Camilla Krakstad

Molecular Landscape of Endometrial Stromal Tumors

PURPOSE The molecular heterogeneity of endometrial stromal tumors (ESTs) is demonstrated by the presence of the same fusion gene in distinct pathologic entities, such as endometrial nodules and low-grade endometrial stromal sarcoma, both exhibiting the JAZ1::SUZ12 chimeric transcript. Given the limited knowledge on these tumors, which is based on a small number of cases studied with a restricted range of techniques, we analyzed 47 ESTs to explore their methylation and transcriptomic landscapes. MATERIALS AND METHODS Tumor methylation and transcriptomes profiles were investigated. RESULTS The methylation profile showed distinct clusters, which correlated with established histopathologic and molecular subtypes. The highest methylation value was reported for nuclear factor of activated T cytoplasmic 1, and the lowest was detected for miR34C. Two different 5'—C—phosphate—G—3' (CpG) sites of LMX1B ( LMX1B-cg04996334 and LMX1B ), along with miR34C , showed the same methylation pattern in both low-grade and high-grade endometrial stromal sarcoma (HG-ESS). Similarly, CFAP45 , HDAC4 , ACY3 , MOB3A , and XXYLT1 showed identical methylation patterns in HG-ESS and undifferentiated uterine sarcomas, highlighting the similarities between these tumors within the EST spectrum. We identified 13 novel fusion transcripts involving several genes that are active in transcriptional regulation. CONCLUSION In ESTs, the genes involved in chromosomal rearrangements function as transcription regulators, either directly through the formation of zinc finger motifs or indirectly through epigenetic regulation. The methylation signature is different for distinct subgroups of the EST spectrum, with more aggressive tumors, HG-ESS, and undifferentiated uterine sarcoma, clustering together. Some genes showed similar methylation levels in different entities, highlighting the presence of a continuum in the tumor profile. Methylation levels of CpG sites at specific gene loci may serve as valuable biomarkers for these tumors.

Nuclear upregulation of class I phosphoinositide 3-kinase p110β correlates with high 47S rRNA levels in cancer cells

ABSTRACT The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC. This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.

Multiplex single‐cell profiling of putative cancer stem cell markers ALDH1 , SOX9 , SOX2 , CD44 , CD133 and CD15 in endometrial cancer

The presence of cancer stem cells is linked to aggressive disease and higher risk of recurrence, and multiple markers have been proposed to detect cancer stem cells. However, a detailed evaluation of the expression patterns and the prognostic value of markers relevant for endometrial cancer is lacking. As organoid models are suggested to be enriched in cancer stem cells, such models may prove valuable to define tissue‐specific cancer stem cells. To address this, imaging mass cytometry and multiplex single‐cell analyses were performed on an endometrial cancer patient series including both tumor biopsies and corresponding patient‐derived organoids. An antibody panel focused on cancer stem cell markers was used to identify cancer stem cell phenotypes. Over 70% of epithelial cells in the tumor biopsies expressed at least one putative cancer stem cell marker. We identified distinct cancer cell phenotypes with heterogeneous expression within individual patients and between patient samples. Few differences in the distribution of cancer cell phenotypes were observed between tumor biopsies and corresponding organoids. Cells expressing aldehyde dehydrogenase 1 (ALDH1) were more prevalent in high‐grade tumors, while expression of CD44 was more prevalent in grade 1 tumors. Spatial analysis revealed significantly less interaction between ALDH1‐ and CD44‐expressing cells. Gene expression data was used to further investigate selected markers. CD44 gene expression was associated with a favorable prognosis and was further validated using immunohistochemistry. High expression of CD44 was significantly associated with better survival. The general high expression of proposed stem cell markers may indicate alternative roles for these in endometrial cancer.

Longitudinal effects of adjuvant chemotherapy and lymph node staging on patient-reported outcomes in endometrial cancer survivors: a prospective cohort study

Most patients with endometrial cancer with localized disease are effectively treated and survive for a long time. The primary treatment is hysterectomy, to which surgical staging procedures may be added to assess the need for adjuvant therapy. Longitudinal data on patient-reported outcomes comparing different levels of primary treatment are lacking, especially when adjuvant radiotherapy is omitted. We assessed the impact of lymphadenectomy and adjuvant chemotherapy on patient-reported symptoms, function, and quality of life. We hypothesized that these treatment modalities would substantially affect patient-reported outcomes at follow-up. We prospectively included patients with endometrial cancer enrolled in the ongoing MoMaTEC2 study (ClinicalTrials.gov Identifier: NCT02543710). Patients were asked to complete the patient-reported outcome questionnaires European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire EN24 preoperatively and at 1 and 2 years of follow-up. Functional domains and symptoms were analyzed for the whole cohort and by treatment received. To assess the effect of the individual treatment modifications, we used mixed regression models. Baseline data were available for 448 patients. Of these patients, 339 and 219 had reached 1-year follow-up and 2-year follow-up, respectively. Treatment included hysterectomy (plus bilateral salpingo-oophorectomy) alone (n=177), hysterectomy and lymph node staging without adjuvant therapy (n=133), or adjuvant chemotherapy irrespective of staging procedure (n=138). Overall, patients reported improved global health status and quality of life (+9 units; P<.001), increased emotional and social functioning, and increased sexual interest and activity (P<.001 for all) from baseline to year 1, and these outcomes remained stable at year 2. Means of functional scales and quality of life were similar to age- and sex-weighted reference cohorts. Mean tingling and numbness and lymphedema increased after treatment. The group who received adjuvant chemotherapy had a larger mean reduction in physical functioning (-6 vs +2; P=.002) at year 1, more neuropathy (+30 vs +5; P<.001; year 1) at years 1 and 2, and more lymphedema at year 1 (+11 vs +2; P=.007) than the group treated with hysterectomy and salpingo-oophorectomy only. In patients not receiving adjuvant chemotherapy, patient-reported outcomes were similar regardless of lymph node staging procedures. Adjuvant chemotherapy independently increased fatigue, lymphedema, and neuropathy in mixed regression models. Patients with endometrial cancer receiving adjuvant chemotherapy reported significantly reduced functioning and more symptoms up to 2 years after treatment. For patients treated by surgery alone, surgical staging did not seem to affect the quality of life or symptoms to a measurable degree at follow-up. Therefore, subjecting patients to lymph node removal to tailor adjuvant therapy seems justified from the patient's viewpoint; however, efforts should increase to find alternatives to traditional chemotherapy.

High-Grade Cervical Intraepithelial Neoplasia (CIN) Associates with Increased Proliferation and Attenuated Immune Signaling

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

ENDORISK-2: A personalized Bayesian network for preoperative risk stratification in endometrial cancer, integrating molecular classification and preoperative myometrial invasion assessment

ENDORISK is a Bayesian network that can assist in preoperative risk estimation of lymph node metastasis (LNM) risk in endometrial cancer (EC) with consistent performance in external validations. To reflect state of the art care, ENDORISK was optimized by integrating molecular classification and preoperative assessment of myometrial invasion (MI). Variables for POLE, MSI, and preoperative assessment of MI, either by expert transvaginal ultrasound or pelvic magnetic resonance imaging (MRI), were added to develop ENDORISK-2. The p53 biomarker, part of the molecular classification, was already included in ENDORISK. External validation of ENDORISK-2 for LNM prediction was performed in two independent cohorts from: Brno (CZ), (n = 581) and Tübingen (DE), (n = 247). ENDORISK-2 yielded AUCs of 0·85 (95 % CI 0·80-0·90) (CZ) and 0·86 (95 % CI 0·77-0·96) (DE) for predicting LNM. In patients with low-grade histology, 83 % (CZ) and 89 % (DE) were estimated having less than 10 % risk of LNM, with false negative rates (FNR) of 4·3 % (CZ) and 2·2 % (DE). The previously defined set of minimally required variables, i.e.: preoperative tumor grade, three of the four immunohistochemical (IHC) markers, and one clinical marker, could be interchanged with the new variables, with comparable validation metrics, including AUC values of 0·79-0·87 for LNM prediction. Incorporation of molecular data and preoperative MI improved the flexibility of ENDORISK with comparable diagnostic accuracy for estimating LNM as when based on low-cost immunohistochemical biomarkers. In addition, the high diagnostic accuracy in patients with low-grade EC demonstrates how ENDORISK-2 could aid clinicians in identifying patients in whom surgical lymph node assessment may safely be omitted. These results underline its power for clinical use in both high and low resource countries.

Biomarker Guided Treatment in Gynaecological Cancer

MoMaTEC2 aims to test, in clinically oriented studies, the applicability of already identified and promising molecular biomarkers, to promote individualisation of treatment for patients with endometrial cancer. Predominantly, but not exclusively, such biomarkers have shown to be interesting in retrospective analysis of our large prospectively collected MoMaTEC1 series. Part 1: Performance of a phase 4 implementation trial for optimised stratification of surgical treatment, specifically the performance of (para-aortic and pelvic) lymphadenectomy guided by validated biomarkers. Part 2: Performance of a phase 2b clinical biomarker study to evaluate the predictive potential of the biomarker stathmin for taxane treatment response in endometrial and ovarian cancer. In this study stathmin will be used as integrated biomarker.