Marjon A. de Boer

High human papillomavirus oncogene mRNA expression and not viral DNA load is associated with poor prognosis in cervical cancer patients

Purpose: Cervical cancer is now known to be caused by infection with an oncogenic type of the human papillomavirus (HPV). However, little is known about the continued role of HPV once cancer has been established. Here, we describe the quantitative relation between HPV DNA copy number and mRNA expression of the viral oncogenes (E6 and E7) and the prognostic value of both measures in cervical cancer patients. Experimental Design: We studied the number of viral DNA copies and the level of HPV E6/E7 mRNA expression in 75 HPV 16–positive or HPV 18–positive International Federation of Gynecology and Obstetrics stage Ib and IIa cervical cancer patients. Measurements were done with quantitative PCR. DNA copy number analysis was done on pure tumor cell samples enriched with flow sorting. mRNA expression data were compensated for the percentage of tumor cells included. Results: The number of viral DNA copies was not predictive of survival in cervical cancer patients. In contrast, high HPV E6/E7 mRNA expression was strongly related to an unfavorable prognosis (P = 0.006). In a multivariate Cox model for overall survival, including all known prognostic variables and stratified for HPV type, the level of E6/E7 mRNA expression was an independent prognostic indicator, second only to lymph node status. No correlation was observed between DNA copy number and the level of HPV E6/E7 mRNA expression, which reflects that not all DNA copies are equally transcriptionally active. Conclusions: Cervical cancer patients with high HPV E6/E7 oncogene mRNA expression have a worse survival independently from established prognostic factors.

Human papillomavirus type 18 variants: histopathology and E6/E7 polymorphisms in three countries.

In cervical cancer, human papillomavirus type 18 (HPV 18) and HPV 16 are predominantly related to adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs), respectively. Here, we studied whether the geographically distributed HPV intratypic variants are also associated with histologically different tumors. A total of 44 HPV 18‐positive and 91 HPV 16‐positive cervical carcinomas from Indonesian, Surinamese and Dutch patients were histologically classified using hematoxilin and eosin, periodic acid Schiff plus and Alcian Blue staining. Samples were sequenced and intratypic variants were classified into the known phylogenetic branches. The Asian Amerindian HPV 18 variant was observed in 56% of ADCs compared to 15% of SCCs (p < 0.006). The African HPV 18 variant was exclusively found in SCCs. By sequencing the HPV 18 E6 and E7 open reading frames, we found predicted amino acid changes only in 8 samples. Two amino acid changes were consistent throughout the African branch. In HPV 16‐positive tumors, we did not find a specific linkage between intratypic variants and histopathology. We conclude that HPV 18 intratypic variants are differentially associated with adenocarcinoma and squamous cell carcinoma of the cervix. The findings described here stress the biologic significance of intratypic HPV variants and might help explaining differences in the pathogenesis of cervical ADCs and SCCs.

Changing attitudes towards termination of pregnancy for trisomy 21 with non‐invasive prenatal trisomy testing: a population‐based study in Dutch pregnant women

The aim of providing testing for chromosomal conditions is enabling reproductive choice with respect to carrying to term, or terminating the pregnancy of a child with a serious disorder or disability. Except for a few countries such as Denmark and Hong Kong, the uptake of screening for fetal trisomy is relatively low, ranging from 25% (The Netherlands) to around 50% in many other Western countries. Reasons for refraining from screening include a number of perceived disadvantages of current screening programs, of which the risk of iatrogenic miscarriage associated with follow-up testing with invasive diagnostic procedures (0.5 to 1%) is an often reported one. At present, the vast majority of women confronted with a confirmed diagnosis of fetal trisomy request termination of pregnancy (TOP). In the Netherlands, 93% of women receiving the diagnosis fetal T21 terminate the pregnancy (according to the 2010 annual report on prenatal diagnosis), which is similar to published European data. A recently published systematic review presented evidence of decreasing termination rates in the USA (67%), which was speculated to be associated with progress in the medical management of Down syndrome children. Another study underlines women’s strong preference for tests with no risk of miscarriage demonstrating that consideration for safety of the fetus is paramount in decision making. With the newly developed non-invasive prenatal testing (NIPT) approach using cell-free fetal DNA obtained from maternal plasma, decision-making in prenatal screening is likely to change. Both the sensitivity and specificity of NIPT exceed 99%. However, ethical debates revolve around the issue of a possible consequence of this increased testing rate: ‘Will the world be without children with Down syndrome in a few years?’ There is also concern that increased testing with likely reduced numbers of live-born children with T21 may lead to a reduction in scientific progress, and funding, aiming for treatment of children with Down syndrome. We sought to evaluate whether and how the assumed increased rate of detection with the introduction of NIPT would influence the rate of TOP for affected pregnancies. This informationmay aid in the planning of new screening strategies. In two hospitals and nine community midwife practices, selfcomplete questionnaires were administered to pregnant women shortly after women received counseling for first trimester combined test (FCT) by their own midwife or doctor between 1 August 2011 and 31 December 2011. All women received information about prenatal screening for trisomies following the current guidelines. Questionnaires were given to all women, independent from their expressed interest in prenatal screening. All questionnaires were handled anonymously. The questionnaire addressed questions regarding prenatal screening in the current pregnancy and regarding NIPT if available. Background information about NIPT was included prior to questions to determine the attitude of women towards NIPT. Participating women were asked to indicate the likelihood that they would choose the option of terminating their pregnancy should their fetus be diagnosedwith Down syndrome based on a visual analog scale (VAS). The VAS is a graphic tool with a 100mm horizontal line with the left end marked as ‘very uncertain’ and the right endmarked as ‘very certain’. The subject is asked to mark the point that is corresponding most with their feeling about the subject questioned. The last part of the questionnaire included sociodemographic questions (age, educational level, religion, and income). The Dutch legislation does not require informed consent for a prospective study using questionnaires when results are treated anonymously. Data were analyzed using SPSS version 17. Completed questionnaires were received from 147 (43%) of the 340 women who were sent a questionnaire. In this group of responders, 79/147 (54%) opted for FCT in their current pregnancy; 82% (121/147) of the women answered they would elect to undergo NIPT if it were available. There were no

Trial by Dutch laboratories for evaluation of non-invasive prenatal testing. Part II—women's perspectives†

To evaluate preferences and decision‐making among high‐risk pregnant women offered a choice between Non‐Invasive Prenatal Testing (NIPT), invasive testing or no further testing.

Circulating human papillomavirus type 16 specific T-cells are associated with HLA Class I expression on tumor cells, but not related to the amount of viral oncogene transcripts

Human papillomavirus (HPV) is a necessary factor in the pathogenesis of cervical cancer. Circulating HPV‐specific T‐cells responding to the E6 and E7 HPV proteins can be detected only in half of cervical cancer patients. Potential explanations for the absence of this response are lack of sufficient amounts of antigen to activate the immune response or local immune escape mechanisms. We studied the relationship between HPV 16 E6/E7 oncogene mRNA expression, human leukocyte antigen (HLA) expression on tumor cells and the presence of circulating E6‐ and E7‐specific T‐cell responses in cervical cancer patients. The amount of antigen was assessed by HPV E6/E7 mRNA expression levels measured by quantitative polymerase chain reaction. HLA Class I and Class II expression on tumor cells was analyzed by immunohistochemistry. A proliferative HPV‐specific T‐cell response was detected in 15/29 patients. The amount of HPV E6/E7 mRNA was not related to the presence of immune response. HLA Class I expression was downregulated in 19 patients and completely lost in 7 patients. HLA Class II expression was upregulated in 18 patients. HLA Class I expression on tumor cells showed a strong correlation with immunity (p = 0.001). Explicitly, all patients with complete HLA loss lacked HPV specific T‐cell responses. The presence of circulating HPV‐specific T‐cells might reflect ongoing antitumor response that is sustained by CD8+ T‐cells killing HLA Class I positive cancer cells. We hypothesize that HLA Class I expression status on tumor cells might as well influence the response to HPV E6/E7 directed immunotherapy.

What do people want to know about NIPT? Content analysis of questions emailed to national NIPT information websites

Whats already known about this topic? Several countries, including the Netherlands, have implemented non‐invasive prenatal testing (NIPT) in their national prenatal screening program Access to relevant online health information is important as it increases informed decision makingWhat does this study add? Visitors of national NIPT websites mostly request more information about testing beyond the currently available NIPT: a broader range of disorders (such as monogenic disorders) and increasing target group eligibility (such as low‐risk or twin pregnancies)

Low-molecular-weight heparin and aspirin use in relation to pregnancy outcome in women with systemic lupus erythematosus and antiphospholipid syndrome: A cohort study

ABSTRACT Objective: To relate anticoagulant use to pregnancy complications in women with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS). Methods: All ongoing pregnancies, 184, in two Dutch tertiary centers between 2000 and 2015. Results: LMWH and aspirin was prescribed in 15/109 SLE women without antiphospholipid antibodies (aPL), 5/14 with aPL, 11/13 with APS, 45/48 with primary APS. Main complications in the four treatment groups (no anticoagulant treatment, aspirin, LMWH, aspirin and LMWH) included hypertensive disorders of pregnancy (9.4%, 23.3%, 50%, 18.4%, respectively, p = 0.12) and preterm birth (16.7%, 34.3%, 75%, 36.8%, respectively, p < 0.001). Conclusion: Maternal and perinatal complications occurred frequently, despite LMWH and aspirin use.

Post-pregnancy aspirin resistance appears not to be related with recurrent hypertensive disorders of pregnancy

OBJECTIVE The FRUIT-RCT concluded that low-molecular-weight heparin added to aspirin compared to treatment with aspirin alone is beneficial in the prevention of early-onset hypertensive disorders of pregnancy (HD) in women with inheritable thrombophilia and prior HD and/or a small-for-gestational age (SGA) infant leading to delivery before 34 weeks gestation. The aim of this study is to answer the question whether aspirin resistance is associated with recurrent HD. STUDY DESIGN Women with and without recurrent HD matched for age, study arm, and chronic hypertension were invited for this follow-up study 6-16 years after they participated in the FRUIT-RCT. Aspirin resistance was tested after 10days of aspirin intake using three complementary tests: PFA-200, VerifyNow® and serum thromboxane B2 (TXB2). An independent t-test, Mann-Whitney U test, Fishers Exact test and Chi2 test were used for the statistical analyses. RESULTS Thirteen of 24 women with recurrent HD and 16 of 24 women without recurrent HD participated. The prevalence of laboratory aspirin resistance was 34.5% according to the PFA-200, 3.4% according to the VerifyNow® and 24.1% according to TXB2. The prevalence of aspirin resistance by any test was 51.7%. Aspirin resistance per individual test did not differ between women with and without recurrent HD. Aspirin resistance measured by any test occurred more frequently in women without recurrent HD (p<0.01), irrespective of low-molecular-weight heparin. CONCLUSIONS No relation could be demonstrated between recurrent HD and aspirin resistance per test, measured up to 16 years after pregnancy. On the contrary, complementary aspirin resistance measurements were encountered more frequently in women without recurrent HD.

Aspirin adherence during high-risk pregnancies, a questionnaire study

OBJECTIVE Aspirin reduces the risk of recurrent hypertensive disorders of pregnancy (HD) and fetal growth restriction (FGR). This study examined the non-adherence rates of aspirin in women with high-risk pregnancies. STUDY DESIGN All consecutive women between 24 and 36weeks gestation with an indication for aspirin use during pregnancy were invited for this study. A survey was used which included two validated questionnaires, the simplified medication adherence questionnaire (SMAQ) and the Beliefs and Behaviour Questionnaire (BBQ). MAIN OUTCOME MEASURES To determine the non-adherence rates of aspirin, and to identify the beliefs and behavior concerning aspirin. RESULTS Indications for aspirin use during pregnancy were previous HD, FGR, intrauterine fetal death or current maternal disease. Non-adherence rates according to the SMAQ and BBQ were 46.3% and 21.4% respectively. No differences in demographic background or obstetrical characteristics between adherent and non-adherent women could be demonstrated. CONCLUSIONS Adherence for aspirin in this high-risk population cannot be taken for granted. The non-adherence rates in pregnant women are comparable with the non-adherence rates for aspirin in the non-pregnant population.

Safety of Tioguanine During Pregnancy in Inflammatory Bowel Disease

BACKGROUND AND AIMS Conventional thiopurine [azathioprine and mercaptopurine] treatment during pregnancy in patients with inflammatory bowel disease [IBD] is considered to be safe; however data on the safety and teratogenicity of the non-conventional thiopurine tioguanine [TG] in pregnant IBD patients are lacking. We aim to describe the safety and teratogenicity of TG treatment during pregnancy in IBD patients. METHODS This was a retrospective, multicentre descriptive case series of female IBD patients using TG during pregnancy. Data on disease and medication history, pregnancy complications, pregnancy outcome, mode of delivery, preterm birth, birthweight, congenital abnormalities, laboratory signs of myelosuppression or hepatotoxicity, and 6-thioguaninenucleotide [6-TGN] concentrations in mother and neonate were collected. RESULTS In all, 13 patients [77% Crohns disease, 23% ulcerative colitis] used TG [median dose 18 g/day] during pregnancy; 19 pregnancies, including 1 twin pregnancy, were included. Spontaneous abortion occurred in three pregnancies. In 7 of the 16 ongoing pregnancies a caesarean section was performed. One neonate had a mild congenital abnormality [distal shaft hypospadias]. In the singleton pregnancies, the median birthweight was 3410 g at a median of gestational age of 39 weeks. No preterm birth [< 37 weeks] or low birthweight [< 2500 g] was observed in the singleton newborns. In the twin pregnancy an induction of labour was performed at 35 + 1 weeks of gestation because of pre-eclampsia. Both neonates had a low birthweight. CONCLUSIONS This relatively small case series supports safe use of TG in pregnant IBD patients. Still, consideration should be given to the indication and continuation of TG during pregnancy.