Skeletal fluorosis secondary to methoxyflurane use for chronic pain.

Skeletal fluorosis is rare and occurs secondary to chronic high amounts of fluoride consumption, manifesting as diffuse osteosclerosis, skeletal pain, connective tissue calcification, and increased fracture risk. Methoxyflurane is a volatile, fluorinated hydrocarbon-inhaled analgesic, and the maximum recommended dose is 15 mL (99.9 % w/w) per wk. A rodent study found increased skeletal fluoride after methoxyflurane exposure. However, skeletal fluorosis secondary to methoxyflurane use in humans has rarely been reported. We present the case of a 47-yr-old female with diffuse osteosclerosis secondary to fluorosis from methoxyflurane use for chronic pain, presenting with 3 yr of generalized bony pain and multiple fragility fractures. Lumbar spine BMD was elevated. CT and radiographs demonstrated new-onset marked diffuse osteosclerosis, with calcification of interosseous membranes and ligaments, and a bone scan demonstrated a grossly increased uptake throughout the skeleton. Biochemistry revealed an elevated alkaline phosphatase and bone turnover markers, mild secondary hyperparathyroidism with vitamin D deficiency, and mild renal impairment. Zoledronic acid, prescribed for presumed Paget's disease, severely exacerbated bony pain. Urinary fluoride was elevated (7.3 mg/L; reference range < 3.0 mg/L) and the patient revealed using methoxyflurane 9 mL per wk for 8 yr for chronic pain. A decalcified bone biopsy revealed haphazardly arranged cement lines and osteocytes lacunae and canaliculi, which was consistent with an osteosclerotic process. Focal subtle basophilic stippling around osteocyte lacunae was suggestive of fluorosis. Although fluorosis is not a histological diagnosis, the presence of compatible histology features was supportive of the diagnosis in this case with clinical-radiological-pathological correlation. Skeletal fluorosis should be considered as a cause of acquired diffuse osteosclerosis. Methoxyflurane should not be recommended for chronic pain. The risk of repeated low-dose exposure to fluoride from methoxyflurane use as analgesia may be greater than expected, and the maximum recommended dose for methoxyflurane may require re-evaluation to minimize skeletal complications. Abbreviated abstract: Skeletal fluorosis is rare and occurs secondary to chronic high amounts of fluoride consumption, manifesting as diffuse osteosclerosis, skeletal pain, connective tissue calcification, and increased fracture risk. We present the case of a 47-yr-old female with skeletal fluorosis secondary to long-term methoxyflurane for chronic pain. The risk of repeated low-dose exposure to fluoride from methoxyflurane use for analgesia may be greater than expected, and the maximum recommended dose for methoxyflurane may require re-evaluation to minimize skeletal complications.

POU2F3-expressing intraepithelial small-cell carcinoma with mixed small-cell carcinoma and conventional-type urothelial carcinoma of the urinary bladder.

Based on lineage-specific transcription factors, small-cell neuroendocrine carcinoma (SmCC) of the urinary bladder has recently been subtyped into three molecular subtypes: ASCL1, NEUROD1 and POU2F3. The latter is a master transcriptional regulator of tuft cells (TCs) which are rare solitary cells found in various mucosal epithelia such as the gastrointestinal tract, but which have not been reported in the bladder. The POU2F3 subtype shows low or absent neuroendocrine marker expression. A case of mixed SmCC and conventional-type urothelial carcinoma (CUC) of the urinary bladder with POU2F3-expressing intraepithelial small-cell carcinoma in keeping with a tuft cell phenotype, arising in association with intestinal metaplasia (IM) is described. The presence of POU2F3-expressing cells in normal urothelium, cystitis cystica glandularis and IM of the urinary bladder is demonstrated in separate cases of cystitis cystica glandularis with IM. Also, POU2F3 expression is identified in a subset of bladder SmCC.

Identification of aberrant luminal progenitors and mTORC1 as a potential breast cancer prevention target in BRCA2 mutation carriers.

Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2mut/+ tissue, including expansion of aberrant ERBB3lo luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3lo progenitors in BRCA2mut/+ breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3lo progenitors could generate both ER+ and ER- cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.

Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies.

PURPOSE: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. EXPERIMENTAL DESIGN: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. RESULTS: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. CONCLUSIONS: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

DNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

A Matched Molecular and Clinical Analysis of the Epithelioid Haemangioendothelioma Cohort in the Stafford Fox Rare Cancer Program and Contextual Literature Review.

BACKGROUND: Epithelioid haemangioendothelioma (EHE) is an ultra-rare malignant vascular tumour with a prevalence of 1 per 1,000,000. It is typically molecularly characterised by a WWTR1::CAMTA1 gene fusion in approximately 90% of cases, or a YAP1::TFE3 gene fusion in approximately 10% of cases. EHE cases are typically refractory to therapies, and no anticancer agents are reimbursed for EHE in Australia. METHODS: We report a cohort of nine EHE cases with comprehensive histologic and molecular profiling from the Walter and Eliza Hall Institute of Medical Research Stafford Fox Rare Cancer Program (WEHI-SFRCP) collated via nation-wide referral to the Australian Rare Cancer (ARC) Portal. The diagnoses of EHE were confirmed by histopathological and immunohistochemical (IHC) examination. Molecular profiling was performed using the TruSight Oncology 500 assay, the TruSight RNA fusion panel, whole genome sequencing (WGS), or whole exome sequencing (WES). RESULTS: Molecular analysis of RNA, DNA or both was possible in seven of nine cases. The WWTR1::CAMTA1 fusion was identified in five cases. The YAP1::TFE3 fusion was identified in one case, demonstrating unique morphology compared to cases with the more common WWTR1::CAMTA1 fusion. All tumours expressed typical endothelial markers CD31, ERG, and CD34 and were negative for pan-cytokeratin. Cases with a WWTR1::CAMTA1 fusion displayed high expression of CAMTA1 and the single case with a YAP1::TFE3 fusion displayed high expression of TFE3. Survival was highly variable and unrelated to molecular profile. CONCLUSIONS: This cohort of EHE cases provides molecular and histopathological characterisation and matching clinical information that emphasises the molecular patterns and variable clinical outcomes and adds to our knowledge of this ultra-rare cancer. Such information from multiple studies will advance our understanding, potentially improving treatment options.

Prevalence of subclinical lung cancer detected at autopsy: a systematic review.

BACKGROUND: Lung cancer screening in high-risk populations with low-dose computed tomography is supported by international associations and recommendations. Overdiagnosis is considered a risk of screening with associated harms. The aim of this paper is to determine the prevalence of subclinical lung cancer diagnosed post-mortem to better understand the reservoir of subclinical lung cancer. METHODS: We searched EMBASE, PubMed, and MEDLINE databases from inception until March 2022 with no language restrictions. We considered all studies with ≥100 autopsies in adults. Two reviewers independently assessed eligibility of studies, extracted data, and assessed risk of bias of included studies. We performed a meta-analysis using a random-effects model for prevalence of subclinical lung cancer diagnosed post-mortem with sensitivity and subgroup analyses. RESULTS: A total of 13 studies with 16 730 autopsies were included. Pooled prevalence was 0.4% (95% CI 0.20 to 0.82%, I2 = 84%, tau2 = 1.19, low certainty evidence,16 730 autopsies). We performed a sensitivity analysis excluding studies which did not specify exclusion of children in their cohort, with a pooled prevalence of subclinical lung cancer of 0.87% (95% CI 0.48 to 1.57%, I2 = 71%, tau2 = 0.38, 6998 autopsies, 8 studies). CONCLUSIONS: This is the first published systematic review to evaluate the prevalence of post-mortem subclinical lung cancer. Compared to autopsy systematic reviews in breast, prostate and thyroid cancers, the pooled prevalence is lower in lung cancer for subclinical cancer. This result should be interpreted with caution due to the included studies risk of bias and heterogeneity, with further high-quality studies required in target screening populations.

Bridging Massive Acetabular Defects With the Triflange Cup: 10- to 28-Year Results.

BACKGROUND: Massive acetabular bone loss, encountered complex primary or revision total hip arthroplasty, remains a reconstructive challenge. The custom triflange cup reliably achieves both early fixation and longer-term stability. This study presents the 10-year minimum three-surgeon follow-up of acetabular defects managed with a custom triflange component. METHODS: All patients who underwent a custom triflange acetabular component implantation from January 1992 to December 2009 were identified. Demographics, implant data, outcomes, and reoperations were collected and analyzed. Bone defects in all cases were Paprosky type IIIA, IIIB, or IV. A total of 233 patients (241 hips) underwent implantation of a custom triflange during the study period. There were 81 patients (83 hips) who died prior to minimum follow-up and 84 patients (88 hips) had minimum follow-up of 10 years (mean 15.2; range, 10 to 28), or failure prior to 10 years. RESULTS: Complications requiring additional surgery occurred in 43 hips (49%). There were 10 revisions for failure (11.4%); four were due to recurrent infection, three for aseptic loosening, and one for recurrent infection with all revised to a new triflange. There was one patient who was resected to a Girdlestone for infection and one patient was revised for infection to a bipolar hemiprosthesis due to a healed discontinuity. CONCLUSION: To our knowledge, this study represents the largest cohort and longest follow-up in the current literature and demonstrates excellent survivorship and clinical results at an average of 15 years follow-up. The component was retained in 89% of cases.

Methods for assessment of the tumour microenvironment and immune interactions in non-small cell lung cancer. A narrative review.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer death worldwide. Immunotherapy with immune checkpoint inhibitors (ICI) has significantly improved outcomes in some patients, however 80-85% of patients receiving immunotherapy develop primary resistance, manifesting as a lack of response to therapy. Of those that do have an initial response, disease progression may occur due to acquired resistance. The make-up of the tumour microenvironment (TME) and the interaction between tumour infiltrating immune cells and cancer cells can have a large impact on the response to immunotherapy. Robust assessment of the TME with accurate and reproducible methods is vital to understanding mechanisms of immunotherapy resistance. In this paper we will review the evidence of several methodologies to assess the TME, including multiplex immunohistochemistry, imaging mass cytometry, flow cytometry, mass cytometry and RNA sequencing.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Safety and Feasibility of a Novel Externally Cooled Bronchoscopic Radiofrequency Ablation Catheter for Ablation of Peripheral Lung Tumours: A First-In-Human Dose Escalation Study.

BACKGROUND: Radiofrequency ablation (RFA) is an established modality for percutaneous ablation of non-small cell lung cancer (NSCLC) in medically inoperable patients but is underutilized clinically due to side effects. We have developed a novel, completely endobronchial RFA catheter with an externally cooled electrode. OBJECTIVES: The objective of this study was to establish the safety and feasibility of bronchoscopic RFA using a novel, externally cooled catheter for ablation of peripheral NSCLC. METHODS: Patients with stage I biopsy-confirmed NSCLC underwent bronchoscopic RFA of tumour 7 days prior to lobectomy. The RFA catheter was delivered bronchoscopically to peripheral NSCLC lesions, guided by radial endobronchial ultrasound, with positioning confirmed using intra-procedural cone beam CT. Pre-operative CT chest and histologic examination of resected specimens were used to establish distribution/uniformity of ablation and efficacy of tumour ablation. RESULTS: RFA in the first patient was complicated by dispersal of heated saline due to cough, resulting in ICU admission. The patient recovered fully and underwent uncomplicated lobectomy. Subsequently, the protocol was altered to mandate neuromuscular blockade with a pre-determined dose escalation, with algorithm-restricted energy (kJ) and irrigated saline volume (mL) constraints. A further 10 patients consented and seven underwent successful bronchoscopic RFA of peripheral NSCLC. No significant adverse events were noted. Ablation zone included tumour in all cases (proportion of tumour ablated ranged 8-72%), with uniform necrosis of tissue within ablation zones observed at higher energy levels. Ablation zone diameter correlated with RFA energy delivered (R2 = 0.553), with maximum long axis diameter of ablation zone 3.1 cm (22.9 kJ). CONCLUSION: Bronchoscopic RFA using an externally cooled catheter is feasible, appears safe, and achieves uniform ablation within the treatment zone. Uncontrolled escape of heated saline poses a major safety risk but can be prevented procedurally through neuromuscular blockade and by limiting irrigation.

Cell signaling activation and extracellular matrix remodeling underpin glioma tumor microenvironment heterogeneity and organization.

PURPOSE: Tumor cells thrive by adapting to the signals in their microenvironment. To adapt, cancer cells activate signaling and transcriptional programs and migrate to establish micro-niches, in response to signals from neighboring cells and non-cellular stromal factors. Understanding how the tumor microenvironment evolves during disease progression is crucial to deciphering the mechanisms underlying the functional behavior of cancer cells. METHODS: Multiplex immunohistochemistry, spatial analysis and histological dyes were used to identify and measure immune cell infiltration, cell signal activation and extracellular matrix deposition in low-grade, high-grade astrocytoma and glioblastoma. RESULTS: We show that lower grade astrocytoma tissue is largely devoid of infiltrating immune cells and extracellular matrix proteins, while high-grade astrocytoma exhibits abundant immune cell infiltration, activation, and extensive tissue remodeling. Spatial analysis shows that most T-cells are restricted to perivascular regions, but bone marrow-derived macrophages penetrate deep into neoplastic cell-rich regions. The tumor microenvironment is characterized by heterogeneous PI3K, MAPK and CREB signaling, with specific signaling profiles correlating with distinct pathological hallmarks, including angiogenesis, tumor cell density and regions where neoplastic cells border the extracellular matrix. Our results also show that tissue remodeling is important in regulating the architecture of the tumor microenvironment during tumor progression. CONCLUSION: The tumor microenvironment in malignant astrocytoma, exhibits changes in cell composition, cell signaling activation and extracellular matrix deposition during disease development and that targeting the extracellular matrix, as well as cell signaling activation will be critical to designing personalized therapy.

Conventional MRI features can predict the molecular subtype of adult grade 2-3 intracranial diffuse gliomas.

PURPOSE: Molecular biomarkers are important for classifying intracranial gliomas, prompting research into correlating imaging with genotype ("radiogenomics"). A limitation of the existing radiogenomics literature is the paucity of studies specifically characterizing grade 2-3 gliomas into the three key molecular subtypes. Our study investigated the accuracy of multiple different conventional MRI features for genotype prediction. METHODS: Grade 2-3 gliomas diagnosed between 2007 and 2013 were identified. Two neuroradiologists independently assessed nine conventional MRI features. Features with better inter-observer agreement (κ ≥ 0.6) proceeded to consensus assessment. MRI features were correlated with genotype, classified as IDH-mutant and 1p/19q-codeleted (IDHmut/1p19qcodel), IDH-mutant and 1p/19q-intact (IDHmut/1p19qint), or IDH-wildtype (IDHwt). For IDHwt tumors, additional molecular markers of glioblastoma were noted. RESULTS: One hundred nineteen patients were included. T2-FLAIR mismatch (stratified as > 50%, 25-50%, or < 25%) was the most predictive feature across genotypes (p < 0.001). All 30 tumors with > 50% mismatch were IDHmut/1p19qint, and all seven with 25-50% mismatch. Well-defined margins correlated with IDHmut/1p19qint status on univariate analysis (p < 0.001), but this related to correlation with T2-FLAIR mismatch; there was no longer an association when considering only tumors with < 25% mismatch (p = 0.386). Enhancement (p = 0.001), necrosis (p = 0.002), and hemorrhage (p = 0.027) correlated with IDHwt status (especially "molecular glioblastoma"). Calcification correlated with IDHmut/1p19qcodel status (p = 0.003). A simple, step-wise algorithm incorporating these features, when present, correctly predicted genotype with a positive predictive value 91.8%. CONCLUSION: T2-FLAIR mismatch strongly predicts IDHmut/1p19qint even with a lower threshold of ≥ 25% mismatch and outweighs other features. Secondary features include enhancement, necrosis and hemorrhage (predicting IDHwt, especially "molecular glioblastoma"), and calcification (predicting IDHmut/1p19qcodel).

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Real world outcomes and implementation pathways of exome sequencing in an adult genetic department.

PURPOSE: This study aimed to correlate the indications and diagnostic yield of exome sequencing (ES) in adult patients across various clinical settings. The secondary aim was to examine the clinical utility of ES in adult patients. METHODS: Data on demographics, clinical indications, results, management changes, and cascade testing were collected for 250 consecutive patients who underwent ES through an adult genetics department between 2016 and 2021. Data were analyzed using descriptive and inferential statistics. Testing in which traditional gene panels were in standard use, such as in heritable cancers, was excluded. RESULTS: The average age at testing was 43 years (range = 17-80 years). A molecular diagnosis was identified in 29% of patients. Older age at symptom onset did not pre-exclude a substantial diagnostic yield. Patients with syndromic intellectual disability and multiple system disorders had the highest yield. In >50% of patients with an exome diagnosis, the results changed management. Cascade testing occured in at least one family member for 30% of patients with a diagnosis. Diagnostic results had reproductive implications for 26% of patients and 31% of patients' relatives. CONCLUSION: ES has a robust diagnostic yield and clear clinical utility in adult patients across a range of ages and phenotypes.

In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis.

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.

Combined Treatment with a WNT Inhibitor and the NSAID Sulindac Reduces Colon Adenoma Burden in Mice with Truncated APC.

Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apc min/+ and doublecortin-like kinase 1 (Dclk1)Cre/+ ;Apc fl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apc min/+ (P < 0.01, n > 5) and Dclk1 Cre/+ ;Apc fl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1 Cre/+;Apc fl/fl mice (P < 0.01, n > 17) and in Apc min/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apc min/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1 Cre/+;Apc fl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer. Significance: Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

The Diverse Applications of Pancreatic Ductal Adenocarcinoma Organoids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies. While immortalized cancer cell lines and genetically engineered murine models have increased our understanding of PDAC tumorigenesis, they do not recapitulate inter- and intra-patient heterogeneity. PDAC patient derived organoid (PDO) biobanks have overcome this hurdle, and provide an opportunity for the high throughput screening of potential new therapies. This review provides a summary of the PDAC PDO biobanks established to date, and discusses how they have advanced our understanding of PDAC biology. Looking forward, the development of coculturing techniques for specific immune or stromal cell populations will enable a better understanding of the crosstalk that occurs within the tumor microenvironment, and the impact of this crosstalk on treatment response.