Maria Tzardi

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study

BACKGROUND Knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. We aimed to provide novel and comprehensive data about the worldwide genotype distribution in patients with invasive cervical cancer. METHODS Paraffin-embedded samples of histologically confirmed cases of invasive cervical cancer were collected from 38 countries in Europe, North America, central South America, Africa, Asia, and Oceania. Inclusion criteria were a pathological confirmation of a primary invasive cervical cancer of epithelial origin in the tissue sample selected for analysis of HPV DNA, and information about the year of diagnosis. HPV detection was done by use of PCR with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay. Sequence analysis was done to characterise HPV-positive samples with unknown HPV types. Data analyses included algorithms of multiple infections to estimate type-specific relative contributions. FINDINGS 22,661 paraffin-embedded samples were obtained from 14,249 women. 10,575 cases of invasive cervical cancer were included in the study, and 8977 (85%) of these were positive for HPV DNA. The most common HPV types were 16, 18, 31, 33, 35, 45, 52, and 58 with a combined worldwide relative contribution of 8196 of 8977 (91%, 95% CI 90-92). HPV types 16 and 18 were detected in 6357 of 8977 of cases (71%, 70-72) of invasive cervical cancer. HPV types 16, 18, and 45 were detected in 443 of 470 cases (94%, 92-96) of cervical adenocarcinomas. Unknown HPV types that were identified with sequence analysis were 26, 30, 61, 67, 69, 82, and 91 in 103 (1%) of 8977 cases of invasive cervical cancer. Women with invasive cervical cancers related to HPV types 16, 18, or 45 presented at a younger mean age than did those with other HPV types (50·0 years [49·6-50·4], 48·2 years [47·3-49·2], 46·8 years [46·6-48·1], and 55·5 years [54·9-56·1], respectively). INTERPRETATION To our knowledge, this study is the largest assessment of HPV genotypes to date. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 should be given priority when the cross-protective effects of current vaccines are assessed, and for formulation of recommendations for the use of second-generation polyvalent HPV vaccines. Our results also suggest that type-specific high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45.

Impact of KRAS, BRAF, PIK3CA Mutations, PTEN, AREG, EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients

Background To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC) patients treated with cetuximab containing salvage chemotherapy. Methods Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded. Results KRAS, BRAF and PIK3CA mutations were present in 37 (33%), 8 (7.2%) and 11 (9.8%) cases, respectively, PTEN was lost in 21 (19.8%) cases, AREG and EREG were overexpressed in 48 (45%) and 51 (49%) cases. In the whole study population, time to tumor progression (TTP) and overall survival (OS) was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively) or BRAF (p = 0.001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively) or EREG (p = 0.002 and p = 0.004, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively) or EREG (p = 0.0001 and p<0.0001, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). TTP was significantly lower in patients with PIK3CA mutations (p = 0.01) or lost PTEN (p = 0.002). Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001), BRAF mutation (HR: 5.1, p<0.0001), EREG low expression (HR: 1.6, p = 0.021) and absence of severe/moderate skin rash (HR: 4.0, p<0.0001) as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01), BRAF mutation (HR: 3.0, p = 0.001), EREG low expression (HR: 1.7, p = 0.021), absecence of severe/moderate skin rash (HR: 3.7, p<0.0001) and the presence of undifferantited tumours (HR: 2.2, p = 0.001) were revealed as independent prognostic factors for decreased OS. Conclusions These results underscore that KRAS-BRAF mutations and EREG expression can be used as biomarkers to further select patients undergoing anti-EGFR treatment.

Genetic, immunologic, and immunohistochemical analysis of the programmed death 1/programmed death ligand 1 pathway in human systemic lupus erythematosus

OBJECTIVE A putative regulatory intronic polymorphism (PD1.3) in the programmed death 1 (PD-1) gene, a negative regulator of T cells involved in peripheral tolerance, is associated with increased risk for systemic lupus erythematosus (SLE). We undertook this study to determine the expression and function of PD-1 in SLE patients. METHODS We genotyped 289 SLE patients and 256 matched healthy controls for PD1.3 by polymerase chain reaction-restriction fragment length polymorphism analysis. Expression of PD-1 and its ligand, PDL-1, was determined in peripheral blood lymphocytes and in renal biopsy samples by flow cytometry and immunohistochemistry. A crosslinker of PD-1 was used to assess its effects on anti-CD3/anti-CD28-induced T cell proliferation and cytokine production. RESULTS SLE patients had an increased frequency of the PD1.3 polymorphism (30.1%, versus 18.4% in controls; P=0.006), with the risk A allele conferring decreased transcriptional activity in transfected Jurkat cells. Patients homozygous for PD1.3-but not patients heterozygous for PD1.3-had reduced basal and induced PD-1 expression on activated CD4+ T cells. In autologous mixed lymphocyte reactions (AMLRs), SLE patients had defective PD-1 induction on activated CD4+ cells; abnormalities were more pronounced among homozygotes. PD-1 was detected within the glomeruli and renal tubules of lupus nephritis patients, while PDL-1 was expressed by the renal tubules of both patients and controls. PD-1 crosslinking suppressed proliferation and cytokine production in both normal and lupus T cells; addition of serum from patients with active SLE significantly ameliorated this effect on proliferation. CONCLUSION SLE patients display aberrant expression and function of PD-1 attributed to both direct and indirect effects. The expression of PD-1/PDL-1 in renal tissue and during AMLRs suggests an important role in regulating peripheral T cell tolerance.

The programmed death 1/programmed death ligand 1 inhibitory pathway is up‐regulated in rheumatoid synovium and regulates peripheral T cell responses in human and murine arthritis

OBJECTIVE T cells play a major role in the pathogenesis of rheumatoid arthritis (RA). The programmed death 1 (PD-1)/programmed death ligand 1 (PDL-1) pathway is involved in peripheral tolerance through inhibition of T cells at the level of synovial tissue. The aim of this study was to examine the role of PD-1/PDL-1 in the regulation of human and murine RA. METHODS In synovial tissue and synovial fluid (SF) mononuclear cells from patients with RA, expression of PD-1/PDL-1 was examined by immunohistochemistry and flow cytometry, while PD-1 function was assessed in RA peripheral blood (PB) T cells after stimulation of the cells with anti-CD3 and PDL-1.Fc to crosslink PD-1. Collagen-induced arthritis (CIA) was induced in PD-1(-/-) C57BL/6 mice, and recombinant PDL-1.Fc was injected intraperitoneally to activate PD-1 in vivo. RESULTS RA synovium and RA SF were enriched with PD-1+ T cells (mean +/- SEM 24 +/- 5% versus 4 +/- 1% in osteoarthritis samples; P = 0.003) and enriched with PDL-1+ monocyte/macrophages. PD-1 crosslinking inhibited both T cell proliferation and production of interferon-gamma (IFNgamma) in RA patients; PB T cells incubated with RA SF, as well as SF T cells from patients with active RA, exhibited reduced PD-1-mediated inhibition of T cell proliferation at suboptimal, but not optimal, concentrations of PDL-1.Fc. PD-1(-/-) mice demonstrated increased incidence of CIA (73% versus 36% in wild-type mice; P < 0.05) and greater severity of CIA (mean maximum arthritis score 5.0 versus 2.3 in wild-type mice; P = 0.040), and this was associated with enhanced T cell proliferation and increased production of cytokines (IFNgamma and interleukin-17) in response to type II collagen. PDL-1.Fc treatment ameliorated the severity of CIA and reduced T cell responses. CONCLUSION The negative costimulatory PD-1/PDL-1 pathway regulates peripheral T cell responses in both human and murine RA. PD-1/PDL-1 in rheumatoid synovium may represent an additional target for immunomodulatory therapy in RA.

Glomerular expression of toll-like receptor-9 in lupus nephritis but not in normal kidneys: implications for the amplification of the inflammatory response

Toll-like receptors recognising self-derived nucleic acids may participate in the pathogenesis of autoimmune diseases. Following the description of an enhanced population of toll-like receptor-9 (TLR-9) expressing auto-antibody producing B lymphocytes in active lupus, we explored the expression of TLR-9 in the renal tissue of patients with lupus. TLR-9 expression was studied in the kidneys of 12 lupus and 10 control samples from macroscopically unaffected areas of patients with renal adenocarcinoma by immunohistochemistry. A semiquantitative score was assigned separately for tubular, interstitial and glomerular expression. TLR-9 was expressed in the renal tubules and interstitial tissue in both patients with lupus and controls. Six of 12 patients with lupus with proliferative or membranous nephritis – as compared to none of the controls – exhibited both tubulointerstitial and glomerular TLR-9 expression. Biopsies with glomerular TLR-9 expression had a higher activity index (mean ± SD, 6.3 ± 3.5 in the presence of TLR-9 glomerular expression as compared to 1.3 ± 1.8 in its absence, P = 0.015, t-test). This study documents for the first time the up-regulation of TLR-9 within the glomerulus of patients with lupus nephritis. Activation of TLR-9 expressing glomerular cells by endogenous nucleic acids (nucleosomes) may amplify the inflammatory response.

Lymph Node Clearance after Total Mesorectal Excision for Rectal Cancer: Laparoscopic versus Open Approach

Background: Laparoscopic resection of the rectum is still under scrutiny for its adequacy of oncological clearance. Aim: To assess lymph node yield after laparoscopic total mesorectal excision (TME) for rectal cancer as compared to the open approach. Methods: 74 patients with middle and low rectal cancer were prospectively randomized in two groups. Group A included 39 patients who had an open TME (35 with low anterior resection of the rectum (LARR) and 4 with abdominoperineal resection of the rectum (APR)). In group B, there were 34 patients who had a laparoscopic TME (27 with LARR and 7 with APR). 10 of the LARR patients in group A and 14 of the LARR patients in group B had a defunctioning ileostomy. All operations were performed by one surgeon or under his supervision. Results: Gender and age distribution were similar for both groups (group A: 23 males; mean age 69 (41–85); group B: 20 males; mean age 72 (31–84)). The mean distance of the tumor from the dentate line was 7.6 cm (1–12 cm) for group A and 6.1 cm (1–12 cm) for group B. Anastomosis was formed at a mean distance of 5.5 cm (1.5–8.5 cm) from the dentate line in group A and 3.5 cm (1–4.5 cm) in group B. At histology, in group A there were 5 T4 tumors, 9 T3, 10 T3+ (<1 mm distance from the circumferential resection margin), 13 T2 and 2 T1. In group B, there were 3 T4 tumors, 14 T3, 8 T3+, 7 T2 and 2 T1. Differences between groups were not significant.The mean number of lymph nodes retrieved in group A specimens was 19.2 (5–45) and in group B 19.2 (8–41) (p = 0.2). In group A, 3.9 (1–9) regional, 13.9 (3–34) intermediate and 1.5 (1–3) apical lymph nodes were retrieved. The respective values in group B were 3.7 (3–7), 14.4 (4–33) and 1.3 (1–3). Differences between groups were not significant. Also, the incidence of lymph node involvement by the tumor was not significantly different between groups (group A: 23; group B: 19). Conclusions: Laparoscopic resection of the rectum can achieve similar lymph node clearance to the open approach. Also, distribution of the lymph nodes along the resected specimens is similar between the two approaches.

A case of sigmoid endometriosis difficult to differentiate from colon cancer

BackgroundAlthough endometriosis with sigmoid serosal involvement is not uncommon in women of childbearing age, the mucosal involvement is rare and differential diagnosis from colon cancer may be difficult due to the lack of pathognomonic symptoms and the poor diagnostic yield of colonoscopy and colonic biopsies.Case presentationWe present a case of a young woman with sigmoid endometriosis, in which the initial diagnostic workup suggested colon cancer. Histologic evidence, obtained from a second colonoscopy, along with pelvic ultrasound findings led to the final diagnosis of intestinal endometriosis which was confirmed by laparoscopy.ConclusionColonic endometriosis is often a diagnostic challenge and should be considered in young women with symptoms from the lower gastrointestinal tract.

Colonic tuberculosis mimicking Crohn's disease: case report.

BackgroundIntestinal tuberculosis is a rare disease in western countries, affecting mainly immigrants and immunocompromised patients. Intestinal tuberculosis is a diagnostic challenge, especially when active pulmonary infection is absent. It may mimic many other abdominal diseases.Case presentationHere, we report a case of isolated colonic tuberculosis where the initial diagnostic workup was suggestive of Crohns disease. Computed tomography findings however, raised the possibility of colonic tuberculosis and the detection of acid-fast bacilli in biopsy specimens confirmed the diagnosis.ConclusionsIn conclusion, this case highlights the need for awareness of intestinal tuberculosis in the differential diagnosis of chronic intestinal disease

Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer

Metastasis is the main cause of mortality in patients with colorectal cancer. However, most of the targeted therapies and predictive molecular biomarkers were developed based mainly on primary tumors. The current study was conducted to determine the degree of discordance between potential predictive and/or prognostic molecular markers in primary colorectal tumors and corresponding metastases, as this could have an impact on the efficacy of targeted therapies in the advanced colorectal cancer. KRAS, PIK3CA and BRAF mutations were determined by Sanger sequencing and mutant-enriched polymerase chain reaction (PCR) assays in 83 paired samples, MET gene copy number by quantitative PCR in 59, MET expression by immunohistochemistry in 73 and nuclear and cytoplasmic expression of PTEN by immunohistochemistry in 78 and 71 pairs, respectively. A certain degree of discordance between primary tumors and corresponding metastases was demonstrated for all examined biomarkers except BRAF mutations. PIK3CA exon 9 mutations in primary tumors and loss of PTEN nuclear expression in metastases correlated with KRAS mutations. KRAS wild-type status in primary tumors was associated with loss of PTEN cytoplasmic expression and high gene copy number of MET. Survival and clinical data were available for 68 patients. The multiple regression analysis revealed that the right-sided tumor localization and overexpression of MET were associated with shorter overall survival.

Cancer Microenvironment and Inflammation: Role of Hyaluronan

The role of inflammation in the development of cancer was described as early as the nineteenth century. Abundant evidence supports the preposition that various cancers are triggered by infection and chronic inflammatory disease whereas, evading immune destruction has been proposed as one of the new “hallmarks of cancer.” Changes of the tumor microenvironment have been closely correlated to cancer-mediated inflammation. Hyaluronan (HA), an important extracellular matrices component, has become recognized as an active participant in inflammatory, angiogenic, fibrotic, and cancer promoting processes. This review discusses how HA and specific HA-binding proteins participate in and regulate cancer-related inflammatory processes.