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Advanced hypopharyngeal tumours present complex clinical challenges, and where resection is attempted, there is a requirement for major reconstruction. Despite advances in surgical technique, outcomes remain poor for this patient group, and optimum treatment has yet to be established. We aimed to assess the treatment and outcomes of patients in our institution in the context of previous studies. All patients from 2008 to 2018 who underwent surgical management for hypopharyngeal tumours with pharyngo-laryngo-esophagectomy and flap-based reconstruction were included in the study. Demographic and outcome data were collected, and patient-reported outcomes were solicited from surviving patients using the EORTC QLQ HN 43 questionnaire. Thirty patients were assessed, in which 12 had gastric pull-ups, 16 had free jejunum flaps, and 2 had free anterolateral thigh flaps. There was a 38% five-year survival rate. Overall, the rates of stricture (10.7%) and fistula (7.1%) were low. The majority of patients (53.6%) returned to a normal diet within three months with a soft or puree diet in 35.7% of patients. Some form of speech was possible in 92.9% of patients. The average questionnaire score for surviving patients was 87.3, with good outcomes related to eating and swallowing, but poorer outcomes for speech and communication. This study showed that outcomes for patients receiving complex reconstruction following hypopharyngeal tumour resection are improving over time. There is still scope for improvement of patient outcomes and refinement of optimum surgical management strategies.

Background Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888) assessed the performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity. Participants and methods The 6689 participants [2482 cancer (>50 cancer types), 4207 non-cancer] were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization. Results Performance was consistent in training and validation sets. In validation, specificity was 99.3% [95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR)]. Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ∼63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%. Conclusions cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.

Due to an increase in immigration from African countries, U.S. healthcare professionals are encountering women affected by Female Genital Mutilation/Cutting (FGM/C). Little formal education exists in medical, nursing or midwifery schools. Additionally, very few studies assess attitudes and knowledge of U.S. providers to care for these women. In order to eventually offer FGM/C-affected women informed and culturally competent care, assessments of provider readiness and attitudes must guide future educational efforts. A voluntary survey was administered to healthcare providers prior to thirteen separate education sessions on FGM/C at Philadelphia institutions. Of the 229 surveys distributed, 227 were returned > 50% complete. 61.2% reported encountering FGM/C at least once in the past 5 years, yet 13.7% of participants reported receiving formal training in FGM/C; 77.5% felt unprepared and 47.1% uncomfortable serving patients with FGM/C. 34.1% of participants knew the matriarchal influence on the custom. The majority of providers accurately identified the short and long-term clinical consequences of FGM/C, but 67.7% of participants denied familiarity with laws regarding FGM/C. Attitude assessment revealed beliefs that all types of FGM/C are harmful and a practice rooted in tradition. While most providers reported encountering patients with FGM/C, lack of formal training leaves them unprepared and uncomfortable serving them. Results demonstrate misunderstanding of the tradition and motives of FGM/C. Our findings support the need for expansion of provider education to provide culturally competent care for women affected by FGM/C.

Background Angiocentric glioma (AG) is an epileptogenic low grade (World Health Organization grade I) glial tumor with astrocytic and ependymal differentiation, most commonly affecting the pediatric and adolescent population. Despite its infiltrating histological growth kinetics, it is widely accepted that AG has a low potential for aggressive behavior. Case Description We present the case of a 42-year-old man who represents the first documented case of not only extracranial manifestation of AG, but also spinal metastatic dissemination. Our patient initially presented with a generalized tonic clonic seizure; following a biopsy, he was diagnosed with a low-grade supratentorial astrocytoma and subsequently received fractionated radiotherapy. He presented 10 months later with worsening dorsal column symptoms and was found to have a contrast-enhancing intradural extramedullary lesion that was surgically resected and histologically confirmed as an AG. Conclusion Further research is required to examine the microenvironment and potential for malignant change in this tumor.

INTRODUCTION Papillary thyroid carcinoma (PTC) is the most common form of well-differentiated thyroid carcinoma. Despite its well-differentiated microscopic features, PTC may be minimally or overtly invasive. We present the case of a patient in whom thoracic spinal cord compression was the first presentation of metastatic PTC. CASE REPORT A 65-year-old woman presented with deteriorating mobility over a 2-month period. Her past history was significant for a right hemithyroidectomy 10 years prior. Examination revealed bilateral lower limb weakness, worse on the right side. Investigations showed a large extradural contrast-enhancing mass at the T7 spinal level. Laminectomy and debulking of this lesion was undertaken. Histopathological examination revealed PTC. The patient proceeded to further treatment with external beam radiotherapy. DISCUSSION Spinal metastases are an unusual presentation of PTC. Further research into the outcomes for these patients is imperative to bettering our ability to care for these patients through a multi-modality approach. .

Background "Ondine's curse" or central hypoventilation, induces an apparently spontaneous failure of automatic respiratory drive, henceforth necessitating a conscious effort to breathe and sleep induced hypoventilation. It is typically seen in congenital central hypoventilation syndrome, but may be acquired. Objectives To highlight Ondine's curse as part of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) secondary to microtubule associated protein tau (MAPT) variants. Methods We describe the clinical and neuropathological findings in two patients with fatal Ondine's curse associated with FTDP-17 and secondary to MAPT variants (FTDP-17t). We discuss neuroanatomical correlates. We review two prior reports of central hypoventilation associated with MAPT variants suggesting that Ondine's curse occurs uncommonly in FTDP-17t. Results Despite variants affecting different regions of MAPT and a degree of heterogeneity in pathological findings, the patients reviewed all experienced central hypoventilation during their disease course. Conclusion Tauopathy should be considered in patients with adult-onset Ondine's curse.

This is the fourteenth case of an intracranial and dural-based osteosarcoma in the literature to date. The case involves a forty-year old woman with a previous history of a brain tumour, which was treated with surgery, chemotherapy and radiotherapy six years previously. The hypothesis is that this rare malignancy is secondary to the radiation exposure given the growth of the lesion in the previous radiation field

Introduction Metastatic breast cancer (MBC) is the main source of mortality in breast cancer patients largely due to lack of effective treatments. Our previous results suggest that tumour transcriptional heterogeneity drives therapy resistance and cancer relapse. While traditional in vitro human cancer cell line models have been widely used for disease modelling, they do not faithfully recapitulate the pathophysiology of MBC. Method In this study we developed patient-derived tumour organoid cultures from frozen patient-derived (PDX) models of MBC. Using those models we performed preclinical drug screening of investigational and FDA approved therapeutics previously uncovered by us as potentially clinically actionable in MBC. Result Our results reveal high heterogeneity in the responses to various targeted therapies among tested MBC organoids, which makes them a valuable tool for studying intra-tumor heterogeneity and drug response. Moreover, drug screening identified a divergent set of the breast to brain metastatic MBC organoids that showed high sensitivity to a new class of tyrosine kinase receptors, RET. Conclusion Taken together, our novel MBC models and methodology applied here provides an important modelling tool to assess the contribution of intra-tumour heterogeneity and microenvironment to drug response as they recapitulate the cellular, structural and biochemical complexity previously observed in our genomic characterisation of MBCs. Application of this type of translational research will enhance the development of new targeted precision medicine strategies and prelude stratification for clinical trials. Abbreviations MBC- Metastatic Breast Cancer; PDX- Patient-Derived Xenografts; FDA- Food and Drug Administration; RET- Receptor Tyrosine Kinase Take-home message Patient-derived tumour organoid cultures provide an important modelling tool to assess the contribution of intra-tumour heterogeneity and microenvironment to drug response as they recapitulate the cellular, structural and biochemical complexity previously observed in genomic characterisation of metastatic breast cancer. SURGICAL EDUCATION AND TRAINING

Funder: Breast Cancer Ireland GR 14-0883, Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER+ metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER+ metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.

Background: Ewing’s sarcoma (ES) is a malignant mesenchymal tumor, most often found in the long bones, and usually affecting children and adolescents in the second decade of life. ES of the spine is a clinical rarity. Case Description: A 45-year-old male presented with a 3-month history of lower back pain which acutely worsened in conjunction with urinary retention. The magnetic resonance imaging revealed a mass extending from L5 to S2 with additional extension through the left S2-3 neural foramen. The metastatic workup was negative. At surgery, the lesion was both intradural and extradural. Following complete surgical resection, the patient was later treated with radiation and chemotherapy. Conclusion: Here, we report an adult male who acutely presented with low back pain attributable to primary intradural/extradural sacral ES.

Pituitary adenomas are the commonest sellar tumours. Pituitary metastases are very rare, with the most common primaries being breast and lung cancers. We report the case of an 83-year-old man with a history of breast carcinoma who presented with recent-onset headaches and progressive deterioration of visual acuity. MRI brain showed a large sellar and suprasellar mass compressing the optic chiasm and involving the pituitary stalk. Transsphenoidal debulking resulted in symptomatic relief and visual recovery. Specimen examination revealed a combination of a gonadotroph pituitary adenoma that was infiltrated by metastatic breast carcinoma. He had no symptoms of diabetes insipidus. He was subsequently treated with pituitary radiotherapy. This is a very rare presentation of a pituitary mass with mixed pathology. To our knowledge, this is the third description of a breast carcinoma metastasis into a gonadotroph cell pituitary adenoma. Learning points: Infiltrating metastases into pituitary adenomas are very rare but do occur. To our knowledge this is the third case of breast adenocarcinoma metastasising to a gonadotroph pituitary adenoma. Pituitary metastases should always be considered in rapidly evolving pituitary symptoms in a cancer patient. Not all complex pituitary lesions are associated with panhypopituitarism. Early invasive local management (TSS and post TSS radiotherapy) can provide rapid satisfactory outcomes.

Background Central nervous system (CNS) lymphoma is rare, representing 2% of all brain tumors. The commonest subtype is diffuse large B-cell lymphoma (DLBCL), with primary T-cell lymphomas (PCNSTL) accounting for ~ 2%. Objective To determine the frequency and describe the key features of CNS lymphoma over a 10-year period in an Irish population. Materials and methods This retrospective review was carried out using the neuropathology database in Beaumont hospital, the largest of two national neurosurgical centers, to identify all cases of CNS lymphoma from 2007 to 2017. Clinical, radiological, morphological, immunophenotype, and molecular information was recorded where available. Results We identified 149 cases of CNS lymphoma from 2007 to 2017, which equated to a cumulative incidence rate of 0.4/100,000 persons. Median age at diagnosis was 66 years, and 46% were male. 86% were classified as DLBCL (n = 128), 10% immunodeficiency-associated CNS lymphoma (n = 15), 3% PCNSTL (n = 4), and 1% (n = 2) cases of intravascular large B-cell lymphoma. Location in declining frequency was as follows: supratentorial (n = 125), infratentorial (n = 22), spinal (n = 1), and orbital (n = 1). Conclusion This is the first study in an Irish population to determine a cumulative incidence rate of CNS lymphoma, which is in line with larger international population-based registries. No significant trends in incidence rate have been observed from 2007 to 2017. DLBCL is the commonest subtype encountered. Rare variants including PCNSTL can pose a significant diagnostic challenge, and in this setting, molecular studies can be useful to confirm diagnoses.

Patient: Male, 32-year-old Final Diagnosis: Granulomatous amebic encephalitis Symptoms: — Medication:— Clinical Procedure: — Specialty: — Objective: Challenging differential diagnosis Background: Acanthamoeba are free-living amoebae with potential to infect immunocompromised hosts. The mortality rate of granulomatous amebic encephalitis (GAE) due to Acanthamoeba exceeds 90% and there are currently no reports of survival of this infection in recipients of hematopoietic stem cell transplant. Case Report: We report herein the case of a 32-year-old man presenting to our service with abrupt neurological deterioration and seizures 5 months after allogeneic stem cell transplantation for Hodgkin lymphoma. Clinical and imaging findings were non-specific at presentation. Multiple circumscribed, heterogenous, mass-like lesions were identified on MRI. Brain biopsy was performed and revealed multiple cysts and trophozoites suggesting a diagnosis of granulomatous amebic encephalitis. PCR testing confirmed Acanthamoeba. Treatment with miltefosine, metronidazole, azithromycin, fluconazole, pentamidine isethionate, and co-trimoxazole was instituted and the patient survived and shows continued improvement with intensive rehabilitation. Conclusions: We report the first successful outcome in this setting. The diagnosis would have been missed on cerebrospinal fluid analysis alone, but was rapidly made by histological analysis of brain biopsy. This diagnostically challenging infection is likely under-recognized. Early brain biopsy and commencement of a prolonged miltefosine-containing anti-ameba regimen can be curative.

Preoperative fasting guidelines emerged in the 1800s, prompted by perioperative aspiration deaths.[1][1] Without objective data, modern fasting guidelines for some non-clear liquids, such as orange juice (OJ) with pulp, rely on expert opinion. Gastric point-of-care ultrasound (POCUS) can

Knowledge of the clinical outcome in tumefactive demyelination remains limited. This study aims to characterise the natural history of biopsy-proven, pathogen-free, cerebral demyelination in an adult Irish population. We identified all patients with biopsy-proven demyelination in a single neuropathology centre between 1999 and 2017. A baseline, and at least one follow-up MRI scan was available in each instance (mean of 3 scans per patient), together with both the presenting and most recent clinical details including disability level and disease-modifying drugs. In 21 patients, white matter biopsies showed the following: macrophages with myelin debris, myelin-axonal dissociation, reactive astrocytes and occasional lymphocytes. During a mean follow-up time of 8 years (± 4.4), 17 patients developed MS, confirmed both clinically and on MRI, using the 2010 McDonald criteria: 11 relapsing remitting (RR) MS, four secondary progressive and two primary progressive MS. Four patients had a monophasic illness with lesion regression, without clinical or radiological evidence of any further disease activity on follow-up. The patients with progressive MS had significantly higher levels of physical disability than either the RRMS or monophasic patients. Uniform white matter subacute demyelination is associated with a diverse clinical course ranging from a monophasic illness to progressive MS, suggesting that extraneous factors distinct from the basic pathology significantly influence the clinical course in MS.

We present a case of a 38-year-old male who sustained a laceration from a knife to the volar aspect of his left index and middle fingers. He had clinical injury to his flexor digitorum profundus tendons to both digits. He underwent operative exploration and repair of the tendons under general anaesthetic. An arm tourniquet was inflated to allow for haemostasis in the operative field. A few minutes after inflation, the patient's hand went into carpal spasm. The tourniquet was deflated and the spasm resolved. Intraoperative serum calcium and carbon dioxide levels were normal. The operation proceeded with the tourniquet deflated. Postoperatively serum calcium and magnesium levels were within normal limits, as was serum vitamin D and parathyroid hormone levels. It has been reported that carpal spasm can occur with tourniquet use in the anxious patient due to hyperventilation and resultant metabolic alkalosis. This however is the first reported case of carpal spasm in the setting of tourniquet use and normal serum electrolytes and respiratory parameters in an intubated patient.

Concentrated land spreading of poultry litter has the potential to cause nutrient build-up in soils, eutrophication of water bodies, air pollution and the spread of pathogens. As a result, alternative routes for the disposal of poultry litter are being sought. A consequential life cycle assessment was conducted to examine several scenarios where biogas produced from poultry litter is used to generate heat and electricity or is upgraded to biomethane which can substitute natural gas. For all of the scenarios considered in this study, diverting poultry litter to anaerobic digestion leads to reduced environmental impacts for global warming, fine particulate matter formation and terrestrial acidification. However, the extent to which environmental impacts are reduced varies significantly across the scenarios. Displaced processes including electricity or natural gas, peat moss production and avoided land spreading of litter contribute the most to reducing environmental impacts. Consequential life cycle assessment is a suitable tool to inform decision-makers about the impact of introducing a new multifunctional technology like anaerobic digestion when considering the systems which are displaced. The results show the range of potential environmental outcomes, rather than predicting a single most-likely outcome. The results of this study indicate that anaerobic digestion is a suitable disposal route for poultry litter which may lead to reduced environmental impacts. This type of analysis is recommended when considering alternative feedstocks and valorisation pathways in the circular economy.

The presence of oligodendroglioma-like areas in pilocytic astrocytoma may give rise to pathologic diagnostic uncertainty. This study aims to determine if the oligodendroglioma-like areas present in some pilocytic astrocytomas (PA) possess the signature 1p/19q codeletion that is characteristic of classical oligodendroglioma. Array comparative genomic hybridization was carried out on 12 PA samples, from which oligodendroglioma-like areas were microdissected and used as the template DNA source. 1p/19q codeletions were not found in any of the oligodendroglioma areas in PAs. We conclude that PAs with oligodendroglioma-like areas do not share the same molecular genetics as classic oligodendroglioma. .

The aim of this study is to identify, in our center, all cases of foreign-body reactions to hemostatic agents or other prostheses resulting in a radiological suspicion of tumor recurrence. We interrogated our internal database to identify all such cases and systematically evaluated the MRI brain scans of patients: (i) at the time of initial tumor diagnosis, (ii) postoperatively, (iii) and at the time of suspected tumor recurrence. In addition, we reviewed each patient’s operative notes and reviewed the histology of all cases following a second surgical intervention. In total, we identified 8 patients, 7 of whom had a WHO grade II glioma at initial surgery. We did not identify any distinguishing radiological abnormalities from the initial diagnostic brain scan to the suspected recurrence, and histologically all cases were characterized by extensive gliosis; with both macrophages and reactive astrocytes present throughout. The cause of gliosis was identified as being relating to hemostatic agents in 4 cases; in the other 4 cases, the foreign-body reaction was presumed to be caused be materials used in a craniotomy or cranioplasty. This study highlights the difficulty in radiologically diagnosing a foreign-body reaction and also identifies that such a gliotic reaction may occur as a consequence of exogenous materials used in a craniotomy or cranioplasty.

We report the first clinical-radiological-genetic-molecular-pathological study of a kindred with c.823-10G>T MAPT intronic variant (rs63749974) associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We describe the clinical spectrum within this family and emphasize the association between MAPT gene variants and motor neuron disease. This report of a second family with FTDP-17 associated with c.823-10G>T MAPT variant strongly supports pathogenicity of the variant and confirms it is a 4-repeat (4R) tauopathy. This intronic point mutation, probably strengthens the polypyrimidine tract and alters the splicing of exon 10 (10 nucleotides into intron 9) close to the 3' splice site.

This paper reviews CNS involvement secondary to malignant-mixed Müllerian tumor or uterine carcinosarcoma, a rare aggressive biphasic Müllerian tumor. We report a cerebellar metastasis with epithelial and mesenchymal components, demonstrating heterologous rhabdomyogenic and chondroblastic differentiation. The patient had undergone total abdominal hysterectomy and bilateral salpingo-oophorectomy for palliation of symptomatic chemotherapy-resistant node-positive disease. CNS involvement is rare, and prognostically poor, and suggestively poorer in predominantly sarcomatous metastases. Multimodal therapy is indicated; in solitary metastases, surgical resection or stereotactic radiosurgery is included, followed by whole brain radiotherapy. In unresectable brain metastases, stereotactic radiosurgery and whole brain radiotherapy warrant consideration in up to 2–3 metastases. In multiple metastases, palliative steroid therapy or cranial irradiation may be considered. Combination or platinum-based chemotherapy (i.e., ifosfamide-paclitaxel or carboplatin-paclitaxel) is indicated in all stages, with a role in both disease cure and control-directed management. Targeted therapeutics have thus far not demonstrated significant clinical efficacy.

Summary We present the case of a 23-year-old female with severe cyclical pelvic pain and amenorrhea with multiple emergency room visits and hospitalization. She already had diagnoses of VATER, right renal agenesis, neurogenic bladder, and scoliosis of thoracolumbar region. Imaging demonstrated right sided hematometra, right and possibly left uterine horn, right renal agenesis, and hematosalpinx. Because of outflow obstruction, and resultant retrograde menstrual flow, causing severe pain, surgical management was indicated. Robot-assisted laparoscopy demonstrated endometriosis, significant adhesive disease, bilateral enlarged fallopian tubes, ovaries bilaterally, two uterine horns without midline fusion and no identifiable cervical tissue. Some surgical findings were anticipated, such as absent right ureter, endometrial implants, adhesive disease, enlarged right uterine horn. Other findings were unanticipated such as left uterine horn adjacent to the left round ligament inguinal insertion, obscured and atypical right-sided pelvic sidewall vasculature, and the additional embryologic tissue of unclear origin discovered adjacent to the right uterine horn and vagina. No cervical tissue was identified in either uterine horn and pathology confirmed this. Pathologic diagnosis of the additional embryonic tissue separate from the right uterine horn indicated Mullerian origin. Surgery was without complications, patient was discharged home without incident. Intended Objectives We display the challenges inherent in surgical management of cyclic pelvic pain due to retrograde bleeding from obstruction in a patient with multiple congenital anomalies. Co-occurrence of the vertebral defect, anal atresia, cardiac defect, tracheoesophageal fistula/esophageal atresia, renal defect, and limb defect association (VATER) and mullerian abnormalities is rare and not well-characterized. We describe and demonstrate surgical findings in a patient with the rarely associated VACTERL/VATER spectrum and Mullerian anomaly. Familiarity with the embryological origins of pelvic structures was crucial to decision-making and guided surgery. Robotic assistance facilitated visualization of planes and allowed precise dissection. Because each case of embryologic anomaly presents with unique pre-op and intra-op findings, formulating a sound thought process and surgical technique is crucial to prevent injury and achieve the goals of surgery.

Accurate tissue of origin (TOO) prediction is crucial for effective clinical follow-up in early cancer detection from blood. In the second substudy of the Circulating Cell-free Genome Atlas (CCGA; NCT02889978), we trained logistic regression classifiers under cross-validation to detect and localize cancer. Input features were methylation states from a targeted cfDNA assay of 2023 participants. TOO classification accuracy was 89% across 20 pre-specified prediction classes. We subsequently sought to understand the causes of the remaining TOO errors. 45% of the errors fell into clusters reflecting similarities in developmental biology, histology, or oncological drivers. Here, we analyzed tissues that may be affected by HPV-driven cancers; these accounted for 21% of TOO errors. The original classifier, which used only human epigenetic states as input, demonstrated cross-scoring between likely HPV-driven cancers of the anus (N = 14) and cervix (N = 11), as well as confirmed HPV-positive head & neck (H&N) cancers (37/62). We also observed HPV-associated vulva (N = 9) and penis (N = 1) cancers, which were not directly trained as TOO classes, were assigned high H&N scores. To test the hypothesis of HPV-driven TOO confusion, we assessed HPV cfDNA and HPV-driven methylation in human peripheral blood cfDNA, and used a specialist classifier restricted to HPV-associated cancers to resolve errors. We corroborated putative HPV-positive participants using targeted sequencing of HPV16 and HPV18 cfDNA fragments, and showed that the number of unique HPV-derived fragments in a sample matched with expected cancer localizations, HPV subtypes, and HPV status. Consistent with the literature, we found little evidence of HPV viremia in non-cancer participants despite the high prevalence of transient HPV infections in the US population. At 99.8% specificity, a cross-validated cutoff on the number of HPV cfDNA fragments in a sample achieved sensitivities of 78.6% (11/14), 36.3% (4/11), 66.6% (6/9), 100% (1/1), and 81.0% (30/37), for anus, cervix, vulva, penis, and confirmed HPV-positive H&N cancers, respectively. These sensitivities were similar to those achieved by the epigenetic classifier. Finally, we trained a cross-validated specialist classifier using the same features as the TOO classifier, but restricted to HPV-driven cancers. This improved TOO accuracy for detected anal cancers from 11% (1/9) to 100% (9/9), with little effect on other classes. These data support that HPV presence may explain observed cross-scoring patterns between H&N, cervix, and anus TOO prediction classes, which were driven by recurrent epigenomic changes in participants with HPV-positive cancers as detected by this assay. This suggests that modelling axes of shared biology across cancer types can be useful for accurate cfDNA TOO classification, which is critical to direct diagnostic work-up of diverse cancer types in a multi-cancer early detection test. Citation Format: Robert Calef, Oliver Venn, M. Cyrus Maher, John F. Beausang, Earl Hubbell, Aman Patel, Alexander P. Fields, Joerg Bredno, Arash Jamshidi, Alexander M. Aravanis. HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2114.

Chronic traumatic encephalopathy (CTE) is a postmortem diagnosis. Consensus postmortem, but not antemortem, diagnostic criteria have been established. A key factor in these criteria is evidence of phosphorylated-tau (p-tau) around sulcal vessels in the cortex. However, this sign has been observed anecdotally in a diverse range of neurodegenerative diseases (NDD). We therefore hypothesise that this criterion may lack specificity. To test this, we assessed patients with NDD, but no documented history of brain trauma, for sulcal p-tau. Tissue was retrieved from Dublin Brain Bank (known NDD n = 17; control with no diagnosed NDD n = 6; CTE n = 1), and slides were prepared from three sites with a predilection for trauma: superior frontal gyrus, temporal pole, and superior temporal gyrus. We stained the resulting anonymised slides with both hemotoxylin and eosin (H&E) and p-tau. Three neuropathologists, blinded to the clinical history and neuropathological diagnosis in each instance, evaluated each case for sulcal p-tau. We calculated the interrater agreement, using Fleiss’s kappa, and the specificity of this neuropathological sign. Sulcal p-tau was highly specific to diagnosed CTE cases (specificity 0.98), with moderate interrater agreement (κ = 0.45). In conclusion, therefore, we observed sulcal p-tau to be a sign highly specific to CTE when compared with NDD cases in the absence of head trauma.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the effects of neoadjuvant treatments in adults with American Joint Committee on Cancer (AJCC) stage III or stage IV melanoma.

Chronic postsurgical pain (CPSP) is a distressing disease process that can lead to long-term disability, reduced quality of life, and increased health care spending. Although the exact mechanism of development of CPSP is unknown, nerve injury and inflammation may lead to peripheral and central sensitization. Given the complexity of the disease process, no novel treatment has been identified. The preoperative use of multimodal analgesia has been shown to decrease acute postoperative pain, but it has no proven efficacy in preventing development of CPSP.

Neuroendocrine tumors (NETs) arising from tailgut cysts are a rare but increasingly reported entity with gene expression profiles that may be indicative of the gastrointestinal cell of origin. We present a case report describing the unique pathological and genomic characteristics of a tailgut cyst NET that metastasized to liver. The histologic and immunohistochemical findings were consistent with a well-differentiated NET. Genomic testing indicates a germline frameshift in BRCA1 and a few somatic mutations of unknown significance. Transcriptomic analysis suggests an enteroendocrine L cell in the tailgut as a putative cell of origin. Genomic profiling of a rare NET and metastasis provides insight into its origin, development, and potential therapeutic options.

Significance The recent advances in cancer immunotherapy motivated us to investigate the clonal structure of the T cell receptor repertoire in breast tumors, normal breast, and blood in the same individuals. We found quantitatively distinct clonal structures in all three tissues, which enabled us to predict whether tissue is normal or tumor solely by comparing the repertoire of the tissue with blood. T cell receptor sequences shared between patients’ tumors are rare and, in general, do not appear to be specific to the cancer., Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance “enriched” sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment.

Epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT) promoter via methylation in a glioblastoma (GBM), has been correlated with a more favourable response to alkylating chemotherapeutic agents such as temozolomide. The use of global methylation surrogates such as Long Interspersed Nucleotide Element 1 (LINE1) may also be valuable in order to fully understand these highly heterogeneous tumours. In this study, we analysed both original and recurrent GBMs in 22 patients (i.e. 44 tumours), for both MGMT and LINE1 methylation status. In the 22 patients: 14 (63.6%) displayed MGMT methylation stability in the recurrent GBM versus 8 (36.4%), with instability of methylation status. No significant differences in overall and progression free survival was evident between these two groups. LINE1 methylation status remained stable for 12 (54.5%) of recurrent GBM patients versus 9 (41%) of the patients with instability in LINE1 methylation status (p = 0.02), resulting in an increase in overall survival of the stable LINE1 group (p = 0.04). The results obtained demonstrated major epigenetic instability of GBMs treated with temozolomide as part of the STUPP protocol. GBMs appear to undergo selective evolution post-treatment, and have the ability to recur with a newly reprogrammed epigenetic status. Selective targeting of the altered epigenomes in recurrent GBMs may facilitate the future development of both prognostic biomarkers and enhanced therapeutic strategies.

Tumor infiltrating T-cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T-cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T-cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T-cell beta chain repertoire in 16 patients with early stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing approximately 3-fold more T-cells, but with a lower fraction of unique sequences and higher clonality compared to normal breast. The clonal structure of T-cells in blood and normal breast is more similar than between blood and tumor and can be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T-cells overlap between tissues from the same patient, including approximately 50% of T-cells between tumor and normal breast. Both solid tissues contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T-cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T-cells in both tumor and normal breast. Enriched T-cell sequences are typically unique to each patient, but there is a subset of sequences that are shared between many different patients. We show that most of these are commonly generated sequences and thus unlikely to play an important role in the tumor microenvironment.

The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of “orphan” T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile identification of tumor antigens through unbiased screening.

243 Background: Circulating Cell-free Genome Atlas (CCGA; NCT02889978) is a multi-center, case-control, observational study with longitudinal follow-up to develop a cfDNA assay in which classifiers were trained on whole-genome bisulfite sequencing (WGBS) and targeted methylation (TM) sequencing data for detection of multiple cancer types. Previously, we showed that the fraction of ctDNA fragments (TF) was a stronger predictor of cancer detection than clinical stage and an equivalent predictor for survival. Given that CRC tumors can be described via surface area (TSA) and microscopic tumor extent (microinvasion), CRC was used as a model to examine the biophysical determinants of TF. Methods: Detection of multiple cancers with WGBS at 98% and TM at > 99% specificity, and methods for determining TF, were previously reported. A model to predict the presence of detectable cfDNA fragments for CRC adenocarcinomas of stages I, II, and III included TSA and microinvasion beyond the subserosa. Predictors were combined assuming a linear increase of cfDNA shedding with tumor size, with scaling factors depending on microinvasion. Model parameters were determined for 27 participants (7, 11, 9 for stages I, II, III, resp.) with WGBS and applied to 40 participants (12, 15, 13 for I, II, III, resp.) with TM assay and information on tumor size and microinvasion. Results: CRC detection at stages I/II/III was 33/46, 61/73, 57/74% for WGBS/TM. TF predicted detection with AUC = 97.6. The model predicted TF as TSA multiplied by 3.81*10−6 / mm2 for tumors that invaded beyond the subserosa (p < 0.001). This was 4.4x higher than estimates for tumors below the subserosa. The model trained on the WGBS assay predicted CRC detection in the TM assay with an AUC of 0.844. Conclusions: This model used TSA (number of tumor cells) and microinvasion (bloodstream access) to predict the fraction of CRC ctDNA fragments in blood without needing to account for stage. Tumors not penetrating the subserosa had low ctDNA shedding that likely limited detection. These findings may generalize to other cancer types, providing principles to predict ctDNA shedding and thus cancer detectability based on microinvasion and surface area. Clinical trial information: NCT02889978.

Though an estimated 16 417 women and girls in Philadelphia, PA are currently living with or at risk of FGM/C, the U.S. has no formal protocols for assessing risk of FGM/C or delivering prevention counseling. Physicians from the Drexel Women's Care Center specialty clinic for FGM/C have developed a model for counseling parents of girls who may be at risk. Designed for use in various pediatric and primary care settings, the model includes the following steps: Establish family's country of origin. 2) Promote discussion about exposure to FGM/C. 3) Offer services related to FGM/C for affected women. 4) Explore and assess risk to female children. 5) Discuss health consequences and U.S. legal repercussions. 6) Refer to child services as needed. To facilitate implementation in practice settings, we have developed a toolkit with the following segments: a list of high prevalence countries, considerations for identifying at-risk patients, information on the health consequences of FGM/C, legal resources, and practices for documentation in medical records. Training pediatricians to screen for FGM/C risk bridges a critical gap in U.S.-based prevention efforts. By implementing a protocol for counseling parents of at-risk girls, providers can play a key role in prevention of FGM/C among future generations.

Background Our previous discovery work identified whole-genome bisulfite sequencing as outperforming whole-genome and targeted sequencing approaches for multi-cancer detection. We developed a targeted methylation assay for multi-cancer detection and tissue of origin (TOO) localization. Methods Participants were from Circulating Cell-free Genome Atlas (CCGA; NCT02889978) and STRIVE (NCT03085888), both prospective, multi-center, observational studies with longitudinal follow-up. cfDNA from 3,583 evaluable samples (1,530 cancer, 2,053 non-cancer) spanned >20 tumor types of all stages; a prespecified subset of these comprised the "multi-cancer" group: anorectal, hormone-receptor (HR)-negative breast, colorectal, esophageal, gallbladder, gastric, head and neck, hepatobiliary, lung, lymphoid leukemia, lymphoma, multiple myeloma, ovarian, and pancreatic (937 cancer [all stages]). A cross-validated targeted methylation test evaluated cfDNA for predictability of cancer presence and TOO; precision defined as the fraction of correct calls. Breast and lung cancer subtypes were also assessed. All analyses targeted 99.4% specificity (0.6% false-positive rate). Results Participants with and without cancer were similar in age. Specificity was set to 99.4%. For the prespecified multi-cancer group, overall sensitivity was 76% (73-78): stage I 32% (25-40), stage II 76% (69-82), stage III 85% (80-89), and stage IV 93% (89-95). Among all samples, overall sensitivity was 55% (52-57%): stage I 19% (15-23%), stage II 43% (38-48%), stage III 78% (73-82%), and stage IV 90% (86-93%). Overall TOO precision for the multi-cancer group and all samples was 89%, and was similar across stages. Squamous-cell and small-cell lung cancer had higher sensitivity than adenocarcinoma: 84% (75-91) vs 83% (69-92) vs 58% (49-67); HR-negative had higher sensitivity than HR-positive breast cancer: 66% (53-77) vs 20% (15-27). Conclusions This targeted methylation assay detected cancer signal across >20 cancer types with a single, fixed, low false positive rate and highly accurate TOO localization. These data support the feasibility of a single test that can screen for multiple cancers. Clinical trial identification NCT02889978, NCT03085888. Editorial acknowledgement Sarah Prins, PhD (GRAIL, Inc.), and Megan P. Hall, PhD (GRAIL, Inc.). Legal entity responsible for the study GRAIL, Inc. Funding GRAIL, Inc. Disclosure G.R. Oxnard: Advisory / Consultancy, Officer / Board of Directors: Inivata; Honoraria (self): Guardant Health; Honoraria (self): Sysmex; Honoraria (self): Bio-Rad; Advisory / Consultancy: DropWorks; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: GRAIL, Inc.. E.A. Klein: Advisory / Consultancy: GRAIL, Inc.; Advisory / Consultancy: Genomic Health; Advisory / Consultancy: GenomeDx Biosciences. M.V. Seiden: Shareholder / Stockholder / Stock options, Full / Part-time employment: McKesson. E. Hubbell: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. O. Venn: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. A. Jamshidi: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc.; Shareholder / Stockholder / Stock options: Illumina. N. Zhang: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. J.F. Beausang: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. S. Gross: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. K.N. Kurtzman: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc.; Shareholder / Stockholder / Stock options: Illumina. E.T. Fung: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. B. Allen: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. A.P. Fields: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. H. Liu: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. A.M. Aravanis: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. A. Hartman: Shareholder / Stockholder / Stock options, Full / Part-time employment: GRAIL, Inc. M.C. Liu: Honoraria (institution): GRAIL, Inc. All other authors have declared no conflicts of interest.

CCGA [NCT02889978] is the largest study of cfDNA-based early cancer detection; the first CCGA learnings from multiple cfDNA assays are reported here. This prospective, multi-center, observational study has enrolled 10,012 of 15,000 demographically-balanced participants at 141 sites. Blood was collected from participants with newly diagnosed therapy-naive cancer (C, case) and participants without a diagnosis of cancer (noncancer [NC], control) as defined at enrollment. This preplanned substudy included 878 cases, 580 controls, and 169 assay controls (n=1627) across 20 tumor types and all clinical stages. All samples were analyzed by: 1) Paired cfDNA and white blood cell (WBC)-targeted sequencing (60,000X, 507 gene panel); a joint caller removed WBC-derived somatic variants and residual technical noise; 2) Paired cfDNA and WBC whole-genome sequencing (WGS; 35X); a novel machine learning algorithm generated cancer-related signal scores; joint analysis identified shared events; and 3) cfDNA whole-genome bisulfite sequencing (WGBS; 34X); normalized scores were generated using abnormally methylated fragments. In the targeted assay, non-tumor WBC-matched cfDNA somatic variants (SNVs/indels) accounted for 76% of all variants in NC and 65% in C. Consistent with somatic mosaicism (i.e., clonal hematopoiesis), WBC-matched variants increased with age; several were non-canonical loss-of-function mutations not previously reported. After WBC variant removal, canonical driver somatic variants were highly specific to C (e.g., in EGFR and PIK3CA, 0 NC had variants vs 11 and 30, respectively, of C). Similarly, of 8 NC with somatic copy number alterations (SCNAs) detected with WGS, 4 were derived from WBCs. WGBS data revealed informative hyper- and hypo-fragment level CpGs (1:2 ratio); a subset was used to calculate methylation scores. A consistent “cancer-like” signal was observed in 99% specificity for invasive cancer, and support the promise of cfDNA assay for early cancer detection. Additional data will be presented on detected plasma:tissue variant concordance and on multi-assay modeling. Citation Format: Alexander A. Aravanis, Geoffrey R. Oxnard, Tara Maddala, Earl Hubbell, Oliver Venn, Arash Jamshidi, Ling Shen, Hamed Amini, John A. Beausang, Craig Betts, Daniel Civello, Konstantin Davydov, Saniya Fazullina, Darya Filippova, Sante Gnerre, Samuel Gross, Chenlu Hou, Roger Jiang, Byoungsok Jung, Kathryn Kurtzman, Collin Melton, Shivani Nautiyal, Jonathan Newman, Joshua Newman, Cosmos Nicolaou, Richard Rava, Onur Sakarya, Ravi Vijaya Satya, Seyedmehdi Shojaee, Kristan Steffen, Anton Valouev, Hui Xu, Jeanne Yue, Nan Zhang, Jose Baselga, Rosanna Lapham, Daron G. Davis, David Smith, Donald Richards, Michael V. Seiden, Charles Swanton, Timothy J. Yeatman, Robert Tibshirani, Christina Curtis, Sylvia K. Plevritis, Richard Williams, Eric Klein, Anne-Renee Hartman, Minetta C. Liu. Development of plasma cell-free DNA (cfDNA) assays for early cancer detection: first insights from the Circulating Cell-Free Genome Atlas Study (CCGA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-343.

LBA8501 Background: Plasma cfDNA genomic analysis is used widely for the care of advanced lung cancer, but its suitability for early stage lung cancer detection is not well established. CCGA (NCT02889978) is a prospective, multi-center, observational study launched for the development of a noninvasive assay for cancer detection. Methods: Blood was prospectively collected (N = 1627) from 749 controls (no cancer diagnosis) and 878 participants (pts) with newly-diagnosed untreated cancer in this preplanned substudy, including 127 pts with lung cancer. Three prototype sequencing assays were performed: paired cfDNA and white blood cell (WBC) targeted sequencing (507 genes, 60,000X) for single nucleotide variants/indels; paired cfDNA and WBC whole genome sequencing (WGS) for copy number variation (30X); and cfDNA whole genome bisulfite sequencing (WGBS) for methylation (30X). For each assay, a classification model using 10-fold cross-validation was developed for all pts with cancer, then evaluated in the pts with lung cancer; sensitivity was estimated at 95% specificity. Results: We evaluated pts with lung cancer (127) and a subset of controls (580) with similar ages (mean±SD yrs: 67±9, 60±13), 85% and 43% were ever-smokers, and 46% and 22% were men, respectively. Of 3055 nonsynonymous mutations detected across 122 evaluable pts with lung cancer, > 50% were detected in WBC consistent with clonal hematopoiesis (CH). Accounting for CH, sensitivity in 63 stage I-IIIA pts evaluable across all 3 assays was 48% (35-61, targeted), 54% (41-67, WGS), and 56% (43-68, WGBS); in 54 stage IIIB-IV pts it was 85% (73-93, targeted), 91% (80-97, WGS), and 93% (82-98, WGBS) . Similar sensitivities were observed across histological subtypes (adenocarcinoma, squamous cell, small cell). Comparison to tumor WGS and multi-assay classification will be reported. Conclusions: Early stage lung cancers are detectable in cfDNA using a genome-wide sequencing approach. For lung cancer detection using targeted assays, CH must be accounted for to minimize false positives. Assay optimization is ongoing to allow further clinical development in the intended use population. Clinical trial information: NCT02889978.

12003Background: CHIP is defined by the presence of age-dependent acquired mutations in hematopoietic progenitor cells and has been reported to occur in up to 30% of individuals 60-70 years of age....