Robust and efficient estimation of nonparametric generalized linear models.

Generalized linear models are flexible tools for the analysis of diverse datasets, but the classical formulation requires that the parametric component is correctly specified and the data contain no atypical observations. To address these shortcomings, we introduce and study a family of nonparametric full-rank and lower-rank spline estimators that result from the minimization of a penalized density power divergence. The proposed class of estimators is easily implementable, offers high protection against outlying observations and can be tuned for arbitrarily high efficiency in the case of clean data. We show that under weak assumptions, these estimators converge at a fast rate and illustrate their highly competitive performance on a simulation study and two real-data examples. Supplementary Information: The online version contains supplementary material available at 10.1007/s11749-023-00866-x.

RNA biomarkers from proximal liquid biopsy for diagnosis of ovarian cancer.

BACKGROUND: Most ovarian cancer patients are diagnosed at an advanced stage and have a high mortality rate. Current screening strategies fail to improve prognosis because markers that are sensitive for early stage disease are lacking. This medical need justifies the search for novel approaches using utero-tubal lavage as a proximal liquid biopsy. METHODS: In this study, we explore the extracellular transcriptome of utero-tubal lavage fluid obtained from 26 ovarian cancer patients and 48 controls using messenger RNA (mRNA) capture and small RNA sequencing. RESULTS: We observed an enrichment of ovarian and fallopian tube specific messenger RNAs in utero-tubal lavage fluid compared to other human biofluids. Over 300 mRNAs and 41 miRNAs were upregulated in ovarian cancer samples compared with controls. Upregulated genes were enriched for genes involved in cell cycle activation and proliferation, hinting at a tumor-derived signal. CONCLUSION: This is a proof-of-principle that mRNA capture sequencing of utero-tubal lavage fluid is technically feasible, and that the extracellular transcriptome of utero-tubal lavage should be further explored in larger cohorts to assess the diagnostic value of the biomarkers identified in this study. IMPACT: Proximal liquid biopsy from the gynecologic tract is a promising source for mRNA and miRNA biomarkers for diagnosis of early-stage ovarian cancer.

Outlier robust modeling of survival curves in the presence of potentially time-varying coefficients.

In time to event studies, censoring often occurs and models that take this into account are wide-spread. In the presence of outliers, standard estimators of model parameters may be affected such that results and conclusions are not reliable anymore. This in turn also hampers the detection of these outliers due to masking effects. To cope with outliers when using proportional hazard models, we propose to use the Brier score as a loss function. Since the coefficients often vary over time, we focus on the piecewise constant hazard model, which can flexibly model time-varying coefficients if a large number of cut-points is used. To prevent overfitting, we add a penalty term that potentially shrinks time-varying effects to constant effects. By fitting the coefficients of the piecewise constant hazard model using a penalized Brier score loss, we obtain a robust model that can handle time-varying coefficients. Its good performance is illustrated in a simulation study and using two datasets from practice.

Melanoma addiction to the long non-coding RNA SAMMSON.

Focal amplifications of chromosome 3p13-3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon. However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses.

Mathematical inference on helminth egg counts in stool and its applications in mass drug administration programmes to control soil-transmitted helminthiasis in public health.

In the present study, we present a hierarchical model based on faecal egg counts (FECs; expressed in eggs per 1g of stool) in which we first describe the variation in FECs between individuals in a particular population, followed by describing the variance due to counting eggs under a microscope separately for each stool sample. From this general framework, we discuss how to calculate a sample size for assessing a population mean FEC and the impact of an intervention, measured as reduction in FECs, for any scenario of soil-transmitted helminth (STH) epidemiology (the intensity and aggregation of FECs within a population) and diagnostic strategy (amount of stool examined (∼sensitivity of the diagnostic technique) and examination of individual/pooled stool samples) and on how to estimate prevalence of STH in the absence of a gold standard. To give these applications the most wide relevance as possible, we illustrate each of them with hypothetical examples.

A statistical basis for harmonization of thyroid stimulating hormone immunoassays using a robust factor analysis model.

BACKGROUND: Between-method equivalence ideally is achieved by calibration against an SI-traceable reference measurement procedure. For measurement of thyroid stimulating hormone (TSH), it is unlikely to accomplish this goal in mid-term. Therefore, we investigated a statistical alternative based on a factor analysis (FA) model. METHODS: The FA model was applied to TSH results for 94 samples generated by 14 immunoassays (concentration range: 0.0005-78 mIU/L). The dataset did not fulfill the assumption of a homogeneous sample from an elliptically symmetric distribution, and, therefore, required standardization prior to application of the FA model. As outliers and missing values also occurred, the key quantities of the FA model had to be estimated with a method that can handle these complications. We selected a robust alternating regressions (RAR) method, which replaces in the minimization criterion of the fitting process the squared differences between results xij and model fit x^ij ${\hat x_{ij}}$ by a weighted absolute difference. The weights are adaptively determined in successive regressions, which down weighs the outliers. The weights for missing values are set to zero. RESULTS: The quality of the estimated targets was reflected by their central position in the distributions, and description of the relationship between results and targets by a simple two-parameter regression equation with high correlation coefficients and low SDs of the percentage-residuals. Mathematical recalibration eliminated the method differences and improved the between-method CV from 11% to 6%. CONCLUSIONS: RAR applied to a multimethod comparison dataset hampered by outliers and missing values, is fit to the purpose of harmonization.

Simulation of between repeat variability in real time PCR reactions.

While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in C(q) values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats' plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond.

Xenotransplantation by injection of a suspension of isolated preantral ovarian follicles and stroma cells under the kidney capsule of nude mice.

OBJECTIVE: To develop and test a novel approach to xenotransplantation of isolated preantral follicles underneath the kidney capsule of immunodeficient mice. DESIGN: Prospective experimental animal study. SETTING: Academic research unit. ANIMAL(S): Healthy adult nude mice. INTERVENTION(S): Bovine ovaries from fetuses (n = 3) and calves (n = 3) were enzymatically disaggregated and subsequently filtered. Isolated preantral follicles were suspended in phosphate buffered saline, and granulosa and stroma cells originating from the ovarian digest served as embedding matrix. The suspension was injected under the kidney capsule of adult nude mice. MAIN OUTCOME MEASURE(S): Fourteen days after transplantation, follicular survival and proliferation in grafts was assessed by histology and proliferating cell nuclear antigen (PCNA) immunostaining, and was compared with ungrafted control tissue. RESULT(S): Primordial follicles decreased from 58.2% in control tissue to 17.1% in transplants in the fetal group, and from 76.0% to 17.2% in the calf group. Concomitantly, primary follicles increased from 13.4% to 62.2% in the fetal group, and from 5.4% to 63.5% in the calf group. Follicular proliferation measured by PCNA immunolabeling exhibited an increase from 40.6% growing follicles to 81.9% in the fetal group, and from 21.0% to 80.7% in the calf group. CONCLUSION(S): The massive follicular activation following transplantation indicates that isolated preantral follicles are able to survive and grow 14 days after renal subcapsular xenotransplantation.

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing.

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.

Lack of fluorophotometric evidence of aqueous-vitreous barrier disruption after posterior capsulorhexis.

PURPOSE: To evaluate the integrity of the aqueous-vitreous barrier by assessing the flow of fluorescein from the anterior chamber to the anterior vitreous using fluorophotometry in eyes with a posterior continuous curvilinear capsulorhexis (PCCC) and in eyes without a PCCC. SETTING: University Hospital Antwerp, Edegem, Belgium. METHODS: Ten patients had bilateral extracapsular cataract extraction with implantation of an intraocular lens. In 1 eye, a PCCC was performed; the other eye served as a negative control. The eyes of 2 other patients who had complicated cataract surgery with posterior capsule and anterior hyaloid membrane rupture served as positive controls. All patients had fluorophotometry of both eyes 12 to 18 months after surgery to measure the flow of fluorescein from the anterior chamber to the anterior vitreous. RESULTS: There were no statistically significant differences in the distribution pattern of fluorescein between eyes with PCCC and eyes without PCCC. In contrast, enhanced flow was detected in both eyes with rupture of the posterior capsule and the anterior hyaloid. CONCLUSIONS: In this fluorophotometry study, a PCCC did not seem to disrupt the aqueous-vitreous barrier. Results indicate that an intact anterior vitreous membrane is crucial to maintain the barrier function between the anterior and the posterior segments of the eye.