Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

BCR-ABL1 transcript doubling time as a predictor for treatment-free remission failure after imatinib discontinuation in chronic myeloid leukaemia in chronic phase.

The doubling time (DT) of the BCR-ABL1 quantitative polymerase chain reaction (qPCR) transcript level reflects the re-growing fraction of leukaemic cells after discontinuation of tyrosine kinase inhibitor (TKI). The present study analyzed monthly DT within six months after imatinib discontinuation in 131 patients. Monthly DT was calculated as x = ln(2)/K, where x is the DT and K is the fold BCR-ABL1 change from the previous value divided by the number of days between each measurement. The optimal DT value was determined as 12·75 days at two months using a recursive partitioning method. The patients were stratified into three groups: the high-risk group (DT<12·75 days but >0, with rapidly proliferating chronic myeloid leukaemia (CML) cells; n = 26) showed the lowest molecular relapse-free survival (mRFS) of 7·7% at 12 months, compared to 53·6% in the intermediate-risk group (DT≥12·75 days, with slowly proliferating CML cells; n = 16) or 90·0% in the low-risk group (DT≤0, i.e., without proliferating CML cells; n = 71; P < 0·001). Monthly assessment of DT helps identify high-risk patients for treatment-free remission failure with an imminent risk of molecular recurrence, and to define low-risk patients who can be spared the frequent monitoring of monthly molecular tests.

Optimal duration of imatinib treatment/deep molecular response for treatment-free remission after imatinib discontinuation from a Canadian tyrosine kinase inhibitor discontinuation trial.

Although total duration of tyrosine kinase inhibitor (TKI) therapy and of molecular response at 4 log reduction or deeper (MR4) correlates with treatment-free remission (TFR) success after TKI discontinuation, the optimal cut-off values of the duration remain unresolved. Thus, 131 patients were enrolled into the Canadian TKI discontinuation study. The molecular relapse-free survival (mRFS) was defined from imatinib discontinuation till molecular recurrence, that is, major molecular response (MMR) loss and/or MR4 loss. We evaluated mRFS at 12 months after imatinib discontinuation, analyzed it according to the imatinib treatment duration and MR4 duration, and calculated P value, positive (PPV) and negative predictive value (NPV) in the yearly cut-off period of time. The shortest cut-off was sought that met the joint criteria of a P value ≤ 0·05, PPV ≥ 60% and NPV ≥ 60%. We propose six years as the shortest imatinib duration cut-off with a P value 0·01, PPV 68% and NPV 62%: The patients treated with imatinib duration ≥ 6 years showed a superior mRFS rate (61·8%) compared to those with less treatment (36·0%). Also, 4·5 years MR4 duration as the shortest cut-off with a P value 0·003, PPV 63% and NPV 61%: those with MR4 duration ≥ 4·5 years showed a higher mRFS rate (64·2%) than those with a shorter MR4 duration (41·9%).

Measurable residual disease monitoring provides insufficient lead-time to prevent morphologic relapse in the majority of patients with core-binding factor acute myeloid leukemia.

Core-binding factor acute myeloid leukemia is characterized by t(8;21) or inv(16) and the fusion proteins RUNX1-RUNX1T1 and CBFB-MYH11. International guidelines recommend monitoring for measurable residual disease every 3 months for 2 years after treatment. However, it is unknown if serial molecular monitoring can predict and prevent morphologic relapse. We conducted a retrospective single-center study of 114 patients in complete remission who underwent molecular monitoring with RT-qPCR of RUNX1-RUNX1T1 or CBFB-MYH11 transcripts every 3 months. Morphologic relapse was defined as re-emergence of >5% blasts and molecular relapse as ≥1 log increase in transcript level between 2 samples. Over a median follow-up time of 3.7 years (range 0.2-14.3), remission persisted in 71 (62.3%) patients but 43 (37.7%) developed molecular or morphologic relapse. Patients who achieved <3 log reduction in RUNX1-RUNX1T1 or CBFB-MYH11 transcripts at end of chemotherapy had a significantly higher risk of relapse compared to patients who achieved ≥3 log reduction (61.1% vs. 33.7%, p=0.004). The majority of relapses (74.4%, n=32) were not predicted by molecular monitoring and occurred rapidly with <100 days from molecular to morphologic relapse. Molecular monitoring enabled the detection of impending relapse and permitted pre-emptive intervention prior to morphologic relapse in only 11 (25.6%) patients. The current practice of molecular monitoring every 3 months provided insufficient lead-time to identify molecular relapses and prevent morphologic relapse in the majority of patients with core-binding factor acute myeloid leukemia treated at our institution. Further research is necessary to determine the optimal monitoring strategies for these patients.

The Prevent Ovarian Cancer Program (POCP): Identification of women at risk for ovarian cancer using complementary recruitment approaches.

BACKGROUND: Up to 20% of high-grade serous ovarian carcinomas (HGSOC) are hereditary; however, historical uptake of genetic testing is low. We used a unique combination of approaches to identify women in Ontario, Canada, with a first-degree relative (FDR) who died from HGSOC without prior genetic testing, and offer them multi-gene panel testing. METHODS: From May 2015-Sept 2019, genetic counseling and testing was provided to eligible participants. Two recruitment strategies were employed, including self-identification in response to an outreach campaign and direct targeting of FDRs of deceased HGSOC patients treated at our institution. The rate of pathogenic variants (PV) in established/potential ovarian cancer risk genes and the benefits/challenges of each approach were assessed. RESULTS: A total of 564 women enrolled in response to our outreach campaign (n = 473) or direct recruitment (n = 91). Mean age at consent was 52 years and 96% did not meet provincial testing criteria. Genetic results were provided to 528 individuals from 458 families. The rate of PVs in ovarian cancer risk genes was highest when FDRs were diagnosed with HGSOC <60 years (9.4% vs. 3.9% ≥ 60y, p = 0.0160). Participants in the outreach vs. direct recruitment cohort had a similar rate of PVs; however, uptake of genetic testing (97% vs. 89%; p = 0.0036) and study completion (95% vs. 87%; p = 0.0062) rates were higher in the former. Eleven participants with pathogenic variants have completed risk-reducing gynecologic surgery, with one stage I HGSOC and two breast cancers identified. CONCLUSION: Overall PV rates in this large cohort were lower than expected; however, we provide evidence that genetic testing criteria in Ontario should include individuals with a deceased FDR diagnosed with HGSOC <60 years of age.

Inconsistencies in histone acetylation patterns among different HD model systems and HD post-mortem brains.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene. Emerging evidence shows that additional epigenetic factors can modify disease phenotypes. Harnessing the ability of the epigenome to modify the disease for therapeutic purposes is therefore of interest. Epigenome modifiers, such as histone deacetylase inhibitors (HDACi), have improved pathology in a range of HD models. Yet in clinical trials, HDACi have failed to alleviate HD symptoms in patients. This study investigated potential reasons for the lack of translation of the therapeutic benefits of HDACi from lab to clinic. We analysed histone acetylation patterns of immuno-positive nuclei from brain sections and tissue microarrays from post-mortem human control and HD cases alongside several well-established HD models (OVT73 transgenic HD sheep, YAC128 mice, and an in vitro cell model expressing 97Q mutant huntingtin). Significant increases in histone H4 acetylation were observed in post-mortem HD cases, OVT73 transgenic HD sheep and in vitro models; these changes were absent in YAC128 mice. In addition, nuclear labelling for acetyl-histone H4 levels were inversely proportional to mutant huntingtin aggregate load in HD human cortex. Our data raise concerns regarding the utility of HDACi for the treatment of HD when regions of pathology exhibit already elevated histone acetylation patterns and emphasize the importance of searching for alternative epigenetic targets in future therapeutic strategies aiming to rescue HD phenotypes.

HPV-independent Vulvar Squamous Cell Carcinoma is Associated With Significantly Worse Prognosis Compared With HPV-associated Tumors.

Vulvar squamous cell carcinomas (VSCC) represent the most common carcinoma of the female external genitalia, with increasing incidence. Although high-risk human papillomavirus (HPV) infection has long been implicated in the majority of cervical and anal squamous cell carcinomas, there is uncertainty about its prevalence and prognostic impact in VSCC. In this study, we conducted a retrospective integrated morphologic and multimodal HPV analysis of a cohort of 114 VSCC cases treated at the Princess Margaret Cancer Centre/University Health Network, Toronto, Canada between 2000 and 2010. VSCC histology was reviewed. We analyzed the cohort for HPV using polymerase chain reaction based method, and tissue microarray DNA and RNA in situ hybridization (ISH), and p16 immunohistochemistry. Among the 114 cases (age 70±16 yr), 36.7% of cases were classified as having histomorphology of HPV infection. HPV was detected in 31.9% (polymerase chain reaction), 14.0% (DNA ISH), and 27.3% (RNA ISH) of cases. p16 immunohistochemistry was positive in 37.8% of cases. On univariate analysis, HPV morphology (P=0.009), p16+ (P=0.00013), DNA ISH+ (P=0.021), and RNA ISH+ (P=0.00061) were associated with better 5-yr progression-free survival. DNA ISH+ (P=0.049) was associated with better 5-yr overall survival. On multivariate analysis, HPV morphology (P=0.033), p16+ (P=0.01), and RNA ISH+ (P=0.035) were associated with better 5-yr progression-free survival. In conclusion, a subset of VSCC is associated with HPV, which correlates with better outcome. Relatively inexpensive tests such as histomorphologic evaluation, p16 immunohistochemistry, and HPV RNA ISH can be used to predict outcome in VSCC. Therefore, routine reporting of HPV status in VSCC is recommended.

Brain urea increase is an early Huntington's disease pathogenic event observed in a prodromal transgenic sheep model and HD cases.

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.

Identifying actionable variants using next generation sequencing in patients with a historical diagnosis of undifferentiated pleomorphic sarcoma.

There are limited data regarding the molecular characterization of undifferentiated pleomorphic sarcomas (UPS; formerly malignant fibrous histiocytoma). This study aimed to investigate the utility of next generation sequencing (NGS) in UPS to identify subsets of patients who harbour actionable mutations. Patients diagnosed with UPS underwent pathological re-evaluation by a pathologist specializing in sarcoma. Tumor DNA was isolated from archived fresh frozen tissue samples and genotyped using NGS with the Illumina MiSeq TruSeq Amplicon Cancer Panel (48 genes, 212 amplicons). In total, 95 patients initially classified with UPS were identified. Following pathology re-review the histological subtypes were reclassified to include: Myxofibrosarcoma (MFS, N = 44); UPS(N = 18); and Others (N = 27; including undifferentiated spindle cell sarcoma (N = 15) and dedifferentiated liposarcoma (N = 6)). Seven cases were excluded from further analysis for other reasons. Baseline demographics of the finalized cohort (N = 88) showed a median age of 66 years (32-95), primarily with stage I-III disease (92%) and high-grade (86%) lesions. Somatic mutations were identified in 31 cases (35%)(Total mutations = 36: solitary mutation(n = 27); two mutations( =n = 3); three mutations(n = 1)). The most commonly identified mutations were in TP53 (n = 24), ATM (n = 3) and PIK3CA (n = 2). Three of 43 patients with MFS and one of 18 patients with UPS had clinically relevant mutations, mainly related to biomarkers of prediction of response; however few had targetable driver mutations. Somatic mutation status did not influence disease free or overall survival. Based on the small number of clinically relevant mutations, these data do not support the routine use of targeted NGS panels outside of research protocols in UPS.

Impact of multi-gene mutational profiling on clinical trial outcomes in metastatic breast cancer.

PURPOSE: Next-generation sequencing (NGS) has identified recurrent genomic alterations in metastatic breast cancer (MBC); however, the clinical utility of incorporating routine sequencing to guide treatment decisions in this setting is unclear. We examine the frequency of genomic alterations in MBC patients from academic and community hospitals and correlate with clinical outcomes. METHODS: MBC patients with good performance status were prospectively recruited at the Princess Margaret Cancer Centre (PM) in Canada. Molecular profiling on DNA extracted from FFPE archival tissues was performed on the Sequenom MassArray platform or the TruSeq Amplicon Cancer Panel (TSACP) on the MiSeq platform. Clinical trial outcomes by RECIST 1.1 and time on treatment were reviewed retrospectively. RESULTS: From January 2012 to November 2015, 483 MBC patients were enrolled and 440 were genotyped. At least one somatic mutation was identified in 46% of patients, most commonly in PIK3CA (28%) or TP53 (13%). Of 203 patients with ≥ 1 mutation(s), 15% were treated on genotype-matched and 9% on non-matched trials. There was no significant difference for median time on treatment for patients treated on matched vs. non-matched therapies (3.6 vs. 3.8 months; p = 0.89). CONCLUSIONS: This study provides real-world outcomes on hotspot genotyping and small targeted panel sequencing of MBC patients from academic and community settings. Few patients were matched to clinical trials with targeted therapies. More comprehensive profiling and improved access to clinical trials may increase therapeutic options for patients with actionable mutations. Further studies are needed to evaluate if this approach leads to improved clinical outcomes.

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain.

Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.

Predictive value of molecular remissions postconsolidation chemotherapy in patients with Core Binding Factor Acute Myeloid Leukemia (CBF-AML) - a single center analysis.

We analyzed the outcome of 80 sequential patients with core binding factor acute myeloid leukemia and evaluated the influence of molecular monitoring by quantitative reverse transcriptase polymerase chain reaction. With a median follow-up of 5 years, the estimated 5-year relapse-free survival and overall survival were 58% and 66%, respectively. Patients who were in molecular remission at the completion of consolidation chemotherapy had a 21% risk of relapse, while the relapse risk for those in molecular remission at the end of 2 years was 5.5%. Our data indicate that postconsolidation molecular remission does not necessarily preclude disease relapse and further monitoring is required. In contrast, molecular negativity by quantitative reverse transcriptase polymerase chain reaction at the end of 2 years is associated with a low risk of relapse.

Recurrent genomic alterations in sequential progressive leukoplakia and oral cancer: drivers of oral tumorigenesis?

A significant proportion (up to 62%) of oral squamous cell carcinomas (OSCCs) may arise from oral potential malignant lesions (OPMLs), such as leukoplakia. Patient outcomes may thus be improved through detection of lesions at a risk for malignant transformation, by identifying and categorizing genetic changes in sequential, progressive OPMLs. We conducted array comparative genomic hybridization analysis of 25 sequential, progressive OPMLs and same-site OSCCs from five patients. Recurrent DNA copy number gains were identified on 1p in 20/25 cases (80%) with minimal, high-level amplification regions on 1p35 and 1p36. Other regions of gains were frequently observed: 11q13.4 (68%), 9q34.13 (64%), 21q22.3 (60%), 6p21 and 6q25 (56%) and 10q24, 19q13.2, 22q12, 5q31.2, 7p13, 10q24 and 14q22 (48%). DNA losses were observed in >20% of samples and mainly detected on 5q31.2 (35%), 16p13.2 (30%), 9q33.1 and 9q33.29 (25%) and 17q11.2, 3p26.2, 18q21.1, 4q34.1 and 8p23.2 (20%). Such copy number alterations (CNAs) were mapped in all grades of dysplasia that progressed, and their corresponding OSCCs, in 70% of patients, indicating that these CNAs may be associated with disease progression. Amplified genes mapping within recurrent CNAs (KHDRBS1, PARP1, RAB1A, HBEGF, PAIP2, BTBD7) were selected for validation, by quantitative real-time PCR, in an independent set of 32 progressive leukoplakia, 32 OSSCs and 21 non-progressive leukoplakia samples. Amplification of BTBD7, KHDRBS1, PARP1 and RAB1A was exclusively detected in progressive leukoplakia and corresponding OSCC. BTBD7, KHDRBS1, PARP1 and RAB1A may be associated with OSCC progression. Protein-protein interaction networks were created to identify possible pathways associated with OSCC progression.

Clinical impact of mutation fraction in epidermal growth factor receptor mutation positive NSCLC patients.

BACKGROUND: We examined clinical outcomes in a population-based cohort of EGFR mutant advanced NSCLC patients, exploring the potential role of factors including tumour EGFR mutation fraction and cellularity in predicting outcomes. METHODS: A cohort of patients with EGFR mutant advanced NSCLC was identified (N =2 93); clinical outcomes, pathologic and treatment details were collected. Tumour response was determined from radiology and clinical notes. Association between demographic and pathologic variables EGFR TKI response, time to treatment failure (TTF) and overall survival (OS) was examined using logistic regression and proportional hazards regression. EGFR TKI response rates were summarised by percent mutation fraction to explore their association. RESULTS: Higher mutation fraction was associated with greater EGFR TKI response rate (odds ratio 1.58, 95% CI = 1.21-2.07, P = 0.0008), longer TTF (hazard ratio 0.80, 95% CI = 0.68-0.92, P = 0.003) and better OS (hazard ratio 0.81, 95% CI = 0.67-0.99, P = 0.04). However, even in patients with ⩽ 5% mutation fraction, response rate was 34%. Females had longer TTF (P = 0.02). CONCLUSIONS: EGFR mutation fraction in tumour samples was significantly associated with response, TTF and OS. Despite this, no lower level of mutation fraction was detected for which EGFR TKI should be withheld in those with activating EGFR mutations.

A classification system for clinical relevance of somatic variants identified in molecular profiling of cancer.

PURPOSE: Interpretation systems for clinical laboratory reporting of genetic variants for inherited conditions have been widely published. By contrast, there are no existing systems for interpretation and classification of somatic variants found from molecular testing of cancer. METHODS: We designed an assessment protocol and classification system for somatic variants identified through next-generation sequencing molecular profiling of tumor-derived samples and applied these to a pilot dataset of somatic variants found by next-generation sequencing profiling of 158 tumor samples derived from advanced cancer patients examined at the Princess Margaret Cancer Centre. RESULTS: We present a classification system to interpret the significance of genetic variants in molecular analysis of cancer, including the following key factors: (i) known or predicted pathogenicity of the variant; (ii) primary site and tumor histology in which the variant is found; (iii) recurrence of the variant; and (iv) evidence of clinical actionability. We used these factors to develop a five-category somatic variant classification for simplified reporting of variant interpretations to treating oncologists. CONCLUSION: Our somatic variant classification can be of practical value to other clinical molecular laboratories performing cancer genetic profiling by promoting consistent reporting of somatic variants and permitting harmonization of variant data among laboratories and clinical studies.

Extracorporeal photopheresis in solid organ transplant-associated acute graft-versus-host disease.

BACKGROUND: Extracorporeal photopheresis (ECP) culls pathogenic T lymphocytes, be these the clones of cutaneous T-cell lymphoma, or mediators of chronic graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT-GVHD). Whether or not ECP may have an effect in the rarer instances of solid organ transplantation-associated GVHD (SOT-GVHD) is unclear. Mortality rates in SOT-GVHD rival those of transfusion-associated GVHD, with fatalities preceded by pancytopenia and peripheral blood chimerism (PBC) levels exceeding 20%. ECP has been described in two SOT-GVHD cases to date, with one surviving. STUDY DESIGN AND METHODS: Clinicolaboratory features (including HLA relationships) in a case of multivisceral transplantation were reviewed from the time of surgery to the onset and progression of SOT-GVHD. ECP, which was introduced as a less immunosuppressive and more selective intervention, was assessed for its effect on serial PBC (as measured by short-tandem-repeat analysis) and clinical outcome. RESULTS: Multivisceral SOT-GVHD manifested with erythroderma, neutropenic sepsis, and PBC increasing from 6% on Posttransplant Day (PTD) 38 to 78% by PTD 60 (at a doubling time of 6 days despite corticosteroids). ECP was administered on PTDs 62 and 67 and was associated with the first evidence of PBC decay to 67% on PTD 69. Death nevertheless ensued on the last day of salvage antithymocyte globulin (PTDs 69-73) despite further PBC reduction to 41%. CONCLUSION: Further study is needed to determine if the sooner or more frequent application of ECP might attenuate the high case fatality rates of SOT-GVHD.

Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

Pathologic complete response to intralesional interleukin-2 therapy associated with improved survival in melanoma patients with in-transit disease.

PURPOSE: Melanoma patients with in-transit disease have a high mortality rate despite various treatment strategies. The aim of this study was to validate the role of intralesional interleukin (IL)-2, to understand its mechanism of action, and to better understand factors that may influence its response. METHODS: We retrospectively collected the clinicopathological data of 31 consecutive patients who presented to a tertiary care cancer center for treatment of in-transit melanoma with intralesional IL-2. Kaplan-Meier survival curves and multivariable Cox regression analysis were performed. Immunohistochemistry (IHC) was used to better understand the immune response to localized IL-2 therapy. Targeted next-generation sequencing was performed to genomically characterize the tumors. RESULTS: Ten patients (10/31, 32 %) achieved a pathologic complete response (pCR), 17/21 (55 %) had a partial response, and 4/21 (19 %) had progressive disease on treatment. pCR to IL-2 therapy was associated with overall survival (log-rank p = 0.004) and improved progression-free survival (PFS) [adjusted hazard ratio (HR) 0.11; 95 % CI 0.02-0.47; p = 0.003). A higher CD8+ T cell infiltrate was identified in in-transit lesions with a pCR compared with the other lesions (mean IHC score 3.78 vs. 2.61; p = 0.01). Patients with an elevated CD8+ infiltrate demonstrated an improved PFS (unadjusted HR 0.08; 95 % CI 0.01-0.52; p = 0.008). CONCLUSIONS: Thirty-two percent of patients achieved pCR with intralesional IL-2 therapy and had a significantly improved PFS compared with the rest of the cohort, which may be explained by a systemic CD8+ T-cell response.

Malignant Melanoma of Vulva and Vagina: A Histomorphological Review and Mutation Analysis--A Single-Center Study.

OBJECTIVES: The aim of this work was to determine molecular characteristics and specifically, the frequency of BRAF, C-KIT, and NRAS mutations in vulvar and vaginal melanomas. METHODS: A retrospective review of all cases of vulvar and vaginal melanoma between 2002 and 2013 was performed. We reviewed the clinical and histological characteristics of all cases and performed genotyping studies on cases that had tissue available for the study, using next-generation sequencing. RESULTS: We identified 33 vulvar and 11 vaginal melanomas in women with mean ages 58 and 61 years, respectively. Next-generation sequencing analysis on 20 cases (15 vulvar and 5 vaginal) identified a BRAF mutation in 7.6%, C-KIT mutation in 27.6%, NRAS mutation in 27.6%, and TP53 mutation in 7.6% of the vulvar cases. We detected only a single TP53 mutation in the vaginal cases. We did not identify any statistically significant relationship between the mutation status and patients' outcome, depth of invasion, ulceration, stage at presentation, or lymph node metastasis. CONCLUSIONS: BRAF mutations are infrequent, whereas C-KIT and NRAS mutations are seen with higher frequency in vulvar melanomas than melanomas of other sites. These mutations can be considered as potential therapeutic targets in patients harboring them. Further studies are necessary to increase our understanding of mutational events occurring in melanoma of the lower female genital tract and their relationship with clinical parameters/outcome.