parameters were evaluated for association with cytological high-grade squamous intraepithelial lesion (HSIL) posttransplantation to identify risk factors for cervical dysplasia. Multivariate analysis with logistic regression was used to identify independent risk factors for cervical dysplasia after adjusting for confounding factors. Sixty-one of the 89 patients (68.5%) had cervical cytological abnormalities of varying grades, including atypical squamous cells of undetermined significance (ASC-US; 31.5%; 28 of 89), low-grade squamous intraepithelial lesion (LSIL; 10.1%; 9 of 89), and HSIL (27%; 24 of 89). HPV status was available for 43 patients, 12 of whom (27.9%) were HPV-positive. Among the 69 patients with normal cytological cervical smear findings pretransplantation, the incidence of cytological HSIL was 23.2% (16 of 69) posttransplantation. After adjusting for confounding factors, only unrelated HLA-matched donor and the presence of vulvovaginal chronic graft-versus-host disease (cGVHD) were independent risk factors for cervical cytology HSIL after transplantation, with the highest risk among patients with vulvovaginal cGVHD (adjusted odds ratio, 31.97). We conclude that long-term survivors of allogeneic stem cell transplantation are at high risk for cervical cytological abnormalities. Vulvovaginal cGVHD and unrelated HLA-matched donor were the only independent risk factors for cervical cytological HSIL in patients with normal cervical cytology before transplantation. Regular surveillance by gynecologic examination, including cervical cytological testing, in these patients allows for early diagnosis and effective management of cervical abnormality and decreases the burden of this potentially fatal, but treatable, condition.

Numerical aberrations of chromosome 17 in interphase cell nuclei of breast carcinoma cells: lack of correlation with abnormal expression of p53, neu and nm23 protein

The genes for p53, neu (c‐erbB‐2) and nm23 are all located on chromosome 17. Abnormal expression of their protein products is an important prognostic parameter. The aim of this study was to investigate if numerical aberrations of chromosome 17 are reflected in the expression of these markers. The immunohistochemical expression was analysed on histological specimens from 33 breast carcinomas. In situ hybridization (ISH) was performed on interphase cell nuclei in air‐dried fine‐needle aspirates from the same cases using a digoxigenin‐labelled α‐satellite probe for chromosome 17. ISH for chromosome 6, 7 and 12 was used additionally to give an estimate of ploidy. Of the carcinomas 76% were aneuploid, and numerical abnormalities of chromosome 17 were found in 34%. Abnormal p53 protein was expressed in 15% (five cases). All of these were aneuploid, but only one of them revealed aneusomy of chromosome 17. Neu overexpression was found in 18% of the tumours (six cases). Five of these were aneuploid, whereas two were aneusome for chromosome 17. Four cancers showed full (normal) expression of nm23 protein, whereas 29 had reduced expression. Reduced expression was found in 23 of 25 aneuploid tumours. Numerical aberrations of chromosome 17 were found equally in carcinomas with reduced and full nm23 protein expression. Abnormal numbers of chromosome 17 seem only to have a minor impact on these markers and are not reflected significantly in their expression.