Suzette Sørensen

Generation of a Predictive Melphalan Resistance Index by Drug Screen of B-Cell Cancer Cell Lines

Background Recent reports indicate that in vitro drug screens combined with gene expression profiles (GEP) of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM) a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information. Materials and Methods Microarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA), sparse partial least squares (SPLS) and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis. Principal Findings Both cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value. Conclusion The present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to better understand the mechanism underlying the preparedness to melphalan resistance.

Human B-cell cancer cell lines as a preclinical model for studies of drug effect in diffuse large B-cell lymphoma and multiple myeloma

Drug resistance in cancer refers to recurrent or primary refractory disease following drug therapy. At the cellular level, it is a consequence of molecular functions that ultimately enable the cell to resist cell death-one of the classical hallmarks of cancer. Thus, drug resistance is a fundamental aspect of the cancer cell phenotype, in parallel with sustained proliferation, immortality, angiogenesis, invasion, and metastasis. Here we present a preclinical model of human B-cell cancer cell lines used to identify genes involved in specific drug resistance. This process includes a standardized technical setup for specific drug screening, analysis of global gene expression, and the statistical considerations required to develop resistance gene signatures. The state of the art is illustrated by the first-step classical drug screen (including the CD20 antibody rituximab, the DNA intercalating topoisomerase II inhibitor doxorubicin, the mitotic inhibitor vincristine, and the alkylating agents cyclophosphamide and melphalan) along with the generation of gene lists predicting the chemotherapeutic outcome as validated retrospectively in clinical trial datasets. This B-cell lineage-specific preclinical model will allow us to initiate a range of laboratory studies, with focus on specific gene functions involved in molecular resistance mechanisms.

Human Papillomavirus Infection as a Possible Cause of Spontaneous Abortion and Spontaneous Preterm Delivery

Based on the current literature, we aimed to provide an overview on Human Papillomavirus prevalence in normal pregnancies and pregnancies with adverse outcome. We conducted a systematic literature search in PubMed and Embase. Data extracted from the articles and used for analysis included HPV prevalence, pregnancy outcome, geographical location, investigated tissue types, and HPV detection methods. The overall HPV prevalence in normal full-term pregnancies was found to be 17.5% (95% CI; 17.3–17.7) for cervix, 8.3% (95% CI; 7.6–9.1) for placental tissue, 5.7% (95% CI; 5.1–6.3) for amniotic fluid, and 10.9% (95% CI; 10.1–11.7) for umbilical cord blood. Summary estimates for HPV prevalence of spontaneous abortions and spontaneous preterm deliveries, in cervix (spontaneous abortions: 24.5%, and preterm deliveries: 47%, resp.) and placenta (spontaneous abortions: 24.9%, and preterm deliveries: 50%, resp.), were identified to be higher compared to normal full-term pregnancies (P < 0.05 and P < 0.0001). Great variation in HPV prevalence was observed between study populations of different geographical locations. This review demonstrates an association between spontaneous abortion, spontaneous preterm delivery, and the presence of HPV in both the cervix and the placenta. However, a reliable conclusion is difficult to draw due to the limited number of studies conducted on material from pregnancies with adverse outcome and the risk of residual confounding.

Clinical Impact of Leukemic Blast Heterogeneity at Diagnosis in Cytogenetic Intermediate-Risk Acute Myeloid Leukemia

Individual cellular heterogeneity within the acute myeloid leukemia (AML) bone marrow samples can be observed by multi parametric flow cytometry analysis (MFC) indicating that immunophenotypic screening for leukemic blast subsets may have prognostic impact.

HPV16 E6 and E7 Upregulate Interferon-Induced Antiviral Response Genes ISG15 and IFIT1 in Human Trophoblast Cells

Human papillomavirus (HPV) is suggested to infect trophoblasts in the placenta, and HPV infections are reported to be more prevalent in pregnancies with adverse outcomes. Results are however controversial, and studies investigating the molecular consequences of placental HPV infections are lacking. We studied HPV DNA localization in the placenta in cases of spontaneous abortion/spontaneous preterm delivery as well as in elective abortion/normal full-term delivery. Using in vitro assays, we investigated downstream effects of HPV16 E6 and E7 expression in trophoblast cells at the gene expression level in order to gain increased biological insight into the interaction between HPV and the cellular host. Fluorescent in situ hybridization (FISH), combined with fluorescent immunohistochemistry (FIHC) to target the trophoblast marker CK7 clearly showed, that HPV DNA resides within syncytiotrophoblast cells in the placenta. In vitro HPV16 E6 and E7-transfected trophoblasts were analyzed by RNA sequencing, and results were validated by reverse transcription real time polymerase chain reaction (RT-qPCR) for selected genes in cell lines, as well as in patient material. We show that HPV16 E6 and E7 upregulate interferon-induced antiviral response genes ISG15 and IFIT1 in a human trophoblast cell line two-days post-transfection. This is a response that is not observed when assessing the gene expression levels of the same genes in HPV16-positive placenta samples. Investigations on viral activity find that HPV16 E6 and E7 are not transcribed in patients, possibly suggesting that HPV16 syncytiotrophoblast infection may be latent. We conclude that HPV localizes to syncytiotrophoblast cells of the placenta, and that active expression of HPV16 E6 and E7 induce an immediate interferon-induced antiviral response in trophoblast cells, which is not present in HPV-positive placenta samples, suggesting latent infection.

Human papillomavirus infects placental trophoblast and Hofbauer cells, but appears not to play a causal role in miscarriage and preterm labor

Recently, an association between human papillomavirus infection and both spontaneous abortion and spontaneous preterm delivery was suggested. However, the reported human papillomavirus prevalence in pregnant women varies considerably and reliable conclusions are difficult. We aimed to investigate human papillomavirus infection in placental tissue of a Danish study cohort. Furthermore, we studied the cellular localization of human papillomavirus.

OP23.06: Effects of method of conception and gestational age on first trimester biochemical markers

Objectives: First-trimester levels of PAPP-A and free β-hCG are decreased in pregnancies conceived as a result of assisted reproduction, especially in-vitro fertilization (IVF). The aim of this study was to evaluate if sub-fertility, measured as increased timeto-pregnancy (TTP) in spontaneously conceived pregnancies, affects the levels of PAPP-A and free β-hCG. Methods: We included 13,935 singleton pregnant women, who attended the prenatal screening program between January 2005 and December 2010 and gave birth at Aarhus University Hospital, Denmark. TTP was evaluated through a questionnaire completed by the pregnant women in the first trimester. PAPP-A and free β-hCG were measured between gestational week 8+0 and 13+6. IVF pregnancies (n = 962) were used for comparison, and we excluded unintended pregnancies (n = 2.797) and pregnancies with no information about TTP (n = 460), which left 9,507 spontaneous pregnancies with information about TTP. Results: The median PAPP-A MoM was significantly lower in pregnant women with a TTP ≥24 months compared with the reference group with a TTP < 6 months (0.96 MoM vs. 1.06 MoM, P = 0.003). The levels were not as low as in IVF pregnancies (0.89 MoM). The median level of free β-hCG was also significantly lower in pregnancies with a TTP ≥24 months compared with pregnancies with a TTP < 6 months (1.04 MoM vs. 1.12 MoM, P = 0.03). TTP at 6–11 months and TTP at 12–23 months did not significantly affect levels of PAPP-A or free β-hCG. Conclusions: Increased time-to-pregnancy in spontaneously conceived pregnancies is associated with decreased levels of PAPP-A and free β-hCG measured in the first trimester, suggesting that some of the well-known decrease in IVF-pregnancies could be related to sub-fertility instead of the infertility treatment per se. OP23.06 Effects of method of conception and gestational age on first trimester biochemical markers