High-resolution mapping of essential maternal and child health service coverage in Nigeria: a machine learning approach.

BACKGROUND: National-level coverage estimates of maternal and child health (MCH) services mask district-level and community-level geographical inequities. The purpose of this study is to estimate grid-level coverage of essential MCH services in Nigeria using machine learning techniques. METHODS: Essential MCH services in this study included antenatal care, facility-based delivery, childhood vaccinations and treatments of childhood illnesses. We estimated generalised additive models (GAMs) and gradient boosting regressions (GB) for each essential MCH service using data from five national representative cross-sectional surveys in Nigeria from 2003 to 2018 and geospatial socioeconomic, environmental and physical characteristics. Using the best-performed model for each service, we map predicted coverage at 1 km2 and 5 km2 spatial resolutions in urban and rural areas, respectively. RESULTS: GAMs consistently outperformed GB models across a range of essential MCH services, demonstrating low systematic prediction errors. High-resolution maps revealed stark geographic disparities in MCH service coverage, especially between rural and urban areas and among different states and service types. Temporal trends indicated an overall increase in MCH service coverage from 2003 to 2018, although with variations by service type and location. Priority areas with lower coverage of both maternal and vaccination services were identified, mostly located in the northern parts of Nigeria. CONCLUSION: High-resolution spatial estimates can guide geographic prioritisation and help develop better strategies for implementation plans, allowing limited resources to be targeted to areas with lower coverage of essential MCH services.

Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype.

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

MYC drives platinum resistant SCLC that is overcome by the dual PI3K-HDAC inhibitor fimepinostat.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with an appalling overall survival of less than 5% (Zimmerman et al. J Thor Oncol 14:768-83, 2019). Patients typically respond to front line platinum-based doublet chemotherapy, but almost universally relapse with drug resistant disease. Elevated MYC expression is common in SCLC and has been associated with platinum resistance. This study evaluates the capacity of MYC to drive platinum resistance and through screening identifies a drug capable of reducing MYC expression and overcoming resistance. METHODS: Elevated MYC expression following the acquisition of platinum resistance in vitro and in vivo was assessed. Moreover, the capacity of enforced MYC expression to drive platinum resistance was defined in SCLC cell lines and in a genetically engineered mouse model that expresses MYC specifically in lung tumors. High throughput drug screening was used to identify drugs able to kill MYC-expressing, platinum resistant cell lines. The capacity of this drug to treat SCLC was defined in vivo in both transplant models using cell lines and patient derived xenografts and in combination with platinum and etoposide chemotherapy in an autochthonous mouse model of platinum resistant SCLC. RESULTS: MYC expression is elevated following the acquisition of platinum resistance and constitutively high MYC expression drives platinum resistance in vitro and in vivo. We show that fimepinostat decreases MYC expression and that it is an effective single agent treatment for SCLC in vitro and in vivo. Indeed, fimepinostat is as effective as platinum-etoposide treatment in vivo. Importantly, when combined with platinum and etoposide, fimepinostat achieves a significant increase in survival. CONCLUSIONS: MYC is a potent driver of platinum resistance in SCLC that is effectively treated with fimepinostat.

HIV Knowledge and Perceived Risk Among Black Men and Women Who Are Incarcerated in Kentucky.

In the United States, Black men and women who are incarcerated bear a disproportionate and inequitable burden of HIV infection. While HIV knowledge does not consistently predict HIV risk behaviors, HIV knowledge can inform one's perceptions of their risk for HIV. We examined gender differences in HIV knowledge and perceived risk of contracting HIV (N = 424) among Black men and women who were incarcerated and nearing community reentry from seven prisons in Kentucky. Our results demonstrated that women reported greater levels of HIV knowledge and perceived greater risk for contracting HIV than their male counterparts. Implications for HIV prevention interventions are discussed.

SILAC kinase screen identifies potential MASTL substrates.

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

Deletion of kif3a in CK19 positive cells leads to primary cilia loss, biliary cell proliferation and cystic liver lesions in TAA-treated mice.

BACKGROUND & AIMS: Loss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood. METHODS: Primary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3af/f;CK19CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays. RESULTS: At the end of 20 weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro. CONCLUSIONS: TAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Radical resection of large metastatic non-functioning pancreatic neuroendocrine carcinoma complicated by splenic vein thrombosis and sinistral portal hypertension.

INTRODUCTION AND IMPORTANCE: There are limited reports in the literature of radical surgical resection for pancreatic neuroendocrine carcinoma (PNEC). In patients with non-functioning PNEC (NF-PNEC) within the tail of the pancreas tumours can cause splenic vein thrombosis (SVT) and subsequent sinitral portal hypertension (SPH). Radical surgical resection in such patients with concomitant liver metastasis has not previously been reported. CASE PRESENTATION: We present a 67-year old female patient who presented with a large NF-PNEC within the tail of the pancreas with liver metastasis. We performed a distal pancreatectomy, splenectomy, partial gastrectomy and liver resection to achieve radical resecton. DISCUSSION: All patients with NF-PNEC within the tail of the pancreatic should be considered for radical surgical resection. In the presence of multi-visceral involvement and complications such as SVT and/or SPH multi-speciality surgical expertise is likely to be required. CONCLUSION: Radical multi-visceral resection for large NF-PNEC can be safely performed in the presence of SVT and SPH.

p53 and RB1 regulate Hedgehog responsiveness via autophagy-mediated ciliogenesis.

Loss of tumor protein p53 (p53) and RB transcriptional corepressor 1 (RB1) in developmental and small cell lung cancer models promotes primary cilia formation and hyper-responsiveness to Hedgehog ligand. This is mediated by impaired transcription of p53 and RB1 target genes involved in autophagic degradation of primary cilia.

Analysis of pulsed cisplatin signalling dynamics identifies effectors of resistance in lung adenocarcinoma.

The identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has previously been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely mimics the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA-damage and apoptotic responses across a panel of human lung adenocarcinoma cell lines. By coupling this data to real-time, single-cell imaging of cell cycle and apoptosis we provide a fine-grained stratification of response, where a P70S6K-mediated signalling axis promotes resistance on a TP53 wildtype or null background, but not a mutant TP53 background. This finding highlights the value of in vitro models that match the physiological pharmacokinetics of drug exposure. Furthermore, it also demonstrates the importance of a mechanistic understanding of the interplay between somatic mutations and the signalling networks that govern drug response for the implementation of any consistently effective, patient-specific therapy.

Lung adenocarcinoma is the most common type of lung cancer, and it emerges because of a variety of harmful genetic changes, or mutations. Two lung cancer patients ­ or indeed, two different sets of cancerous cells within a patient ­ may therefore carry different damaging mutations. A group of drugs called platinum-based chemotherapies are currently the most effective way to treat lung adenocarcinoma. Yet, only 30% of patients actually respond to the therapy. Many studies conducted in laboratory settings have tried to understand why most cases are resistant to treatment, with limited success. Here, Hastings, Gonzalez-Rajal et al. propose that previous research has been inconclusive because studies done in the laboratory do not reflect how the treatment is actually administered. In patients, platinum-based drugs are cleared from the body within a few hours, but during experiments, the treatment is continually administered to cells growing in a dish. Hastings, Gonzalez-Rajal et al. therefore developed a laboratory method that mimics the way cells are exposed to platinum-based chemotherapy in the body. These experiments showed that the lung adenocarcinoma cells which resisted treatment also carried high levels of a protein known as P70S6K. Pairing platinum-based chemotherapy with a drug that blocks the activity of P70S6K killed these resistant cells. This combination also treated human lung adenocarcinoma tumours growing under the skin of mice. However, it was ineffective on cancerous cells that carry a mutation in a protein called p53, which is often defective in cancers. Overall, this work demonstrates the need to refine how drugs are tested in the laboratory to better reflect real-life conditions. It also underlines the importance of personalizing drug combinations to the genetic background of each tumour, a concept that will be vital to consider in future clinical trials.

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

Randomized, double-blind, placebo-controlled phase II study of istiratumab (MM-141) plus nab-paclitaxel and gemcitabine versus nab-paclitaxel and gemcitabine in front-line metastatic pancreatic cancer (CARRIE).

BACKGROUND: Preclinical data suggest that dual blockade of the insulin-like growth factor-1 receptor (IGF-1R) and HER3 pathways has superior activity to IGF-1R blockade alone in pancreatic ductal adenocarcinoma (PDAC). We tested whether istiratumab, an IGF-1R- and ErbB3-bispecific antibody, can enhance the efficacy of standard of care (SOC) chemotherapy in patients with metastatic PDAC selected for high IGF-1 serum levels. PATIENTS AND METHODS: CARRIE was an international, randomized, double-blind, placebo-controlled phase II study for patients with previously untreated metastatic PDAC. In part 1, 10 patients were evaluated for pharmacokinetics and safety. In part 2, patients with high free serum IGF-1 levels were randomized 1 : 1 to receive either istiratumab [2.8 g intravenously (i.v.) every 2 weeks] or placebo combined with gemcitabine/nab-paclitaxel at approved dose schedule. The co-primary endpoints were progression-free survival (PFS) in patients with high IGF-1 levels and PFS in patients with both high serum IGF-1 levels and heregulin (HRG)+ tumors. Key secondary endpoints were overall survival (OS), objective response rate (ORR) by RECIST v.1.1, and adverse events (AEs) rate. RESULTS: A total of 317 patients were screened, with 88 patients randomized in part 2 (experimental arm n = 43; control n = 45). In the high IGF-1 cohort, median PFS was 3.6 and 7.3 months in the experimental versus control arms, respectively [hazard ratio (HR) = 1.88, P = 0.027]. In the high IGF-1/HRG+ subgroup (n = 44), median PFS was 4.1 and 7.3 months, respectively (HR = 1.39, P = 0.42). Median OS and ORR for the overall population were similar between two arms. No significant difference in serious or grade ≥3 AEs was observed, although low-grade AEs leading to early discontinuation were higher in the experimental (39.5%) versus control arm (24.4%). CONCLUSIONS: Istiratumab failed to improve the efficacy of SOC chemotherapy in this patient setting. High serum IGF-1 levels did not appear to be an adverse prognostic factor when compared with non-biomarker-selected historic controls. CLINICAL TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov: NCT02399137; EUDRA CT: 2014-004572-34.

GEAMP, a novel gastroesophageal junction carcinoma cell line derived from a malignant pleural effusion.

Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate preclinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 nonsynonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a subpopulation with multipotent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.

A Sexually Dimorphic Role for STAT3 in Sonic Hedgehog Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children and represents 20% of all pediatric central nervous system neoplasms. While advances in surgery, radiation and chemotherapy have improved overall survival, the lifelong sequelae of these treatments represent a major health care burden and have led to ongoing efforts to find effective targeted treatments. There is a well-recognized male bias in medulloblastoma diagnosis, although the mechanism remains unknown. Herein, we identify a sex-specific role for the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) in the Sonic Hedgehog (SHH) medulloblastoma subgroup. Specific deletion of Stat3 from granule cell precursors in a spontaneous mouse model of SHH medulloblastoma completely protects male, but not female mice from tumor initiation. Segregation of SHH medulloblastoma patients into high and low STAT3 expressing cohorts shows that low STAT3 expression correlates with improved overall survival in male patients. We observe sex specific changes in IL-10 and IL-6 expression and show that IL-6 stimulation enhances SHH-mediated gene transcription in a STAT3-dependent manner. Together these data identify STAT3 as a key molecule underpinning the sexual dimorphism in medulloblastoma.

Renal epithelial cells retain primary cilia during human acute renal allograft rejection injury.

OBJECTIVES: Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection. RESULTS: Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1 week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection.

Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases.

Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 years using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.

Chiral DNA sequences as commutable controls for clinical genomics.

Chirality is a property describing any object that is inequivalent to its mirror image. Due to its 5'-3' directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide-order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin genetic analysis, including PCR amplification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences mirroring clinically relevant or analytically challenging regions of the human genome are ideal controls for clinical genomics. The addition of synthetic chiral sequences (sequins) to patient tumor samples can prevent false-positive and false-negative mutation detection to improve diagnosis. Accordingly, we propose that sequins can fulfill the need for commutable internal controls in precision medicine.