Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Molecular immuno-imaging improves tumor detection in head and neck cancer.

Detection and accurate delineation of tumor is important for the management of head and neck squamous cell carcinoma (HNSCC) but is challenging with current imaging techniques. In this study, we evaluated whether molecular immuno-imaging targeting myeloperoxidase (MPO) activity, an oxidative enzyme secreted by many myeloid innate immune cells, would be superior in detecting tumor extent compared to conventional contrast agent (DTPA-Gd) in a carcinogen-induced immunocompetent HNSCC murine model and corroborated in human surgical specimens. In C57BL/6 mice given 4-nitroquinoline-N-oxide (4-NQO), there was increased MPO activity in the head and neck region as detected by luminol bioluminescence compared to that of the control group. On magnetic resonance imaging, the mean enhancing volume detected by the MPO-targeting agent (MPO-Gd) was higher than that by the conventional agent DTPA-Gd. The tumor volume detected by MPO-Gd strongly correlated with tumor size on histology, and higher MPO-Gd signal corresponded to larger tumor size found by imaging and histology. On the contrary, the tumor volume detected by DTPA-Gd did not correlate as well with tumor size on histology. Importantly, MPO-Gd imaging detected areas not visualized with DTPA-Gd imaging that were confirmed histopathologically to represent early tumor. In human specimens, MPO was similarly associated with tumors, especially at the tumor margins. Thus, molecular immuno-imaging targeting MPO not only detects oxidative immune response in HNSCC, but can better detect and delineate tumor extent than nonselective imaging agents. Thus, our findings revealed that MPO imaging could improve tumor resection as well as be a useful imaging biomarker for tumor progression, and potentially improve clinical management of HNSCC once translated.

Leucocyte-derived micro-RNAs as candidate biomarkers in Brugada syndrome.

AIMS: Risk stratification for sudden cardiac death in patients with Brugada syndrome remains a major challenge. Contemporary risk prediction models have only modest predictive value. The aim of this study was to assess the role of micro-RNAs from peripheral blood as candidate biomarkers in Brugada syndrome. METHODS AND RESULTS: In this prospective study, Brugada patients and unaffected control individuals were enrolled for analysis of leucocyte-derived microRNAs (miRNAs) levels. Expression levels of 798 different circulating miRNAs were analysed on the NanoString® nCounter platform. All results were cross-validated by using a quantitative polymerase chain reaction. Micro-RNA expression levels of Brugada patients were compared with clinical data. A total of 21 definite Brugada patients (38% with a history of ventricular arrhythmia or cardiac arrest) and 30 unaffected control individuals were included in the study. Micro-RNA analysis showed a distinct expression profile in Brugada patients with 42 differentially expressed markers (38 up-regulated, 4 down-regulated miRNAs). The symptom status of Brugada patients was associated with a distinct miRNA signature. Micro-RNAs 145-5p and 585-3p were significantly up-regulated in symptomatic Brugada patients (P = 0.04). Incorporating miRNAs 145-5p and 585-3p into a multivariable model demonstrated significantly increased symptom prediction (area under the curve = 0.96; 95% confidence interval: 0.88-1.00). CONCLUSION: Brugada patients display a distinct miRNA expression profile compared with unaffected control individuals. There is also evidence that certain miRNAs (miR-145-5p and miR-585-3p) are associated with the symptom status of Brugada patients. The results suggest the principal utility of leucocyte-derived miRNAs as prognostic biomarkers for Brugada syndrome.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 µg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Discovery of a putative blood-based protein signature associated with response to ALK tyrosine kinase inhibition.

BACKGROUND: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. METHODS: Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. RESULTS: Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 < PFS < 24 months (n = 10)] and poor responders [PFS ≤ 3 months (n = 5)]. Several proteins were identified as differentially expressed between long-term responders and poor responders, including DPP4, KIT and LUM. Next, using machine learning algorithms, we evaluated the classification potential of 40 proteins. Finally, by integrating the different analytic methods, we selected 22 proteins as potential candidates for a blood-based prognostic signature of response to crizotinib in NSCLC patients harboring ALK fusion. CONCLUSION: In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy.Trial registration ClinicalTrials.gov, NCT02041468. Registered 22 January 2014, https://clinicaltrials.gov/ct2/show/NCT02041468?term=NCT02041468&rank=1.

Tumour budding predicts increased recurrence after curative resection for T2N0 colorectal cancer

Background: Tumour budding is defined as the presence of a cluster of fewer than 5 cells along the invasive margin. It may confer a worse prognosis in colorectal cancer, but its importance in pT2N0 colorectal cancer is unknown. This study aimed to determine the prognostic value of tumour budding in pT2N0 colorectal cancer. Methods: This was a retrospective cohort study with prospective assessment of tumour budding by 2 pathologists. We included all patients who underwent elective curative resection for pT2N0 colorectal cancer except those with hereditary colorectal cancer syndromes, inflammatory bowel disease or positive resection margins, those who received neoadjuvant or adjuvant therapy and those who died within 90 days of operation. Patients were classified as having high-grade tumour budding (≥ 10 budding foci per high-power field) or low-grade tumour budding (< 9 budding foci per high-power field). The main outcome measure was locoregional or distant recurrence. Results: Of 85 patients, 36 had high-grade tumour budding and 49 had low-grade tumour budding. The overall recurrence rate was 11% (9/85) and median follow-up was 41.0 months (interquartile range 22.0­68.0). Interrater reliability for tumour budding assessment was excellent (κ = 0.86, 95% confidence interval [CI] 0.76­0.96). There were more recurrences in patients with high-grade tumour budding (7/36, 19.4% v. 2/49, 4.1%; p = 0.020). On multivariate analysis, after we adjusted for confounders, the presence of high-grade tumour budding was independently associated with recurrence (hazard ratio 5.11, 95% CI 1.01­25.9). Conclusion: Tumour budding was independently associated with increased recurrence after pT2N0 colorectal cancer resection. It offers additional prognostic information that may affect treatment strategy.

Contexte: Le bourgeonnement tumoral se définit par la présence d'un amas de 5 cellules ou moins le long de la marge invasive. Il pourrait conférer un pronostic plus sombre dans le cancer colorectal, mais on ignore sa portée dans le cancer colorectal de stade pT2N0. Cette étude visait à déterminer la valeur pronostique du bourgeonnement tumoral dans un cancer colorectal de stade pT2N0. Méthodes: Il s'agit d'une étude de cohorte rétrospective avec évaluation prospective du bourgeonnement tumoral par 2 pathologistes. Nous avons inclus tous les patients ayant subi une résection curative non urgente pour un cancer colorectal de stade pT2N0, sauf ceux qui présentaient un syndrome de cancer colorectal héréditaire, une maladie inflammatoire de l'intestin ou des marges de résection positives ceux qui avaient reçu des traitements néoadjuvants ou adjuvants et ceux qui étaient décédés dans les 90 jours suivant l'intervention. Les patients ont été classés selon qu'ils avaient un bourgeonnement tumoral de haut grade (≥ 10 foyers bourgeonnants par champ à fort grossissement) ou bourgeonnement tumoral de bas grade (< 9 foyers bourgeonnants par champ à fort grossissement). Le principal paramètre était la récurrence locorégionale ou distante. Résultats: Sur 85 patients, 36 présentaient un bourgeonnement tumoral de haut grade, et 49, de bas grade. Le taux de récurrence global a été de 11 % (9/85) et le suivi médian a été de 41,0 mois (intervalle interquartile 22,0­68,0). La fiabilité interévaluateur de l'évaluation du bourgeonnement tumoral a été excellente (κ = 0,86, intervalle de confiance [IC] de 95 % 0,76­0,96). Les récurrences ont été plus nombreuses chez les patients qui avaient un bourgeonnement tumoral de haut grade (7/36, 19,4 % c. 2/49, 4,1 %; p = 0,020). À l'analyse multivariée, après ajustement pour tenir compte des variables de confusion, la présence de bourgeonnement tumoral de haut grade s'est révélée indépendamment liée à la récurrence (risque relatif 5,11, IC de 95 % 1,01­25,9). Conclusion: Le bourgeonnement tumoral s'est révélé indépendamment lié à l'augmentation des récurrences après la résection pour cancer colorectal de stade pT2N0. Il offre une information pronostique additionnelle qui pourrait influer sur la stratégie thérapeutique.

Do clinical criteria reflect pathologic complete response in rectal cancer following neoadjuvant therapy?

BACKGROUND: Clinical complete response (cCR) in rectal cancer is being evaluated as a tool to identify patients who would not require surgery in the curative management of rectal cancer. Our study reviews mucosal changes after neoadjuvant therapy for rectal cancer in patients treated at our center. METHODS: Pathology reports were retrieved for patients treated with neoadjuvant chemoradiation therapy (CRT) or high-dose rate brachytherapy (HDRBT). The macroscopic appearance of the specimen was compared with pathologic staging. RESULTS: This study included 282 patients: 88 patients underwent neoadjuvant CRT and 194 patients underwent HDRBT; all patients underwent total mesorectal excision (TME). There were 160 male and 122 female patients with a median age of 65 years (range 29-87). The median time between neoadjuvant therapy and surgery was 50 and 58 days. Sixty patients (21.2%) were staged as ypT0N0, 21.2% had a pathologic complete response (pCR), and only 3.2% had a cCR. Of the 67 patients with initial involvement of the circumferential radial margin (CRM), 44 converted to pathologic CRM-. Two hundred seventy-three patients (96.8%) had mucosal abnormalities. Of the 222 patients with residual tumor, 70 patients had no macroscopic tumor visualized but an ulcer in its place. CONCLUSION: Most patients undergoing neoadjuvant therapy for rectal cancer have residual mucosal abnormalities which preclude to a cCR as per published criteria from Brazil. Further studies are required to optimize clinical evaluation and MRI imaging in selected patients.

Cutaneous melanoma.

In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. Surgery for localised melanoma and regional lymph node metastases is the standard of care. Sentinel-node biopsy provides precise staging, but has not been reported to affect survival. The effect of lymph-node dissection on survival is a topic of investigation. Two distinct approaches have emerged to try to extend survival in patients with metastatic melanoma: immunomodulation with anti-CTLA4 monoclonal antibodies, and targeted therapy with BRAF inhibitors or MEK inhibitors for BRAF-mutated melanoma. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Response patterns differ substantially-anti-CTLA4 immunotherapy can induce long-term responses, but only in a few patients, whereas targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. Anti-PD1 and anti-PDL1 antibodies have emerged as breakthrough drugs for melanoma that have high response rates and long durability. Biomarkers that have predictive value remain elusive in melanoma, although emerging data for adjuvant therapy indicate that interferon sensitivity is associated with ulceration of the primary melanoma. Intense investigation continues for clinical and biological markers that predict clinical benefit of immunotherapeutic drugs, such as interferon alfa or anti-CTLA4 antibodies, and the mechanisms that lead to resistance of targeted drugs.

Real-World Evidence of the Impact of the COVID-19 Pandemic on Lung Cancer Survival: Canadian Perspective.

Background: The effect of COVID-19 on treatment outcomes in the literature remains limited and is mostly reported either as predictive survival using prioritization and modeling techniques. We aimed to quantify the effect of COVID-19 on lung cancer survival using real-world data collected at the Jewish General Hospital, Montreal. Methods: This is a retrospective chart review study of patients diagnosed between March 2019 and March 2022. We compared three cohorts: pre-COVID-19, and 1st and 2nd year of the pandemic. Results: 417 patients were diagnosed and treated with lung cancer at our centre: 130 in 2019, 103 in 2020 and 184 in 2021. Although the proportion of advanced/metastatic-stage lung cancer remained the same, there was a significant increase in the late-stage presentation during the pandemic. The proportion of M1c (multiple extrathoracic sites) cases in 2020 and 2021 was 57% and 51%, respectively, compared to 31% in 2019 (p < 0.05). Median survival for early stages of lung cancer was similar in the three cohorts. However, patients diagnosed in the M1c stage had a significantly increased risk of death. The 6-month mortality rate was 53% in 2021 compared to 47% in 2020 and 29% in 2019 (p = 0.004). The median survival in this subgroup of patients decreased significantly from 13 months in 2019 to 6 months in 2020 and 5 months in 2021 (p < 0.001). Conclusions: This study is, to our knowledge, the largest single-institution study in Canada looking at lung cancer survival during the COVID-19 pandemic. Our study looks at overall survival in the advanced/metastatic setting of NSCLC during the COVID-19 pandemic. We have previously reported on treatment pattern changes and increased wait times for NSCLC patients during the pandemic. In this study, we report that the advanced/metastatic subgroup had both an increase in the 6-month mortality rate and worsening overall survival during this same time period. Although there was no statistical difference in the proportion of patients with advanced disease, there was a concerning trend of increased M1c disease in cohorts 2 and 3. The higher M1c disease during the COVID-19 pandemic (cohorts 2 and 3) likely played a crucial role in increasing the 6-month mortality rate and leading to a reduced overall survival of lung cancer patients during the pandemic. These findings are more likely to be better identified with longer follow-up.

The Impact of Next-generation Sequencing on Interobserver Agreement and Diagnostic Accuracy of Desmoplastic Melanocytic Neoplasms.

Next-generation sequencing (NGS) is increasingly being utilized as an ancillary tool for diagnostically challenging melanocytic neoplasms. It is incumbent upon the pathology community to perform studies assessing the benefits and limitations of these tools in specific diagnostic scenarios. One of the most challenging diagnostic scenarios faced by skin pathologists involves accurate diagnosis of desmoplastic melanocytic neoplasms (DMNs). In this study, 20 expert melanoma pathologists rendered a diagnosis on 47 DMNs based on hematoxylin and eosin sections with demographic information. After submitting their diagnosis, the experts were given the same cases, but this time with comprehensive genomic sequencing results, and asked to render a diagnosis again. Identification of desmoplastic melanoma (DM) improved by 7%, and this difference was statistically significant ( P <0.05). In addition, among the 15 melanoma cases, in the pregenomic assessment, only 12 were favored to be DM by the experts, while after genomics, this improved to 14 of the cases being favored to be DM. In fact, some cases resulting in metastatic disease had a substantial increase in the number of experts recognizing them as DM after genomics. The impact of the genomic findings was less dramatic among benign and intermediate-grade desmoplastic tumors (BIDTs). Interobserver agreement also improved, with the Fleiss multirater Kappa being 0.36 before genomics to 0.4 after genomics. NGS has the potential to improve diagnostic accuracy in the assessment of desmoplastic melanocytic tumors. The degree of improvement will be most substantial among pathologists with some background and experience in bioinformatics and melanoma genetics.

Analysis of surrogate gene expression markers in peripheral blood of melanoma patients to predict treatment outcome of adjuvant pegylated interferon alpha 2b (EORTC 18991 side study).

We analysed mRNA levels of interferon response genes (ISG15, STAT1, CXCL10) of inhibitors of the JAK/STAT pathway (STAT3, SOCS1, SOCS3) and of cytokines (TNFα, IL10, TGFß1) in peripheral blood of 91 stage III melanoma patients enrolled in EORTC 18991 trial to find biomarkers indicative for disease stage and predictive for efficacy of pegylated interferon alpha-2b (PEG-IFNα-2b) therapy. mRNA levels were analysed at baseline and after 6 months. Univariate and multivariate analyses were performed to estimate the prognostic and predictive role of mRNA levels for distant metastasis-free survival (DMFS) and relapse-free survival (RFS). Compared to healthy controls, melanoma patients showed significantly higher TGFß1 mRNA levels. In a multivariate model, increasing SOCS1 and SOCS3 mRNA levels were associated with worse RFS (P = 0.02 and P = 0.04, respectively) and DMFS (P = 0.05 and P = 0.05, respectively) due to negative correlation between, respectively, SOCS1/SOCS3 mRNA levels and ulceration or Breslow thickness. No impact of PEG-IFNα-2b on mRNA levels was observed except for ISG15 mRNA levels, which decreased in the treatment arm (P = 0.001). It seems that patients with a decrease >60 % of ISG15 mRNA levels during 6 months PEG-IFNα-2b had inferior outcome.

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine.

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Barriers and facilitators of adherence to medical advice on skin self-examination during melanoma follow-up care.

BACKGROUND: Melanoma is the fastest growing tumor of the skin, which disproportionately affects younger and middle-aged adults. As melanomas are visible, recognizable, and highly curable while in early stages, early diagnosis is one of the most effective measures to decrease melanoma-related mortality. Skin self-examination results in earlier detection and removal of the melanoma. Due to the elevated risk of survivors for developing subsequent melanomas, monthly self-exams are strongly recommended as part of follow-up care. Yet, only a minority of high-risk individuals practices systematic and regular self-exams. This can be improved through patient education. However, dermatological education is effective only in about 50% of the cases and little is known about those who do not respond. In the current literature, psychosocial variables like distress, coping with cancer, as well as partner and physician support are widely neglected in relation to the practice of skin self-examination, despite the fact that they have been shown to be essential for other health behaviors and for adherence to medical advice. Moreover, the current body of knowledge is compromised by the inconsistent conceptualization of SSE. The main objective of the current project is to examine psychosocial predictors of skin self-examination using on a rigorous and clinically sound methodology. METHODS/DESIGN: The longitudinal, mixed-method study examines key psychosocial variables related to the acquisition and to the long-term maintenance of skin self-examination in 200 patients with melanoma. Practice of self-exam behaviors is assessed at 3 and 12 months after receiving an educational intervention designed based on best-practice standards. Examined predictors of skin self-exam behaviors include biological sex, perceived self-exam efficacy, distress, partner and physician support, and coping strategies. Qualitative analyses of semi-structured interviews will complement and enlighten the quantitative findings. DISCUSSION: The identification of short and long-term predictors of skin self-examination and an increased understanding of barriers will allow health care professionals to better address patient difficulties in adhering to this life-saving health behavior. Furthermore, the findings will enable the development and evaluation of evidence-based, comprehensive intervention strategies. Ultimately, these findings could impact a wide range of outreach programs and secondary prevention initiatives for other populations with increased melanoma risk.

Generalizability of Machine Learning Models: Quantitative Evaluation of Three Methodological Pitfalls.

Purpose: To investigate the impact of the following three methodological pitfalls on model generalizability: (a) violation of the independence assumption, (b) model evaluation with an inappropriate performance indicator or baseline for comparison, and (c) batch effect. Materials and Methods: The authors used retrospective CT, histopathologic analysis, and radiography datasets to develop machine learning models with and without the three methodological pitfalls to quantitatively illustrate their effect on model performance and generalizability. F1 score was used to measure performance, and differences in performance between models developed with and without errors were assessed using the Wilcoxon rank sum test when applicable. Results: Violation of the independence assumption by applying oversampling, feature selection, and data augmentation before splitting data into training, validation, and test sets seemingly improved model F1 scores by 71.2% for predicting local recurrence and 5.0% for predicting 3-year overall survival in head and neck cancer and by 46.0% for distinguishing histopathologic patterns in lung cancer. Randomly distributing data points for a patient across datasets superficially improved the F1 score by 21.8%. High model performance metrics did not indicate high-quality lung segmentation. In the presence of a batch effect, a model built for pneumonia detection had an F1 score of 98.7% but correctly classified only 3.86% of samples from a new dataset of healthy patients. Conclusion: Machine learning models developed with these methodological pitfalls, which are undetectable during internal evaluation, produce inaccurate predictions; thus, understanding and avoiding these pitfalls is necessary for developing generalizable models.Keywords: Random Forest, Diagnosis, Prognosis, Convolutional Neural Network (CNN), Medical Image Analysis, Generalizability, Machine Learning, Deep Learning, Model Evaluation Supplemental material is available for this article. Published under a CC BY 4.0 license.

BORIS/CTCFL-mediated chromatin accessibility alterations promote a pro-invasive transcriptional signature in melanoma cells.

Melanoma is the deadliest form of skin cancer, due to its tendency to metastasize early. Brother of regulator of imprinted sites (BORIS), also known as CCCTC binding factor-like (CTCFL), is a transcription regulator that becomes ectopically expressed in melanoma. We recently showed that BORIS contributes to melanoma phenotype switching by altering the gene expression program of melanoma cells from an intermediate melanocytic state toward a more mesenchymal-like state. However, the mechanism underlying this transcriptional switch remains unclear. Here, ATAC-seq was used to study BORIS-mediated chromatin accessibility alterations in melanoma cells harboring an intermediate melanocytic state. The gene set that gained promoter accessibility, following ectopic BORIS expression, showed enrichment for biological processes associated with melanoma invasion, while promoters of genes associated with proliferation showed reduced accessibility. Integration of ATAC-seq and RNA-seq data demonstrated that increased chromatin accessibility was associated with transcriptional upregulation of genes involved in tumor progression processes, and the aberrant activation of oncogenic transcription factors, while reduced chromatin accessibility and downregulated genes were associated with repressed activity of tumor suppressors and proliferation factors. Together, these findings indicate that BORIS mediates transcriptional reprogramming in melanoma cells by altering chromatin accessibility and gene expression, shifting the cellular transcription landscape of melanoma cells toward a mesenchymal-like genetic signature.

Future Role of Health Technology Assessment for Genomic Medicine in Oncology: A Canadian Laboratory Perspective.

Genome-based testing in oncology is a rapidly expanding area of health care that is the basis of the emerging area of precision medicine. The efficient and considered adoption of novel genomic medicine testing is hampered in Canada by the fragmented nature of health care oversight as well as by lack of clear and transparent processes to support rapid evaluation, assessment, and implementation of genomic tests. This article provides an overview of some key barriers and proposes approaches to addressing these challenges as a potential pathway to developing a national approach to genomic medicine in oncology.

Next-generation sequencing of non-small cell lung cancer at a Quebec health care cancer centre.

BACKGROUND: Lung cancer is the leading cause of cancer death in both men and women. Quebec has the highest lung cancer mortality out of all provinces in Canada, believed to be caused by higher smoking rates. Molecular testing for lung cancer is standard of care due to the discovery of actionable driver mutations that can be targeted with tyrosine kinase inhibitors. To date, no detailed molecular testing characterization of Quebec patients with lung cancer using next generation sequencing (NGS) has been performed. MATERIALS AND METHODS: The aim of this study was to describe the genomic landscape of patients with lung cancer (n = 997) who underwent NGS molecular testing at a tertiary care center in Quebec and to correlate it with clinical and pathology variables. RESULTS: Compared to 10 other NGS studies found through a structured search strategy, our cohort had a higher prevalence of KRAS mutations (39.2%) compared to most geographical locations. Additionally, we observed a significant positive association between decreasing age and a higher proportion of KRAS G12C mutations. CONCLUSION: Overall, it remains important to assess institutional rates of actionable driver mutations to help guide governing bodies, fuel clinical trials and create benchmarks for expected rates as quality metrics.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.