Jaume Ordi

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.

The impact of placental malaria on gestational age and birth weight.

Maternal malaria is associated with reduced birth weight, which is thought to be effected through placental insufficiency, which leads to intrauterine growth retardation (IUGR). The impact of malaria on preterm delivery is unclear. The effects of placental malaria-related changes on birth weight and gestational age were studied in 1177 mothers (and their newborns) from Tanzania. Evidence of malaria infection was found in 75.5% of placental samples. Only massive mononuclear intervillous inflammatory infiltration (MMI) was associated with increased risk of low birth weight (odds ratio ¿OR, 4.0). Maternal parasitized red blood cells and perivillous fibrin deposition both were associated independently with increased risk of premature delivery (OR, 3.2; OR, 2.1, respectively). MMI is an important mechanism in the pathogenesis of IUGR in malaria-infected placentas. This study also shows that placental malaria causes prematurity even in high-transmission areas. The impact of maternal malaria on infant mortality may be greater than was thought previously.

Massive chronic intervillositis of the placenta associated with malaria infection

Massive chronic intervillositis (MCI) is an infrequently recognized placental lesion thought to be of immunologic origin that has been associated with poor fetal outcome. It is characterized by a prominent inflammatory infiltrate in the intervillous space, composed mainly of monocytes and macrophages that can simulate a maternal malignant disorder involving the placenta. The villi are characteristically spared. We report 74 cases of placental malarial infection with morphologic features of MCI. In all cases, the massive inflammatory infiltrate was limited to the intervillous space, which appeared largely obliterated. Increased fibrin deposition and prominent syncytial knots were frequent associated findings. Inflammatory cells were CD45 and CD68 positive, consistent with a monocyte-macrophage population. Some polymorphonuclear leukocytes and scattered T and B lymphocytes were also present. Villi were not inflamed. Malarial pigment was present in all cases, and parasitized maternal erythrocytes were evident in 73 of 74 patients. The histologic pattern of MCI was observed in 17.6% of placentas with malarial parasites. Malarial MCI affected predominantly primigravida women (77%) and was associated with a reduced birth weight, which in 39 (53%) of the infants was less than 2500 g, and a low gestational age. None of the infants with placentas with MCI died in the early neonatal period. Morphologic changes of MCI are seen in a significant percentage of placentas with malarial infection, especially in primigravida women, and are associated with a low birth weight. Malarial infection should therefore be considered in the differential diagnosis of massive intervillous infiltrates.

Placental pathology in malaria: A histological, immunohistochemical, and quantitative study

To characterize the histological changes in malarial placentas and their relationship with parity and maternal and cord parasitemias, we conducted a histological study on 1,179 placentas from Ifakara, Tanzania, an area with intense and perennial malaria transmission. Immunohistochemical and quantitative studies for CD45, fibrin, and villous area were performed in 60 cases. Four hundred fifteen placentas (35.2%) showed parasites (active infections); in 303 of them, parasites co-existed with pigment covered by fibrin (chronic infections), and in 112 only parasites were detected (acute infections). Four hundred seventy-five cases (40.3%) showed hemozoin deposition without parasites (past infections). Of women with parasitized placentas, 46.3% did not show parasites in the peripheral blood. Basal membrane thickening (P = .002), fibrinoid necrosis (P = .004), and prominence of syncytial knots (P = .031) were associated with active malarial infection. No quantitative differences for perivillous fibrin deposition or villous area were found. The most significant association with active malarial infection was intervillous infiltration by mononuclear inflammatory cells (P < .001). Chronic infections were associated with the most severe changes, particularly intervillous mononuclear inflammation (OR, 28.7; 95% CI = 16.0 to 51.5, P< .001). Past infections showed only minimal differences with noninfected placentas. Primiparas showed chronic infections more frequently than multiparas (52% v 15%, P < .001). They also showed significantly higher placental parasitemias and intervillous inflammatory infiltrate. In conclusion, placental histology is more sensitive than peripheral blood examination in detecting malarial infection during pregnancy. Most malarial infections recover during pregnancy, leaving few residual changes in the placenta. Intervillous inflammation is the most frequent finding associated with malaria and is especially severe in primiparas, suggesting that mechanisms other than immunosuppression are responsible for the high susceptibility in this group.

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BACKGROUND Human papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. METHODS Histologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16(INK4a) by immunohistochemistry (CINtec histology kit, ROCHE). An IVC was considered HPV driven if both HPV-DNA and p16(INK4a) overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). RESULTS Of 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16(INK4a) positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16(INK4a) positive (AP=69.5%, CI=63.6-74.8) versus KSCC (AP=11.5%, CI=9.7-13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). CONCLUSION Combined data from HPV-DNA and p16(INK4a) testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.

CD10 expression in epithelial tissues and tumors of the gynecologic tract: a useful marker in the diagnosis of mesonephric, trophoblastic, and clear cell tumors.

We tested 417 cases of formalin-fixed, paraffin-embedded normal or hyperplastic gynecologic tissues as well as neoplasms involving the gynecologic tract with a monoclonal antibody against CD10 (clone 56C6), with special emphasis on epithelial and epithelial-like structures and tumors. CD10 was always expressed in mesonephric remnants (mesonephric remnants of the uterine cervix, epoophoron, rete ovarii) and tumors (mesonephric adenocarcinoma of the uterine cervix, tumors of wolffian origin of the broad ligament and ovary). CD10 was also positive in the syncytiotrophoblast, cytotrophoblast, and intermediate trophoblast of normal gestations, partial and complete moles, choriocarcinoma, and placental site trophoblastic tumors. Finally, CD10 was positive in several metastatic neoplasms to the gynecologic tract (100% in metastatic renal clear cell and intestinal carcinomas and melanomas). In contrast, CD10 was almost invariably negative in müllerian epithelia of the female genital tract and in their corresponding tumors, with the exception of focal expression found in squamous epithelia and tumors with squamous differentiation. Thus, the expression of CD10 may be useful in the establishing the diagnosis of mesonephric and trophoblastic tumors and in the differential diagnosis between gynecologic clear cell carcinoma (always negative) and metastatic clear cell carcinoma of renal origin.

Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67 Dual-stained cytology.

OBJECTIVE Testing for human papillomavirus (HPV) has been shown to increase the sensitivity and negative predictive value for detection of high-grade cervical intraepithelial neoplasia (CIN2+), either when used in conjunction with Pap cytology testing or alone. However, there is no satisfying clinical management algorithm for women testing Pap negative/HPV positive. We therefore evaluated the clinical utility of a novel dual biomarker-based approach (p16/Ki-67 Dual-stained cytology) for the identification of CIN2+ in women with Pap negative/HPV positive screening results, without the need to refer all women to immediate colposcopy. METHODS All women aged ≥30 enrolled during 2007/2008 into a regional prospective Pap/HPV co-testing screening pilot project and tested Pap negative, but positive for HPV (n=425) were included in the analysis. p16/Ki-67 Dual-stained cytology was performed from residual cellular material available from the liquid-based cytology vial collected during the initial Pap/HPV co-testing screening visit. Results were correlated to the presence of CIN2+ confirmed during preliminary follow-up. RESULTS p16/Ki-67 Dual-stained cytology tested positive at baseline in 108 out of 425 (25.4%) Pap negative/HPV positive cases. Sensitivity of Dual-stain testing for the detection of biopsy-confirmed CIN2+ during preliminary follow-up within the group of Pap negative/HPV positive women was 91.9% for CIN2+ (34/37 cases), and 96.4% for CIN3+ (27/28 cases). Specificity was 82.1% for CIN2+ on biopsy, and 76.9% for CIN3+, respectively. CONCLUSIONS Triaging Pap negative/HPV positive screening test results with p16/Ki-67 Dual-stained cytology may identify women with a high probability of underlying CIN2+ and may efficiently complement HPV-based screening programs to prevent cervical cancer.

A Randomized Placebo-Controlled Trial of Intermittent Preventive Treatment in Pregnant Women in the Context of Insecticide Treated Nets Delivered through the Antenatal Clinic

Background Current recommendations to prevent malaria in African pregnant women rely on insecticide treated nets (ITNs) and intermittent preventive treatment (IPTp). However, there is no information on the safety and efficacy of their combined use. Methods 1030 pregnant Mozambican women of all gravidities received a long-lasting ITN during antenatal clinic (ANC) visits and, irrespective of HIV status, were enrolled in a randomised, double blind, placebo-controlled trial, to assess the safety and efficacy of 2-dose sulphadoxine-pyrimethamine (SP). The main outcome was the reduction in low birth weight. Findings Two-dose SP was safe and well tolerated, but was not associated with reductions in anaemia prevalence at delivery (RR, 0.92 [95% CI, 0.79–1.08]), low birth weight (RR, 0.99 [95% CI, 0.70–1.39]), or overall placental infection (p = 0.964). However, the SP group showed a 40% reduction (95% CI, 7.40–61.20]; p = 0.020) in the incidence of clinical malaria during pregnancy, and reductions in the prevalence of peripheral parasitaemia (7.10% vs 15.15%) (p<0.001), and of actively infected placentas (7.04% vs 13.60%) (p = 0.002). There was a reduction in severe anaemia at delivery of borderline statistical significance (p = 0.055). These effects were not modified by gravidity or HIV status. Reported ITNs use was more than 90% in both groups. Conclusions Two-dose SP was associated with a reduction in some indicators, but these were not translated to significant improvement in other maternal or birth outcomes. The use of ITNs during pregnancy may reduce the need to administer IPTp. ITNs should be part of the ANC package in sub-Saharan Africa. Trial Registration ClinicalTrials.gov NCT00209781

p16 overexpression identifies HPV-positive vulvar squamous cell carcinomas.

Two types of vulvar squamous cell carcinomas (VSCCs) are recognized according to their relationship to human papillomavirus (HPV). Basaloid or warty carcinomas are considered HPV-associated tumors, whereas differentiated keratinizing neoplasms are considered non-HPV–associated. Recently, immunohistochemical detection of p16 and p53 has been proposed to differentiate these 2 types of VSCCs. We conducted a histologic study with immunohistochemical evaluation of p16 and p53 and HPV detection and typing by polymerse chain reaction using 2 different sets of primers in 92 cases of VSCCs to evaluate the usefulness of immunohistochemistry in the classification of VSCCs and to describe the clinico-pathologic characteristics of both types of VSCCs. HPV was detected in 16/92 (17.4%) specimens, HPV16 being identified in 75% of positive cases. A significant number of discrepancies between histology and HPV detection were observed, with 37.5% of HPV-positive tumors being keratinizing and 9.2% of HPV-negative carcinomas showing basaloid or warty features. Diffuse positivity for p16 and p53 was observed in 100% and 6.2% of HPV-positive tumors and in 2.3% and 64.5% of HPV-negative neoplasms, respectively. The sensitivity and specificity of p16 immunostaining to detect HPV-associated carcinomas (100% and 98.7%, respectively) were better than those of histologic criteria (93.8% and 35.5%) and of p53 negative stain (62.5% and 93.4%). Vulvar intraepithelial neoplasia grade 3 of basaloid/warty type was identified in 53.8% HPV-positive tumors, including 3 keratinizing tumors. All these cases were p16 positive and p53 negative. Vulvar intraepithelial neoplasia grade 3 of differentiated type was observed in 45.6% of HPV-negative cases; 90.8% of them were positive for p53 but all were negative for p16. No differences in age, stage, or development of recurrence were observed between HPV-positive and negative tumors. In summary, the current morphologic criteria to discriminate HPV-positive and negative VSCCs have a significant overlap. Immunostaining for p16 is a reliable marker for HPV-positive VSSCs, which improves the results of histologic classification.

Immunohistochemical staining for p16 and p53 in premalignant and malignant epithelial lesions of the vulva.

Summary:Two distinct types of vulvar squamous cell carcinomas and their precursors, vulvar intraepithelial neoplasias (VIN), which differ in terms of clinical presentation and behavior, have been delineated. Human papillomavirus (HPV)-associated carcinomas are of basaloid or warty type, whereas tumors unrelated to HPV are usually keratinizing and differentiated. Thus, the major stratifying factor for vulvar carcinomas and VIN is their etiopathogenetic relationship with HPV. However, because of technical difficulties in confidently detecting HPV in tissues, this diagnosis is usually based on purely morphologic criteria, even though some overlap exists between these histologic types. Recently, the tumor suppressor protein p16 has been shown to be specifically overexpressed in HPV-related carcinomas and premalignant lesions of the uterine cervix, oral cavity, and anus, but the presence of p16 vulvar squamous lesions has not been examined. We have evaluated the immunohistochemical expression of p16 in a series of formalin-fixed, paraffin-embedded vulvar carcinomas and their putative precursors. p16 was strongly positive in all cases of basaloid/condylomatous VIN3 (30/30) and basaloid (7/7) and warty (3/3) carcinomas. In contrast, p16 was almost consistently negative in normal skin, squamous cell hyperplasia (0/20), lichen sclerosus (0/19), differentiated (simplex) VIN3 (0/11), verrucous carcinoma (0/2), and keratinizing squamous cell carcinoma (3/33, 9%). One of the keratinizing squamous cell carcinomas positive for p16 occurred in a 25-year-old woman and the other two were associated with small foci of basaloid VIN3 adjacent to the tumor, suggesting a probable relationship with HPV. p16 was positive in 6 of 10 of basal cell carcinomas. In conclusion, p16 immunostaining is a good discriminator between HPV-associated and HPV-unrelated vulvar carcinomas and VIN, although it cannot differentiate basaloid squamous and basal cell carcinoma.