Emulating Target Trials to Avoid Immortal Time Bias - An Application to Antibiotic Initiation and Preterm Delivery.

BACKGROUND: Randomized trials in pregnancy are extremely challenging, and observational studies are often the only option to evaluate medication safety during pregnancy. However, such studies are often susceptible to immortal time bias if treatment initiation occurs after time zero of follow-up. We describe how emulating a sequence of target trials avoids immortal time bias and apply the approach to estimate the safety of antibiotic initiation between 24 and 37 weeks gestation on preterm delivery. METHODS: The Tsepamo Study captured birth outcomes at hospitals throughout Botswana from 2014 to 2021. We emulated 13 sequential target trials of antibiotic initiation versus no initiation among individuals presenting to care <24 weeks, one for each week from 24 to 37 weeks. For each trial, eligible individuals had not previously initiated antibiotics. We also conducted an analysis susceptible to immortal time bias by defining time zero as 24 weeks and exposure as antibiotic initiation between 24 and 37 weeks. We calculated adjusted risk ratios (RR) and 95% confidence intervals (CI) for preterm delivery. RESULTS: Of 111,403 eligible individuals, 17,009 (15.3%) initiated antibiotics between 24 and 37 weeks. In the sequence of target trials, RRs (95% CIs) ranged from 1.04 (0.90, 1.19) to 1.24 (1.11, 1.39) (pooled RR: 1.11 [1.06, 1.15]). In the analysis susceptible to immortal time bias, the RR was 0.90 (0.86, 0.94). CONCLUSIONS: Defining exposure as antibiotic initiation at any time during follow-up after time zero resulted in substantial immortal time bias, making antibiotics appear protective against preterm delivery. Conducting a sequence of target trials can avoid immortal time bias in pregnancy studies.

Intramuscular Transplantation Improves Engraftment Rates for Esophageal Patient-Derived Tumor Xenografts.

BACKGROUND: Recently, there has been an increase in the availability of targeted molecular therapies for cancer treatment. The application of these approaches to esophageal cancer, however, has been hampered by the relative lack of appropriate models for preclinical testing. Patient-derived tumor xenograft (PDTX) models are gaining popularity for studying many cancers. Unfortunately, it has proven difficult to generate xenografts from esophageal cancer using these models. The purpose of this study was to improve the engraftment efficiency of esophageal PDTXs. METHODS: Fresh pieces of esophageal tumors obtained from endoscopic biopsies or resected specimens were collected from 23 patients. The tumors were then coated in Matrigel and transplanted in immunocompromised mice subcutaneously (n = 6) and/or using a novel implantation technique whereby the tumor is placed in a dorsal intramuscular pocket (n = 18). They are then monitored for engraftment. RESULTS: With the novel intramuscular technique, successful engraftment was achieved for all 18 patient tumors. Among these PDTXs, 13 recapitulated the original patient tumors with respect to degree of differentiation, molecular and genetic profiles, and chemotherapeutic response. Lymphomatous transformation was observed in the other five PDTXs. Successful engraftment was achieved for only one of six patient tumors using the classic subcutaneous approach. DISCUSSION: We achieved a much higher engraftment rate of PDTXs using our novel intramuscular transplant technique than has been reported in other published studies. It is hoped that this advancement will help expedite the development and testing of new therapies for esophageal cancer.

APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma.

OBJECTIVES: p53 is a critical tumour suppressor and is mutated in 70% of oesophageal adenocarcinomas (OACs), resulting in chemoresistance and poor survival. APR-246 is a first-in-class reactivator of mutant p53 and is currently in clinical trials. In this study, we characterised the activity of APR-246 and its effect on p53 signalling in a large panel of cell line xenograft (CLX) and patient-derived xenograft (PDX) models of OAC. DESIGN: In vitro response to APR-246 was assessed using clonogenic survival, cell cycle and apoptosis assays. Ectopic expression, gene knockdown and CRISPR/Cas9-mediated knockout studies of mutant p53 were performed to investigate p53-dependent drug effects. p53 signalling was examined using quantitative RT-PCR and western blot. Synergistic interactions between APR-246 and conventional chemotherapies were evaluated in vitro and in vivo using CLX and PDX models. RESULTS: APR-246 upregulated p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in OAC cells harbouring p53 mutations. Sensitivity to APR-246 correlated with cellular levels of mutant p53 protein. Ectopic expression of mutant p53 sensitised p53-null cells to APR-246, while p53 gene knockdown and knockout diminished drug activity. Importantly, APR-246 synergistically enhanced the inhibitory effects of cisplatin and 5-fluorouracil through p53 accumulation. Finally, APR-246 demonstrated potent antitumour activity in CLX and PDX models, and restored chemosensitivity to a cisplatin/5-fluorouracil-resistant xenograft model. CONCLUSIONS: APR-246 has significant antitumour activity in OAC. Given that APR-246 is safe at therapeutic levels our study strongly suggests that APR-246 can be translated into improving the clinical outcomes for OAC patients.

Characterization of a novel tumorigenic esophageal adenocarcinoma cell line: OANC1.

BACKGROUND: Esophageal adenocarcinoma (EAC) has a very high case fatality rate and is one of the fastest rising cancers worldwide. At the same time, research into EAC has been hampered by a relative lack of pre-clinical models, including representative cell lines. AIM: The purpose of this study was to establish and characterize a new EAC cell line. METHODS: Tumor cells were isolated from EAC tissue by enzymatic digestion. Origin of the cell line was confirmed by microsatellite based genotyping. A panel of cancer-related genes was screened for mutations by targeted deep sequencing, Sanger sequencing and high resolution melting.CDKN2A promoter methylation was assessed by methylation specific high resolution melting. HER2 amplification was assessed by fluorescent in situ hybridization. Immunohistochemistry was used to assess expression of markers in xenografts grown in SCID mice. RESULTS: A novel EAC cell line, OANC1, was derived from a Barrett's-associated EAC. Microsatellite-based genotyping of OANC1 and patient DNA confirmed the origin of the cell line. Sequencing of OANC1 DNA identified homozygous TP53 missense (c.856G[A, p.E286K)and SMAD4 nonsense (c.1333C[T, p.R445X) mutations.OANC1 are tumorigenic when injected sub-cutaneously into SCID mice and xenografts were positive for columnar, glandular and intestinal epithelial markers commonly expressed in EAC. Xenografts exhibited strong p53 expression, consistent with a TP53 mutation. Some proteins, including p16, EGFR and b-catenin, had heterogeneous expression patterns across xenograft cross-sections, indicative of tumor heterogeneity. CONCLUSIONS: OANC1 represents a valuable addition to the limited range of pre-clinical models for EAC. This new cell line will be a useful model system for researchers studying both basic and translational aspects of this disease.

The impact of free antiretroviral therapy for pregnant non-citizens and their infants in Botswana.

INTRODUCTION: In December 2019, the Botswana government expanded free antiretroviral therapy (ART) to include non-citizens. We evaluated the impact of this policy change on antenatal care (ANC), antiretroviral therapy coverage and adverse birth outcomes. METHODS: The Tsepamo Surveillance study collects data at up to 18 delivery sites in Botswana. We compared outcomes in citizens and non-citizens living with HIV before and after antiretroviral therapy expansion to non-citizens. Adverse birth outcomes included preterm delivery (PTD) <37 weeks, very preterm delivery (VPTD) <32 weeks, small for gestational age (SGA) <10th percentile, very small for gestational age (VSGA) <3rd percentile, stillbirth and neonatal death. Log-binomial regression models were constructed to generate risk ratios. RESULTS: From August 2014 to September 2021, 45,576 (96.5%) citizens and 1513 (3.2%) non-citizens living with HIV delivered; 954 (62.9%) non-citizen deliveries were before the antiretroviral therapy expansion, and 562 (37.1%) were after. Non-citizen ANC attendance among pregnant people living with HIV increased from 79.2% pre-expansion to 87.2% post-expansion (p<0.001), and became more similar to citizens (96.0% post-expansion). Non-citizens receiving any antenatal antiretroviral therapy increased from 65.5% pre-expansion to 89.9% post-expansion (p < 0.001), also more similar to citizens (97.2% post-expansion). Infants born to non-citizens with singleton gestations in the pre-expansion period had significantly greater risk of PTD (aRR = 1.28, 95% CI, 1.11, 1.46), VPTD (aRR = 1.89, 95% CI, 1.43, 2.44) and neonatal death (aRR = 1.69, 95% CI, 1.03, 2.60), but reduced SGA risk (aRR = 0.75; 95% CI, 0.62, 0.89) compared with citizens. Post-expansion, greater declines in most adverse outcomes were observed in non-citizens, with largely similar outcomes between non-citizens and citizens. Non-significant differences were observed for non-citizenship in PTD (aRR = 0.84, 95% CI, 0.66, 1.06), VPTD (aRR = 0.57, 95% CI, 0.28, 1.01), SGA (aRR = 0.91, 95% CI, 0.72, 1.13), VSGA (aRR = 0.87, 95% CI, 0.58, 1.25), stillbirth (aRR = 0.71, 95% CI, 0.35, 1.27) and neonatal death (aRR = 1.35, 95% CI, 0.60, 2.62). CONCLUSIONS: Following the expansion of free antiretroviral therapy to non-citizens, gaps narrowed in ANC and antiretroviral therapy use in pregnancy between citizens and non-citizens living with HIV. Disparities in adverse birth outcomes were no longer observed.

Sox9 drives columnar differentiation of esophageal squamous epithelium: a possible role in the pathogenesis of Barrett's esophagus.

The molecular mechanism underlying the development of Barrett's esophagus (BE), the precursor to esophageal adenocarcinoma, remains unknown. Our previous work implicated sonic hedgehog (Shh) signaling as a possible driver of BE and suggested that bone morphogenetic protein 4 (Bmp4) and Sox9 were downstream mediators. We have utilized a novel in vivo tissue reconstitution model to investigate the relative roles of Bmp4 and Sox9 in driving metaplasia. Epithelia reconstituted from squamous epithelial cells or empty vector-transduced cells had a stratified squamous phenotype, reminiscent of normal esophagus. Expression of Bmp4 in the stromal compartment activated signaling in the epithelium but did not alter the squamous phenotype. In contrast, expression of Sox9 in squamous epithelial cells induced formation of columnar-like epithelium with expression of the columnar differentiation marker cytokeratin 8 and the intestinal-specific glycoprotein A33. In patient tissue, A33 protein was expressed specifically in BE, but not in normal esophagus. Expression of Cdx2, another putative driver of BE, alone had no effect on reconstitution of a squamous epithelium. Furthermore, epithelium coexpressing Cdx2 and Sox9 had a phenotype similar to epithelium expressing Sox9 alone. Our results demonstrate that Sox9 is sufficient to drive columnar differentiation of squamous epithelium and expression of an intestinal differentiation marker, reminiscent of BE. These data suggest that Shh-mediated expression of Sox9 may be an important early event in the development of BE and that the potential for inhibitors of the hedgehog pathway to be used in the treatment of BE and/or esophageal adenocarcinoma could be tested in the near future.

Adverse Birth Outcomes in Botswana Among Women With Vertically or Horizontally Acquired Human Immunodeficiency Virus.

BACKGROUND: Women with vertically acquired HIV (VHIV) may have a greater risk of adverse birth outcomes than women with horizontally acquired HIV (HHIV). METHODS: The Tsepamo study performed birth outcomes surveillance at 8 government delivery sites in Botswana from July 2014 through March 2019. Pregnant women diagnosed with HIV before their 11th birthday received VHIV status, and other women had HHIV. Small for gestational age (SGA), preterm delivery (PTD), stillbirth, and neonatal death were compared using χ2 and Fisher's exact tests. Log-binomial regression models determined risk ratios (RRs). RESULTS: VHIV women (n = 402) aged 15-27 years were identified over 4 years of surveillance and compared with HHIV women (n = 8465) of the same age. VHIV women were more likely to use nevirapine (NVP)-based antiretroviral treatment (ART) in pregnancy and to have SGA and very SGA infants, but less likely to have very PTD infants. In unadjusted analyses, VHIV women had a higher risk of any adverse birth outcome combined (RR = 1.21, 95% confidence interval [CI], 1.08-1.36). After adjusting for potential confounders, particularly use of NVP-based regimens, the risk of adverse birth outcomes among VHIV and HHIV women was similar. CONCLUSIONS: NVP-based ART is a primary and modifiable risk factor for adverse birth outcomes. Updating ART regimens could improve birth outcomes for women with HIV.

The Impact of Syndromic Management of Vaginal Discharge Syndrome on Adverse Birth Outcomes in Botswana.

BACKGROUND: Vaginal discharge syndrome (VDS) is a common clinical diagnosis during pregnancy in Botswana; it is treated with broad-spectrum antibiotics using a syndromic approach. We evaluated associations between the syndromic management of VDS and adverse birth outcomes. METHODS: The Tsepamo Study performs birth outcomes surveillance at government hospitals throughout Botswana. Obstetric record data collected from August 2014 to March 2019 were analyzed. Chi-square tests were conducted to compare proportions of maternal characteristics and infant outcomes. To avoid immortal time bias, all analyses were conducted among women who presented to care before 24 weeks gestation, with VDS categorized as present or absent by 24 weeks gestation. Log-binomial regression models were generated to determine associations between treated VDS and infant outcomes. RESULTS: VDS was diagnosed in 36 731 (30.7%) pregnant women, of whom 33 328 (90.7%) received antibiotics. Adjusted analyses yielded a harmful association between treated VDS and very preterm delivery (adjusted risk ratio, 1.11; 95% CI, 1.02-1.21). This association remained when restricting to women with VDS who received the recommended antibiotic treatment regimen. Sensitivity analyses produced nonsignificant associations when women with treated VDS were compared with women without VDS who received antibiotics for other indications. CONCLUSIONS: A clinical diagnosis of VDS is common among pregnant women in Botswana, and the majority receive antibiotics in pregnancy. Although analyses of VDS occurring later in pregnancy are precluded by immortal time bias, a modest association between treated VDS and very preterm delivery was observed among women diagnosed with VDS by 24 weeks gestation.

Identification of Pik3ca Mutation as a Genetic Driver of Prostate Cancer That Cooperates with Pten Loss to Accelerate Progression and Castration-Resistant Growth.

Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in vivo Furthermore, we report that PIK3CA mutation and PTEN loss coexist in patients with prostate cancer and can cooperate in vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.Significance: We show PIK3CA mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, PIK3CA mutation and PTEN loss coexist in prostate cancer and can cooperate in vivo to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. Cancer Discov; 8(6); 764-79. ©2018 AACR.See related commentary by Triscott and Rubin, p. 682This article is highlighted in the In This Issue feature, p. 663.

Prevalence of and Risk Factors for Oral Human Papillomavirus Infection among HIV-Positive and HIV-Negative People Who Inject Drugs.

BACKGROUND: Human papillomavirus (HPV) causes most oropharyngeal cancers in the United States. Oral HPV prevalence is associated with immunosuppression, and drug use can be immunosuppressive, but the epidemiology of oral HPV among people who use drugs is not well described. METHODS: We enrolled men and women with a current or prior history of injection drug use in this cross-sectional sub-study within the AIDS Linked to the Intravenous Experience (ALIVE) cohort. We tested oral rinse samples for 37 types of HPV DNA and collected self-reported risk factor information. We compared oral HPV prevalence across categories using chi-squared statistics and multivariable logistic regression. RESULTS: Among 199 subjects, 32% were HIV-positive (median CD4 count 384 cells/µL), 90% were Black, 56% had less than a high school education, 17% had recently used injection drugs, and the median age was 54 years. Most had performed oral sex (82%) but had fewer than 5 lifetime partners (58%). The prevalence of any oral HPV was 29%, and of any oncogenic oral HPV was 13%. Oral HPV prevalence was high among both heterosexual men (30%) and women (20%). After adjustment, odds of oral HPV were increased among HIV-positive individuals with a low CD4 count (<350 cells/µl, aOR = 2.7, 95%CI = 1.2-6.4, vs. HIV-negative individuals), but not among HIV-positive individuals with a higher CD4 cell count. Odds were also elevated for those who had recently performed oral sex on a woman (aOR = 2.2, 95%CI = 1.01-4.6) and, even after this adjustment, among bisexual/lesbian females (aOR = 5.6, 95%CI = 1.4-23, vs. heterosexual females). Oral HPV prevalence was not associated with vaginal sex, performing oral sex on a man, or recent drug use. CONCLUSIONS: Recent drug use was not associated with oral HPV prevalence in our study. However, despite modest numbers of sexual partners, the prevalence of oral HPV among this largely Black population with lower socioeconomic status was high.

Role of p53 in the progression of gastric cancer.

Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer (GC) but is poorly described in terms of molecular changes. Here, we explored the role of TP53, a commonly mutated gene in GC, to determine if p53 protein expression and/or the presence of somatic mutations in TP53 can be used as a predictive marker for patients at risk of progressing to GC from IM. Immunohistochemistry and high resolution melting were used to determine p53 protein expression and TP53 mutation status respectively in normal gastric mucosa, IM without concurrent GC (IM-GC), IM with concurrent GC (IM+GC) and GC. This comparative study revealed an incremental increase in p53 expression levels with progression of disease from normal mucosa, via an IM intermediate to GC. TP53 mutations however, were not detected in IM but occurred frequently in GC. Further, we identified increased protein expression of Mdm2/x, both powerful regulators of p53, in 100% of the IM+GC cohort with these samples also exhibiting high levels of wild-type p53 protein. Our data suggests that TP53 mutations occur late in gastric carcinogenesis contributing to the final transition to cancer. We also demonstrated involvement of Mdmx in GC.