The histological spectrum and immunoprofile of head and neck NUT carcinoma: A multicentre series of 30 cases.

BACKGROUND AND AIM: Head and neck nuclear protein of testis carcinoma (HN-NUT) is a rare form of carcinoma diagnosed by NUT immunohistochemistry positivity and/or NUTM1 translocation. Although the prototype of HN-NUT is a primitive undifferentiated round cell tumour (URC) with immunopositivity for squamous markers, it is our observation that it may assume variant histology or immunoprofile. METHODS: We conducted a detailed clinicopathological review of a large retrospective cohort of 30 HN-NUT, aiming to expand its histological and immunohistochemical spectrum. RESULTS: The median age of patients with HN-NUT was 39 years (range = 17-86). It affected the sinonasal tract (43%), major salivary glands (20%), thyroid (13%), oral cavity (7%), larynx (7%), neck (7%) and nasopharynx (3%). Although most cases of HN-NUT (63%) contained a component of primitive URC tumour, 53% showed other histological features and 37% lacked a URC component altogether. Variant histological features included basaloid (33%), differentiated squamous/squamoid (37%), clear cell changes (13%), glandular differentiation (7%) and papillary architecture (10%), which could co-exist. While most HN-NUT were positive for keratins, p63 and p40, occasional cases (5-9%) were entirely negative. Immunopositivity for neuroendocrine markers and thyroid transcription factor-1 was observed in 33 and 36% of cases, respectively. The outcome of HN-NUT was dismal, with a 3-year disease specific survival of 38%. CONCLUSIONS: HN-NUT can affect individuals across a wide age range and arise from various head and neck sites. It exhibits a diverse spectrum of histological features and may be positive for neuroendocrine markers, potentially leading to underdiagnosis. A low threshold to perform NUT-specific tests is necessary to accurately diagnose HN-NUT.

Variant classification changes over time in the clinical molecular diagnostic laboratory setting.

BACKGROUND: Variant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB) and benign (B) using the standards and guidelines recommended by the American College of Medical Genetics and the Association for Molecular Pathology, with modifications for specific genes. As the literature continues to rapidly expand, and evidence continues to accumulate, prior classifications can be updated accordingly. In this study, we aim to characterise variant reclassifications in Ontario. METHODS: DNA samples from patients seen at hereditary cancer clinics in Ontario from January 2012 to April 2022 were submitted for testing. Patients met provincial eligibility criteria for testing for hereditary cancer syndromes or polycystic kidney disease. Reclassification events were determined to be within their broader category of significance (B to LB or vice versa, or P to LP or vice versa) or outside of their broader category as significance (ie, significant reclassifications from B/LB or VUS or P/LP, from P/LP to VUS or B/LB, or from VUS to any other category). RESULTS: Of the 8075 unique variants included in this study, 23.7% (1912) of variants were reassessed, and 7.2% (578) of variants were reclassified. Of these, 351 (60.7%) variants were reclassified outside of their broader category of significance. Overall, the final classification was significantly different for 336 (58.1%) variants. Importantly, most reclassified variants were downgraded to a more benign classification (n=245; 72.9%). Of note, most reclassified VUS was downgraded to B/LB (n=233; 84.7%). CONCLUSIONS: The likelihood for reclassification of variants on reassessment is high. Most reclassified variants were downgraded to a more benign classification. Our findings highlight the importance of periodic variant reassessment to ensure timely and appropriate care for patients and their families.

Expanding the histological spectrum of salivary gland neoplasms with HMGA2::WIF1 fusion emphasising their malignant potential: a report of eight cases.

AIMS: Recently, HMGA2::WIF1 fusion has been reported in pleomorphic adenoma (PAs) originating from the parotid gland with a characteristic canalicular adenoma (CAA)-like pattern. However, it is unclear whether HMGA2::WIF1 fusion may occur in salivary gland carcinoma or tumours originating from the minor salivary glands. We herein conducted a detailed clinicopathological review of eight salivary gland tumours harbouring HMGA2::WIF1 fusions. METHODS AND RESULTS: The reviewed diagnoses of salivary gland neoplasms with HMGA2::WIF1 fusion were PA (n = four), myoepithelioma (n = one), myoepithelial carcinoma ex PA (n = two) and high-grade carcinoma with basaloid features (n = one). Two tumours originated from the minor salivary glands. Six tumours (80%) contained areas reminiscent of CAA characterised by interconnected trabeculae/canaliculi of monotonous oncocytic or cuboidal tumour cells associated with a hypocellular, hyalinised to myxoid stroma. Areas typical of PA were seen in four (50%) cases. All tumours showed diffuse S100 and CK7 immunopositivity. Adverse events were detected in two cases, including local recurrence in a patient with PA, and local and distant recurrences and disease-related death in a patient with a high-grade carcinoma of the minor salivary gland of the buccal space, showing tumour necrosis and perineural invasion. CONCLUSION: Salivary gland neoplasms with HMGA2::WIF1 fusion are predominantly characterised by CAA/striated duct adenoma-like histology and a S100+/CK7+ immunoprofile. These tumours are not always benign, as among all reported cases approximately 20% showed malignancy (six of 28) and adverse outcome (three of 15), including recurrence, distant metastasis and disease-specific mortality.

Comprehensive Molecular Characterization of Polymorphous Adenocarcinoma, Cribriform Subtype: Identifying Novel Fusions and Fusion Partners.

Polymorphous adenocarcinoma (PAC) is a common, usually low-grade salivary gland carcinoma. While conventional PACs are most associated with PRKD1 p.E710D hotspot mutations, the cribriform subtype is often associated with gene fusions in PRKD1, PRKD2, or PRKD3. These fusions have been primarily identified by fluorescence in situ hybridization (FISH) analysis, with a minority evaluated by next-generation sequencing (NGS). Many of the reported fusions were detected by break-apart FISH probes and therefore have unknown partners or were negative by FISH altogether. In this study, we aimed to further characterize the fusions associated with PAC with NGS. Fifty-four PACs (exclusively cribriform and mixed/intermediate types to enrich the study for fusion-positive cases) were identified and subjected to NGS. Fifty-one cases were successfully sequenced, 28 of which demonstrated gene fusions involving PRKD1, PRKD2, or PRKD3. There were 10 cases with the PRKD1 p.E710D mutation. We identified a diverse group of fusion partners, including 13 novel partners, 3 of which were recurrent. The most common partners for the PRKD genes were ARID1A and ARID1B. The wide variety of involved genes is unlike in other salivary gland malignancies and warrants a broader strategy of sequencing for molecular confirmation for particularly challenging cases, as our NGS study shows.

Adenoid Cystic Carcinoma With Striking Tubular Hypereosinophilia: A Unique Pattern Associated With Nonparotid Location and Both Canonical and Novel EWSR1::MYB and FUS::MYB Fusions.

The classification of salivary gland tumors is ever-evolving with new variants of tumors being described every year. Next-generation sequencing panels have helped to prove and disprove prior assumptions about tumors' relationships to one another, and have helped refine this classification. Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and occurs at all major and minor salivary gland and seromucous gland sites. Most AdCC are predominantly myoepithelial and basaloid with variable cribriform, tubular, and solid growth. The luminal tubular elements are often less conspicuous. AdCC has largely been characterized by canonical MYB fusions, with MYB::NFIB and rarer MYBL1::NFIB. Anecdotal cases of AdCC, mostly in nonmajor salivary gland sites, have been noted to have unusual patterns, including squamous differentiation and macrocystic growth. Recently, this has led to the recognition of a subtype termed "metatypical adenoid cystic carcinoma." Another unusual histology that we have seen with a wide range of architecture, is striking tubular hypereosinophilia. The hypereosinophilia and luminal cell prominence is in stark contrast to the vast majority of AdCC that are basaloid and myoepithelial predominant. A total of 16 cases with tubular hypereosinophilia were collected, forming morular, solid, micropapillary, and glomeruloid growth, and occasionally having rhabdoid or Paneth-like cells. They were subjected to molecular profiling demonstrating canonical MYB::NFIB (5 cases) and MYBL1::NFIB (2 cases), as well as noncanonical EWSR1::MYB (2 cases) and FUS::MYB (1 case). The remaining 6 cases had either no fusion (3 cases) or failed sequencing (3 cases). All cases were present in nonmajor salivary gland sites, with seromucous glands being the most common. These include sinonasal tract (7 cases), laryngotracheal (2 cases), external auditory canal (2 cases), nasopharynx (1 case), base of tongue (2 cases), palate (1 case), and floor of mouth (1 case). A tissue microarray of 102 conventional AdCC, including many in major salivary gland sites was examined for EWSR1 and FUS by fluorescence in situ hybridization and showed that these novel fusions were isolated to this histology and nonmajor salivary gland location. In summary, complex and striking tubular hypereosinophilia and diverse architectures are present within the spectrum of AdCC, particularly in seromucous gland sites, and may show variant EWSR1/FUS::MYB fusions.

Resident Depression and Burnout During the COVID-19 Pandemic: A Survey of Canadian Laboratory Medicine Trainees.

CONTEXT.­: Resident physicians face a higher rate of burnout and depression than the general population. Few studies have examined burnout and depression in Canadian laboratory medicine residents, and none during the COVID-19 pandemic. OBJECTIVE.­: To identify the prevalence of burnout and depression, contributing factors, and the impact of COVID-19 in this population. DESIGN.­: An electronic survey was distributed to Canadian laboratory medicine residents. Burnout was assessed using the Oldenburg Burnout Inventory. Depression was assessed using the Patient Health Questionnaire 9. RESULTS.­: Seventy-nine responses were collected. The prevalence of burnout was 63% (50 of 79). The prevalence of depression was 47% (37 of 79). Modifiable factors significantly associated with burnout included career dissatisfaction, below average academic performance, lack of time off for illness, stress related to finances, lack of a peer or staff physician mentor, and a high level of fatigue. Modifiable factors significantly associated with depression further included a lack of access to wellness resources, lack of time off for leisure, and fewer hours of sleep. Fifty-five percent (41 of 74) of participants reported direct impacts to their personal circumstances by the COVID-19 pandemic. CONCLUSIONS.­: Burnout and depression are significant issues affecting Canadian laboratory medicine residents. As the COVID-19 pandemic continues, we recommend the institution of flexible work arrangements, protected time off for illness and leisure, ongoing evaluation of career satisfaction, formal and informal wellness programming with trainee input, formal mentorship programming, and a financial literacy curriculum as measures to improve trainee wellness.

Microcribriform Adenocarcinoma of Salivary Glands: A Unique Tumor Entity Characterized by an SS18::ZBTB7A Fusion.

The landscape of salivary gland carcinomas is ever-changing, with a growing list of new tumors and newly elucidated variants of well-known tumor entities. The routine use of next-generation sequencing has been instrumental in identifying novel fusions and tumor entities, which has helped bring the classification to a more objective and evidenced-based model. However, morphology remains critical in assessing the validity of these novel molecular findings, and most importantly, in assessing which of these findings will have an impact on the prognosis and treatment decisions for patients. The recognition of microsecretory adenocarcinoma (MSA) as a distinct low-grade malignancy of salivary glands, underpinned by MEF2C::SS18 , and a single possibly related case of SS18::ZBTB7A , recently expanded this growing list of distinctive tumors. It was not until now, however, that the morphology of the latter case was known to be unique and reproducible. The authors have now seen 4 of these distinctive tumors that show a combination of distinctive oncocytic cells forming compact glandular growth as well as amphophilic cells forming tubular growth, and suggest the appellation "microcribriform adenocarcinoma" (MCA). So far, these tumors appear to preferentially occur in nonoral sites (2 parotid, 1 submandibular gland, and 1 bronchial seromucous glands). By immunohistochemistry, they express S100 and SOX-10 with focal outer myoepithelial cells marked by circumferential p63, p40, and smooth muscle actin staining around some of the nests and tubules. The tumors show infiltrative growth within a hyalinized and myxoid stroma. Cytologically, they appear generally low grade, similar to MSA. The morphologic and molecular uniformity of these 4 microcribriform adenocarcinoma cases warrants their recognition, and while related to MSA, they are sufficiently different to be classified as a distinct tumor. So far, in limited follow-up, these tumors appear to be relatively indolent.

Molecular yield and cytomorphologic assessment of fine needle aspiration specimen supernatants.

INTRODUCTION: Cytology samples are frequently relied upon for the diagnosis of advanced cancer such as lung cancer. As the recommendations for solid malignancies biomarker testing continue to expand, it becomes increasingly important to efficiently utilize limited specimens to minimize the need for additional sampling and its associated risks and costs. MATERIALS AND METHODS: We performed molecular testing on fresh or CytoLyt-fixed supernatants derived from fine needle aspirates (FNAs) and compared its performance against the clinical specimen (including formalin-fixed paraffin-embedded cell blocks, residual PreservCyt and fresh samples). Supernatants were assessed for cellularity using Field-stained Cytospin (CS) preparations. RESULTS: There was overall almost perfect agreement (41/45 cases, K = 0.822) and substantial to almost perfect agreement in molecular testing results of clinically actionable variants between fresh (20/23 cases, Κ = 0.742) and CytoLyt-fixed (21/22 cases, Κ = 0.908) and its clinical specimen counterpart. Interestingly, CS examination of the supernatants revealed viable tumor cells. Centrifugation for 1 minute at 300 rpm is optimal for overall or tumor cellularity recovery. Delayed molecular testing after 3, 4 and 7 days at 4 degrees Celsius showed identical molecular results. CONCLUSIONS: We validated the use of supernatants derived from FNA cytology samples as a substrate for molecular testing using next-generation sequencing and other molecular techniques.

Gene of the month: STK11.

STK11 encodes for the protein liver kinase B1, a serine/threonine kinase which is involved in a number of physiological processes including regulation of cellular metabolism, cell polarity and the DNA damage response. It acts as a tumour suppressor via multiple mechanisms, most classically through AMP-activated protein kinase-mediated inhibition of the mammalian target of rapamycin signalling pathway. Germline loss-of-function mutations in STK11 give rise to Peutz-Jeghers syndrome, which is associated with hamartomatous polyps of the gastrointestinal tract, mucocutaneous pigmentation and a substantially increased lifetime risk of many cancers. In the sporadic setting, STK11 mutations are commonly seen in a subset of adenocarcinomas of the lung in addition to a number of other tumours occurring at various sites. Mutations in STK11 have been associated with worse prognoses across a range of malignancies and may be a predictor of poor response to immunotherapy in a subset of lung cancers, though further studies are needed before the presence of STK11 mutations can be implemented as a routine clinical biomarker.

Case of epithelioid hemangioendothelioma occurring in the postradiation setting for breast cancer.

Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular tumor, which is typically characterized by recurrent fusion genes. EHEs most commonly occur in the lung, liver, bone, and internal organs. EHE has rarely been reported to occur in the post-radiotherapeutic setting, the breast site or in association with breast cancer. The differential diagnosis for radiation-associated vascular lesions of the breast is classically limited to atypical vascular lesion and angiosarcoma and does not include EHE. We present the case of a woman with a history of breast cancer and post-surgical radiotherapy who went on to develop an EHE of the chest wall skin within 3 years of the completion of radiotherapy. Microscopically, the lesion was infiltrative and composed of anastomosing nests of epithelioid-to-spindled cells with eosinophilic and vacuolated cytoplasm. By immunohistochemistry, the cells were positive for ERG, D2-40, and CD31. The diagnosis was confirmed by identification of a characteristic WWTR1-CAMTA1 fusion gene using RNA sequencing. This case expands our understanding of radiation-associated tumors.

Hepatocellular adenoma in the paediatric population: Molecular classification and clinical associations.

Hepatocellular adenomas (HCAs) represent rare, benign liver tumours occurring predominantly in females taking oral contraceptives. In children, HCAs comprise less than 5% of hepatic tumours and demonstrate association with various conditions. The contemporary classification of HCAs, based on their distinctive genotypes and clinical phenotypes, includes hepatocyte nuclear factor 1 homeobox alpha-inactivated HCAs, beta-catenin-mutated HCAs, inflammatory HCAs, combined beta-catenin-mutated and inflammatory HCAs, sonic hedgehog-activated HCAs, and unclassified HCAs. In children, there is a lack of literature on the characteristics and distribution of HCA subtypes. In this review, we summarized different HCA subtypes and the clinicopathologic spectrum of HCAs in the paediatric population.

Therapeutic Gene Editing with CRISPR: A Laboratory Medicine Perspective.

Therapeutic gene editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system offers significant improvements in specificity and programmability compared with previous methods. CRISPR editing strategies can be used ex vivo and in vivo with many theoretic disease applications. Off-target effects of CRISPR-mediated gene editing are an important outcome to be aware of, minimize, and detect. The current methods of regulatory approval for personalized therapies are complex and may be proved inefficient as these therapies are implemented more widely. The role of pathologists and laboratory medicine practitioners is vital to the clinical implementation of therapeutic gene editing.

Immune infiltrates and PD-L1 expression in treatment-naïve acinar prostatic adenocarcinoma: an exploratory analysis.

Tumour-induced immunosuppression plays a role in the development and progression of cancer. Of interest is the interaction between programmed death-1 and programmed death ligand-1 (PD-L1) which can be targeted through immune checkpoint blockade; however, there are limited data surrounding the composition of the immune milieu in prostate cancer. We preliminarily assessed 21 radical prostatectomies in therapy-naïve patients for immune markers and PD-L1 expression. The immune infiltrates were higher in adenocarcinoma than benign prostate (lymphocytes p<0.001, macrophages p=0.010) with 5% of cases being PD-L1 high (≥5% expression). Increased peritumoural CD68 and CD163 expression correlated with lower grade group (GG) (p=0.024 and p=0.014, respectively) with a trend towards increased CD68 expression in lower stage cases (p=0.086). There was also increased CD45 expression in lower GGs (p=0.063). We found the immune infiltrate in acinar prostate cancer to be extremely heterogeneous with an overall immunophenotype unlikely to respond to immune checkpoint blockade.

Reproducibility of PD-L1 immunohistochemistry interpretation across various types of genitourinary and head/neck carcinomas, antibody clones, and tissue types.

Programmed death-ligand 1 (PD-L1) expression by tumor cells is a mechanism for down-regulation of antitumor T-cell responses and is a target for immunotherapy in various cancers. PD-L1 status as a predictor of treatment response has led to the development of multiple platforms with different reference cutoffs. We studied 128 cases of genitourinary and head/neck carcinomas, aiming to assess the frequency of PD-L1 positivity, interobserver reliability of PD-L1 interpretation, and the concordance of PD-L1 scoring between small samples from tissue microarray and whole sections using SP263 and SP142 clones. No prostatic carcinoma (0/21) was PD-L1 positive compared with 15% to 24% PD-L1 positivity in urothelial carcinoma (UC), hypopharyngeal squamous cell carcinoma (HP-SCC), and high-grade salivary gland carcinoma. There was substantial interobserver agreement in determining overall PD-L1 positivity in UC and HP-SCC using SP263 (κ = 0.702) and SP142 (κ = 0.757) antibodies. Subgroup analysis for both antibodies showed excellent agreement in UC (κ = 0.812 and 0.827) and moderate agreement in HP-SCC (κ = 0.469 and 0.591). Moderate to substantial agreement between tissue microarray and whole sections was achieved using SP263 (overall, κ = 0.573; UC, κ = 0.424; and HP-SCC, κ = 0.667) and SP142 (UC, κ = 0.493). PD-L1 interpretation in genitourinary and head/neck carcinomas is reliable and reproducible among pathologists and across different tissue preparations. Tumor PD-L1 staining heterogeneity may lead to discrepant PD-L1 results between small biopsies and large sections from surgical resection in a subset of tumors (19% of UC and 15% of HP-SCC). Retesting in such cases may be required to determine patient suitability for anti-PD-1/PD-L1 therapy.

Prognostic pathological factors in radical cystectomy after neoadjuvant chemotherapy.

AIMS: We undertook a systematic evaluation of the prognostic value of numerous histological factors in 165 radical cystectomies (RCs) of patients with invasive urothelial carcinoma (UC) who underwent surgery after neoadjuvant chemotherapy (NAC). METHODS AND RESULTS: Tumour regression grade (TRG) and therapy-related stromal and epithelial changes were also recorded. Locally advanced disease (≥pT2 and/or pN+) was present in 64% of patients, 22% had no evidence of residual carcinoma (pT0 + pN0), and 28% had no evidence of residual muscle-invasive carcinoma (≤pT1 + N0). TRG1, TRG2 and TRG3 were found in 32%, 15% and 50% of patients, respectively. Histological variants of UC were reported in 25% of cases. The most common therapy-related stromal change was fibroblastic reaction (78%), and the most common epithelial change in residual UC was smudgy and poorly preserved chromatin (28%). Prominent stromal and epithelial changes were noted in 41% and 5% of RCs, respectively. Progression was found in 45% of patients, and cancer-related deaths occurred in 30%. Multivariate analysis showed that the only independent prognostic parameters for progression were T stage, N stage, lymphovascular invasion, and margin status. Similarly, only T stage, N stage and margin status correlated with cancer-related deaths. Neither TRG nor any of the stromal-related or epithelial-related variables correlated with outcome. CONCLUSIONS: We confirm that the traditional and routinely reported histological parameters in RC post-NAC remain the most powerful prognosticators of disease course. The significance of TRG in the bladder remains unconfirmed.

Design and Implementation of a Trauma Care Bundle at a Community Hospital.

The Niagara Health System (NHS) in Ontario, Canada is comprised of three non-designated trauma center (NTC) hospitals which provide primary care to approximately 100 trauma patients annually. NTCs often lack standardized resources such as trauma surgeons, trauma-trained emergency room physicians, Advanced Trauma Life Support certified staff, trauma protocols, and other resources commonly found at designated trauma centers. Studies indicate that these differences contribute to poorer outcomes for trauma patients treated at community hospitals in Ontario, including the NTC hospitals of the NHS. In other settings healthcare checklists and bundles have proven effective in streamlining processes to ensure effective, efficient and timely patient care. Quality Improvement (QI) tools and methods were used to design, implement, and evaluate a trauma care bundle at one of the NHS's community hospitals. We assessed outcome and process measures through a chart audit of all trauma care patients in the NHS from July 2015 - November 2015. A Safety Attitudes Questionnaire (SAQ) was administered to health system staff who were involved in the pilot to assess balancing measures. Between July-November 2015, 39 patients were treated at the St. Catharines Hospital that were identified as either Canadian Triage and Acuity Scale (CTAS) I or CTAS II trauma patients. Of those 39 major trauma patients, 15 received care using the trauma care bundle, representing a 38% uptake. Patients who received care with the trauma bundle had an average Emergency Department (ED) length of stay (LOS) of 1.7 hours, compared with those patients in whom the bundle was not used, whose average ED LOS was 3.4 hours. The SAQ administered to ED physicians who used the bundle (n=10) highlighted the impact on ED patient safety. These early findings suggest that the bundle provides a substantial improvement to the current trauma care process within the Niagara Health System.