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The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based BRCA1 and BRCA2 (BRCA1/2) tumour testing in ovarian cancers. This document was drafted by the members of the Canadian College of Medical Geneticists (CCMG) somatic BRCA Ad Hoc Working Group, and representatives from the Canadian Association of Pathologists. The document was circulated to the CCMG members for comment. Following incorporation of feedback, this document has been approved by the CCMG board of directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada; however, they are not inclusive of all information laboratories should consider in the validation and use of NGS for BRCA1/2 tumour testing in ovarian cancers.

Objectives In precision medicine, where oncologic management is tailored to the individual’s clinical and genetic profiles, advanced diagnostic testing provides prognostic information and guides management in a growing number of malignancies. There is a need to capture the work pathologists perform to meet this demand by providing medically relevant, timely, and accurate testing results. This work includes not only direct patient consults (interpretation of results and issuing reports) but the administrative and medical oversight as well as the research needed to provide the necessary quality assurance, quality control, direction, and framework for the laboratory. Methods An expert panel of Canadian pathologists involved in advanced diagnostics was convened to establish and beta test a model for workload assessment in advanced diagnostics. Results All aspects of the advanced diagnostics workload were detailed and applied to models based on members’ experience, including medical oversight, administration, and the introduction of new testing and platforms. Models for biomarker testing were developed for simple and complex or multiplexed assays, and a detailed model was developed to assess the workload for next-generation sequencing–based assays. Conclusions This paper provides the first detailed proposal for capturing an advanced diagnostic workload to enable appropriate pathologist allotment for performing all the steps required to run an advanced diagnostic service.

AIMS Nodal staging in colorectal cancer (CRC) informs prognosis and guides adjuvant treatment decisions. A standard minimum of 12 lymph nodes is widely used, with additional sampling being performed as required. However, there are few data on how lymph node resampling in this context has an impact on nodal stage. The aims of this study were to evaluate the effectiveness of resampling in detecting metastases and tumour deposits, and the impact on stage. METHODS AND RESULTS A retrospective cohort analysis was performed on CRC resections that underwent resampling because of an initial yield of

Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%.

PurposeHuman papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) is the fastest rising cancer in North America. There is significant interest in treatment de-escalation for these patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to predict the survival of patients with newly diagnosed HPV+ HNSCC.MethodsWe created a prognostic score (UWO3) that was successfully validated in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Transcriptomic data from two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes.ResultsA three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival at 5 years to the immune desert (HR= 9.0, 95% CI 3.2–25.5, P=3.6×10−5) and mixed (HR=6.4, 95%CI 2.2–18.7, P=0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation.ConclusionsThe UWO3 immune score could enable biomarker-driven clinical decision-making for patients with HPV+ HNSCC based on robust outcome prediction across six independent cohorts. The superior survival of immune rich patients supports de-intensification strategies, while the inferior outcomes of the immune desert patients suggest the potential for intensification and/or immunotherapy. Prospective de-escalation and intensification clinical trials are currently being planned.

The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based

Introduction In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. Methods Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. Results Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. Conclusions Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.

Objective. Detection of circulating tumor DNA (ctDNA) in cancer patients can potentially serve as a noninvasive, sensitive test of disease status. The purpose of this study was to determine the ability to detect BRAF (V600E) mutations in the plasma of patients with thyroid nodules, with the goal of distinguishing between benign and malignant nodules. Methods. Consecutive patients with thyroid nodules who consented for surgery were recruited. Plasma samples were obtained preoperatively and one month postoperatively. Quantitative PCR was used to determine the levels of the BRAF (V600E) mutation preoperatively and postoperatively. Results. A total of 109 patients were recruited. On final pathology, 38 (32.8%) patients had benign thyroid nodules, 45 (38.8%) had classical papillary thyroid cancer (PTC), 23 (19.8%) had nonclassical PTC, and 3 (2.6%) had follicular thyroid cancer. 15/109 patients had detectable BRAF (V600E) ctDNA in their preoperative samples—all of them having classical PTC. Higher T-stage and extrathyroidal extension in PTC were associated with positive BRAF (V600E) ctDNA ( p < 0.05 ). Eighty-eight pairs of preoperative and postoperative plasma samples were collected and analyzed. Of these eighty-eight paired samples, a total of 13/88 (14.8%) patients had detectable BRAF (V600E) ctDNA in their preoperative samples—all of them having classical PTC. 12 of these 13 patients had no detectable BRAF (V600E) postoperatively, while one remaining patient had a significant decline in his levels ( p < 0.05 ). Conclusion. BRAF (V600E) circulating thyroid tumor DNA can be detected in plasma and is correlated with a final diagnosis of the classical variant of PTC. Given that a postoperative drop in BRAF (V600E) ctDNA levels was observed in all cases suggests its utility as a tumor marker.

Phosphoinositide 3‐kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K‐PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K‐activated Akt signaling is well established in HNSCC, the significance of mTORC2‐driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2‐specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.

OBJECTIVES The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen. METHODS Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results. RESULTS The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%. CONCLUSIONS Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories.

Background Frequent mutations in the nuclear receptor binding SET domain protein 1 (NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear. Methods Using data from over 500 HNSCC patients from The Cancer Genome Atlas (TCGA), we compared the relative level of mRNA expression of NSD1, NSD2, and NSD3 in HPV+ and HPV- HNSCC. Correlation analyses were performed between T-cell infiltration and the relative level of expression of NSD1, NSD2, and NSD3 mRNA in HPV+ and HPV- HNSCC. In addition, overall survival outcomes were compared for both the HPV+ and HPV- subsets of patients based on stratification by NSD1, NSD2, and NSD3 expression levels. Results Expression levels of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC. Conclusion These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.

Background. Recently, reanalysis of The Cancer Genome Atlas study demonstrated that human papillomavirus (HPV) genotypes in head and neck cancers other than HPV-16 have inferior survival to HPV-16-positive tumors. We aimed to examine the association of HPV subtypes and survival in a large cohort of patient samples from our institution. Methods. Fresh frozen primary site biopsy samples were collected either in clinic or at the time of surgery. Patient demographic, staging, and survival data were also collected. Tumors were tested for HPV subtypes by quantitative polymerase chain reaction (qPCR). Univariable and multivariable analyses were performed using Cox proportional hazards regression. Results. 280 patient biopsy samples were collected between 2011 and 2017. Mean ± standard deviation (SD) age was 61.9 ± 11.1 years and most patients (78%) were male. The majority of cancers were of the oral cavity (60%) or oropharynx (25%) and 30% had HPV-positive disease. Median follow-up was 3.76 years and 96/280 patients (34%) developed recurrences. Patients with p16-positive versus negative disease had significantly improved 5-year overall survival (OS, 77.6% vs. 53.3%; p = 0.009 ) and progression-free survival (PFS, 67.3% vs. 41.0%, p = 0.006 ). Similarly improved 5-year OS and PFS were observed for patients with HPV-positive versus negative disease (65.0% vs. 55.0%, p = 0.084 ; 53.3% vs. 43.2%, p = 0.072 , resp.). Patients with HPV-16 compared to other HPV diseases had worse 5-year OS and PFS (62.1% vs. 88.9%, p = 0.273 ; 49.0% vs. 88.9%, p = 0.081 , resp.). Conclusions. In contrast to the data derived from The Cancer Genome Atlas, patients with HPV-16 tumors trended towards decreased PFS and OS compared with tumors driven by other HPV genotypes. Further larger multi-institutional studies are necessary to understand the relationship between other HPV genotypes and survival in head and neck squamous cell carcinomas.

Background Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs). Methods Five unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance. Results Prolonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20–35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib. Conclusions We have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.

Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSM-LPD) is a rare indolent disorder often associated with a favourable prognosis. It typically presents as a solitary skin lesion, mainly in the head, neck, or upper trunk region. Multifocal PCSM-LPD is a rare entity, with no standard treatment approaches available. In this article, we present the case of a 56-year-old male patient with multifocal biopsy-proven PCSM-LPD that was treated with methotrexate orally at 10 mg/m2 body surface area weekly and successfully achieved full clinical resolution by the 10th week of therapy. A review of the literature indicates the efficacy of combination chemotherapy. However, due to the indolent nature of this disorder and the undesired side effects from combination chemotherapy, our treatment method involved oral methotrexate alone, and it was successful. Oral methotrexate is a potential therapeutic option in the management of multifocal PCSM-LPD and it warrants further investigations.

Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC.SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models.Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p 0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models.HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.

Abtract Background The genomic landscape of head and neck cancer has been reported through The Cancer Genome Atlas project. We attempt to determine if high-risk human papillomavirus (HPV) or frequently mutated genes are correlated with survival in an oral cancer cohort. Methods Patient demographic data along with data from final pathology was collected. Tumor DNA was analyzed using a custom Illumina targeted sequencing panel. Five high-risk HPV types were tested by qPCR. Statistical analyses were used to identify associations between patient outcome and mutational status. Results High-risk HPV types were identified in 7% of cases; HPV status was not associated with survival. Mutations were identified in TP53, TERT promoter, & PIK3CA. Mutations in TP53 were significantly associated with poorer overall survival on multi-variate analysis (p = 0.03). Conclusions Mutations in TP53 were associated with poor patient survival. Expanding our sample size may identify further predictors of outcome to direct customized cancer care.

Primary histiocytic tumors of the CNS are rare. The current WHO classification (2016) included 5 entities: Langerhans cell histiocytosis (LCH), Erdheim-Chester disease (ECD), Rosai-Dorfman disease (RDD), juvenile xanthogranuloma (JXG), and histiocytic sarcoma (HS) (1). The diagnosis usually is made based on the tumor differentiation as to the counterpart in normal histiocyte development. This article is protected by copyright. All rights reserved.

Phosphoinositide 3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signaling is well established in HNSCC, the significance of mTORC2-driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.

Objectives Advances in Next Generation Sequencing (NGS) allow for multiple gene analysis in an efficient, cost-effective manner. Deceased ovarian cancer patients untested for germline BRCA mutations represent a missed opportunity for clinicians to prevent future cancers in their surviving relatives. Families of this lost cohort can benefit through Traceback initiatives. We sought to validate our London Health Sciences custom Hereditary Cancer Panel using formalin fixed paraffin embedded (FFPE) tumor samples to assess BRCA 1/2 status in a cohort of high grade serous ovarian cancer (HGSC) patients. Methods FFPE samples from 150 deceased HGSC patients were assessed using an Illumina MiSeq sequencer with a mean coverage of 1000x and average minimum coverage of 700x when 24 samples were tested per run. Validation of a subset of identified variants was then undertaken using Sanger and Multiplex Ligation-dependent Probe Amplication (MLPA). Results Among 150 HGSC samples, we identified 44 samples (29.3%) with reportable variants with variant allele frequencies from 10.3% - 99.4%. These included 35 point mutations/insertions/deletions, 7 exon/whole gene deletions, and 2 BRCA1 exon 13 duplications. A subset (26) of these variants were then confirmed by targeted assays using Sanger and MLPA. Conclusions Utilizing NGS technology, we reliably identified BRCA mutations in FFPE tumor samples. A validated NGS pipeline provides a valuable clinical tool to conduct Traceback initiatives to the families of deceased ovarian cancer patients never tested for germline mutations.

Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit.

SummaryProliferative control in cancer cells is frequently disrupted by mutations in the RB-pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB-pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in CDKN2A silenced cancer cells when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immune compromised mice RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the pre-existing proliferative control deficit.

Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation and copy number aberration differences between heavy smokers and never smokers were compared within human papillomavirus-positive (HPV-positive) (n = 67) and HPV-negative (n = 431) TCGA cohorts with HNSCC, and the impact of these mutations on survival were assessed. No genes were differentially mutated between smoking and never-smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 WT patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancer patients and a meta-analysis that included 2 additional data sets (688 total patients, hazard ratio for death 0.44, 95% CI, 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers, define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients.

The retinoblastoma tumor suppressor pathway is frequently inactivated in human cancer, enabling unrestrained proliferation. Most cancers, however, maintain expression of a wild-type (WT) retinoblastoma tumor suppressor protein (pRB). It is generally in a hyperphosphorylated state (ppRB) because of mutations in upstream regulators such as p16 and cyclin D. Hyperphosphorylated ppRB is considered inactive, although data are emerging that suggest it can retain some function. To test the clinical relevance of pRB status, we obtained archival tissue sections from 91 cases of lung adenocarcinoma resected between 2003 and 2008. All cases received platinum doublet chemotherapy, and the median survival was 5.9 years. All cases were assessed for pRB and ppRB using immunohistochemistry and quantified based on intensity of signal and proportion of positive cells. pRB expression was lost in 15% of lung adenocarcinoma cases. In tumors that did not express pRB, the survival rate was significantly improved (hazard ratio, 0.21; 95% confidence interval, 0.06-0.69; P = .01) in comparison to tumors that express pRB. pRB status was found to be an independent predictor of overall survival on multivariate analysis (hazard ratio, 0.22; 95% confidence interval, 0.07-0.73; P = .01) along with increased stage and age. pRB status did not alter baseline levels of apoptotic or proliferative markers in these tumors, but the DNA damage response protein 53BP1 was higher in cancers with high levels of pRB. In summary, loss of pRB expression is associated with improved survival in patients treated with surgical resection and chemotherapy. This may be useful in classifying patients at greatest benefit for aggressive treatment regimes.

A 42-year-old man with hemochromatosis and cirrhosis developed aplastic anemia. He underwent liver transplantation from a female donor and splenectomy, and his aplastic anemia spontaneously resolved. A bone marrow examination 6 months after the liver transplant showed 17.5% female cells. He did well for 13 years without the need for any blood product support but then developed bone pain and was found to have metastatic hepatocellular carcinoma in the vertebral bodies. Molecular analysis demonstrated that the tumor cells were from his original liver. No primary liver tumor was identified in the explant. The case demonstrates the application of fluorescent in situ hybridization with X and Y chromosome-specific probes to study chimerism and tumor origin after liver transplantation between individuals of different sex. (Hepatology Communications 2018;2:13-15).

Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack-years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation (SNV) and copy number aberration (CNA) differences between heavy smokers and never smokers were compared within HPV-positive (n=67) and negative (n=431) TCGA HNSCC cohorts, and the impact of these mutations on survival was assessed. No genes were differentially mutated between smoking and never smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 wild-type patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancers and a meta-analysis that included two additional datasets (688 total patients, hazard ratio for death 0.44, 95% CI 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers and define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients. Citation Format: Farhad Ghasemi, Stephenie D. Prokopec, Danielle MacNeil, Neil Mundi, Christopher Howlett, William Stecho, Kevin Fung, John Yoo, Eric Winquist, Steven Gameiro, Axel Sahovaler, Paul Plantinga, Joe S. Mymryk, John W. Barrett, Paul C. Boutros, Anthony C. Nichols. Mutational analysis of head and neck squamous cell carcinoma stratified by smoking status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR02.

Background The clinical-radiographic distinction between idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) is challenging. We sought to investigate the gene expression profiles of IPF and NSIP vs. normal controls. Methods Gene expression from explanted lungs of patients with IPF (n = 22), NSIP (n = 10) and from normal controls (n = 11) was assessed. Microarray analysis included Significance Analysis of Microarray (SAM), Ingenuity Pathway, Gene-Set Enrichment and unsupervised hierarchical clustering analyses. Immunohistochemistry and serology of proteins of interest were conducted. Results NSIP cases were significantly enriched for genes related to mechanisms of immune reaction, such as T-cell response and recruitment of leukocytes into the lung compartment. In IPF, in contrast, these involved senescence, epithelial-to-mesenchymal transition, myofibroblast differentiation and collagen deposition. Unlike the IPF group, NSIP cases exhibited a strikingly homogenous gene signature. Clustering analysis identified a subgroup of IPF patients with intermediate and ambiguous expression of SAM-selected genes, with the interesting upregulation of both NSIP-specific and senescence-related genes. Immunohistochemistry for p16, a senescence marker, on fibroblasts differentiated most IPF cases from NSIP. Serial serum levels of periostin, a senescence effector, predicted clinical progression in a cohort of patients with IPF. Conclusions Comprehensive gene expression profiling in explanted lungs identifies distinct transcriptional profiles and differentially expressed genes in IPF and NSIP, supporting the notion of NSIP as a standalone condition. Potential gene and protein markers to discriminate IPF from NSIP were identified, with a prominent role of senescence in IPF. The finding of a subgroup of IPF patients with transcriptional features of both NSIP and senescence raises the hypothesis that “senescent” NSIP may represent a risk factor to develop superimposed IPF. Electronic supplementary material The online version of this article (10.1186/s12931-018-0857-1) contains supplementary material, which is available to authorized users.

The retinoblastoma protein (pRB) plays a key role in proliferative control and genome stability. For these reasons its functions are considered to be tumor suppressive. Its functional status offers critical insight into proliferative control signaling in tissues and in developing malignancies. In this chapter, we outline basic procedures to detect the retinoblastoma protein in formalin fixed, paraffin embedded tissue sections. In addition, we provide protocols to detect phosphorylation levels of pRB in tissues and offer controls to ensure fidelity of measurement. Importantly, these staining methods utilize broadly available reagents and equipment making them accessible to most biomedical research laboratories.

The retinoblastoma protein (pRB) plays a key role in proliferative control and genome stability. For these reasons its functions are considered to be tumor suppressive. Its functional status offers critical insight into proliferative control signaling in tissues and in developing malignancies. In this chapter, we outline basic procedures to detect the retinoblastoma protein in formalin fixed, paraffin embedded tissue sections. In addition, we provide protocols to detect phosphorylation levels of pRB in tissues and offer controls to ensure fidelity of measurement. Importantly, these staining methods utilize broadly available reagents and equipment making them accessible to most biomedical research laboratories.

PIK3CA is the only frequently-mutated, directly druggable oncogene in head and neck squamous cell carcinoma (HNSCC). However, it is unclear if a molecularly-driven intervention trial can be launched successfully, particularly within a single-institution setting secondary to the infrastructure necessary for mutation detection, mutation prevalence, and patient willingness to participate. This study aimed to evaluate 1) local frequency of PIK3CA activating mutations in HNSCC, 2) timeliness of our mutation-profiling clinical pathway, and 3) patients' willingness to enroll in a novel neoadjuvant drug trial. Tissue biopsies of 25 consecutive cases of HNSCC were tested for activating PIK3CA mutations at three mutational hotspots by real-time polymerase chain reaction. Mutations prevalence and number of working days accrued in determining PIK3CA mutational status were calculated. In addition, 30 HNSCC patients were surveyed prospectively regarding their willingness to participate in a hypothetical drug trial. Survey data were summarized descriptively. 4 of 25 (16 %) tumors harbored a PIK3CA activating mutation, including one at codon E542K, two at codon E545K/D, and one at codon H1047R. On average, this result was obtained in approximately 15 working days (range, 9-24 working days). The majority of patients surveyed (70 %) indicated their willingness to participate in a targeted PIK3CA trial. This study provides evidence that within a single institution, PIK3CA activating mutations can be detected with expected frequency, with sufficient timeliness and sufficient patient interest to mount a targeted intervention trial that may lead to improved tumor response in selected HNSCC patients.

A novel role for ATF3 is identified in acinar-to–duct cell metaplasia during pancreatic injury. ATF3 targets transcriptional regulators that affect acinar cell fate and reduces the severity of pancreatic injury. However, by doing so, ATF3 may also increase the susceptibility for progression to pancreatic adenocarcinoma., Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3–/– pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to–duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

© 2014 Nature America, Inc. All rights reserved., Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA., This work was supported in part by an IDEAS grant from Princess Margaret Hospital, the Head and Neck Translational Research Program (I.W., B.A.C., P.C.B. and J.D.M.), the Ontario Institute for Cancer Research and the government of Ontario (P.C.B. and J.D.M.) and by a Terry Fox Research Institute New Investigator Award (P.C.B.). C.H. and F.-F.L. acknowledge support from the Wharton family, Joe's Team, Gordon Tozer, the Campbell Family Institute for Cancer Research and the Ministry of Health and Long-Term Planning, Canada.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Background: The acute respiratory distress syndrome (ARDS) is a complex pulmonary disorder in which the local release of cytokines and chemokines appears central to the pathophysiology. Objective: Based on the known role of matrix metalloproteinase-3 (MMP3) in inflammatory processes, the objective was to examine the role of MMP3 in the pathogenesis of ARDS through the modulation of pulmonary inflammation. Materials and Methods: Female and male, wild type (MMP3+/+) and knock out (MMP3−/−) mice were exposed to two, clinically relevant models of ARDS including (i) lipopolysaccharide (LPS)-induced lung injury, and (ii) hydrochloric acid-induced lung injury. Parameters of lung injury and inflammation were assessed through measurements in lung lavage including total protein content, inflammatory cell influx, and concentrations of mediators such as TNF-α, IL-6, G-CSF, CXCL1, CXCL2, and CCL2. Lung histology and compliance were also evaluated in the LPS model of injury. Results: Following intra-tracheal LP...

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) is the most common subtype of marginal zone lymphoma (MZL), with stomach being the most frequent primary site, followed by salivary gland, lung and ocular adnexa. Although clinically indolent, MALT lymphoma has the potential of local recurrence and systemic spread. Amyloid deposition is a very unusual complication of MALT lymphoma. In this study, we report clinicopathologic features of 5 cases of MALT lymphomas with associated amyloid deposits. One case showed amyloid deposits in the primary lesion; the other four cases showed amyloid deposits only in recurrences. Previous studies suggest that the amyloid deposits do not implicate worse prognosis. In our study, although amyloid deposits were focal and organ confined, one patient had extensive deposits of amyloid in the large bowel wall leading to bowel perforation and another patient developed significant peripheral neuropathy due to amyloid deposits in the brachial plexus. In conclusion, amyloid deposits in MALT lymphomas are rare and organ/tumour confined. However, complications can be critical and cause considerable morbidity. Therefore, pathologists should be aware of the association between MALT lymphoma and amyloid deposition, and clinical follow up is warranted.