Immune profiling of dedifferentiated liposarcoma and identification of novel antigens for targeted immunotherapy.

Dedifferentiated liposarcoma (DDLS) is an aggressive, recurring sarcoma with limited treatments. T-cell immunotherapies selectively target malignant cells, holding promise against DDLS. The development of successful immunotherapy for DDLS requires a thorough evaluation of the tumor immune microenvironment and the identification and characterization of targetable immunogenic tumor antigens. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens, we used the nCounter NanoString platform, analyzing gene expression profiles across 29 DDLS and 10 healthy adipose tissue samples. Hierarchical clustering of tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumors and 14 non-inflamed tumors, demonstrating tumor heterogeneity within this sarcoma subtype. Among the identified antigens, PBK and TTK exhibited substantial upregulation in mRNA expression compared to healthy adipose tissue controls, further corroborated by positive protein expression by IHC. This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS, and provides a novel targetable antigen in DDLS. The results of this study lay the groundwork for the development of a novel immunotherapy for this highly aggressive sarcoma.

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.

Updates in Pathology for Retroperitoneal Soft Tissue Sarcoma.

Retroperitoneal tumors are extremely rare. More than 70% of primary retroperitoneal soft tissue tumors are malignant. The most common sarcomas in the retroperitoneum include liposarcomas and leiomyosarcoma, however other sarcomas, along with benign mesenchymal tumors, can occur. Sarcomas are a heterogenous group of tumors with overlapping microscopic features, posing a diagnostic challenge for the pathologist. Correct tumor classification has become important for prognostication and the evolving targeted therapies for sarcoma subtypes. In this review, the pathology of retroperitoneal soft tissue sarcomas is discussed, which is important to the surgical oncologist. In addition, less common sarcomas and benign mesenchymal tumors of the retroperitoneum, which may mimic sarcoma clinically and pathologically, are also discussed.

Negative Impact of COVID-19 Associated Health System Shutdown on Patients Diagnosed With Colorectal Cancer: A Retrospective Study From a Large Tertiary Center in Ontario, Canada.

Background: In March 2020, a directive to halt all elective and non-urgent procedures was issued in Ontario, Canada because of COVID-19. The directive caused a temporary slowdown of screening programs including surveillance colonoscopies for colorectal cancer (CRC). Our goal was to determine if there was a difference in patient and tumour characteristics between CRC patients treated surgically prior to the COVID-19 directive compared to CRC patients treated after the slowdown. Methods: CRC resections collected within the Champlain catchment area of eastern Ontario in the 6 months prior to COVID-19 (August 1, 2019-January 31, 2020) were compared to CRC resections collected in the 6 months post-COVID-19 slowdown (August 1, 2020-January 31, 2021). Clinical (e.g., gender, patient age, tumour site, and clinical presentation) and pathological (tumour size, tumour stage, nodal stage, and lymphovascular invasion) features were evaluated using chi-square tests, T-tests, and Mann-Whitney tests where appropriate. Results: Three hundred and thirty-eight CRC specimens were identified (173 pre-COVID-19, 165 post-COVID-19 slowdown). CRC patients treated surgically post-COVID-19 slowdown had larger tumours (44 mm vs. 35 mm; P = 0.0048) and were more likely to have presented emergently (24% vs. 10%; P < 0.001). Although there was a trend towards higher tumour stage, nodal stage, and clinical stage, these differences did not reach statistical significance. Other demographic and pathologic variables including patient gender, age, and tumour site were similar between the two cohorts. Interpretation: The COVID-19 slowdown resulted in a shift in the severity of disease experienced by CRC patients in Ontario. Pandemic planning in the future should consider the long-term consequences to cancer diagnosis and management.

Glial Fibrillary Acidic Protein Expression Helps Distinguish Pleomorphic Adenoma from Histologic Mimics.

Pleomorphic adenoma (PA) is the most common biphasic type of salivary gland tumour to arise in adults. It is a biphasic tumour composed of both luminal (ductal) cells and abluminal (basal and myoepithelial) cells. Other biphasic salivary gland type tumours, both benign and malignant, can mimic PA, especially on small biopsies. Previous studies have shown that glial fibrillary acidic protein (GFAP) is preferentially expressed in PA and can be useful in the distinction from other salivary gland tumours. However, most of these studies were performed on a small subset of tumour types at a time when the classification of salivary gland type tumours was less refined. The purpose of this study was to assess the expression of glial fibrillary acidic protein (GFAP) in a broad group of both benign and malignant salivary gland tumours. The expression of GFAP was assessed in 99 tumours including 54 PAs, 5 basal cell adenomas, 1 myoepitheliomas, 5 adenoid cystic carcinomas, 6 epithelial-myoepithelial carcinomas (EMCA), 6 mucoepidermoid carcinomas, 7 salivary duct carcinomas, 1 adenocarcinomas NOS, 2 myoepithelial carcinomas, 4 basal cell adenocarcinomas, 5 acinic cell carcinomas and 3 polymorphous adenocarcinomas. Of the malignant cases, 8 were classified as carcinomas ex PA. GFAP was also assessed in 19 concurrent biopsy specimens. GFAP was expressed in the resections of 51 PAs examined (94%). Expression was predominantly strong and diffusely seen in myoepithelial cells. Strong and diffuse GFAP expression was also seen in two EMCAs (33%) and one myoepithelial carcinoma (50%). On biopsy specimens, 100% of PAs and basal cell adenomas expressed GFAP. GFAP was also seen in 1 out of 3 carcinomas ex PAs on biopsies. Almost all PAs show strong and diffuse expression of GFAP. In contrast, most malignant neoplasms that can mimic PA on biopsies show only rare, focal expression. Other benign tumours composed of abluminal/myoepithelial cells also show focal expression of GFAP, highlighting the spectrum these tumours share with PA. Overall, the presence of strong and diffuse GFAP expression can favour a benign neoplasm, specifically a PA, on limited biopsy specimens.

Mammary-type myofibroblastoma of the thigh mimicking liposarcoma.

Myofibroblastoma is a rare tumor that was originally described in the breast but has since been found in extra-mammary locations, where it been renamed mammary-type myofibroblastoma (MTM). We describe a case of MTM occurring in the anterior compartment of the thigh, which was initially interpreted as a possible a liposarcoma on MRI. Two subsequent biopsy samples and eventual excision revealed the tumor to have the characteristic pathology of MTM. This benign tumor, albeit rare, needs to be considered in the imaging differential diagnosis of fat containing deep soft tissue masses of the extremity.

Proliferative Verrucous Leukoplakia: An Expert Consensus Guideline for Standardized Assessment and Reporting.

The many diverse terms used to describe the wide spectrum of changes seen in proliferative verrucous leukoplakia (PVL) have resulted in disparate clinical management. The objective of this study was to produce an expert consensus guideline for standardized assessment and reporting by pathologists diagnosing PVL related lesions. 299 biopsies from 84 PVL patients from six institutions were selected from patients who had multifocal oral leukoplakic lesions identified over several years (a minimum follow-up period of 36 months). The lesions demonstrated the spectrum of histologic features described in PVL, and in some cases, patients developed oral cavity squamous cell carcinoma (SCC). An expert working group of oral and maxillofacial and head and neck pathologists reviewed microscopic features in a rigorous fashion, in combination with review of clinical photographs when available. The working group then selected 43 single slide biopsy cases for whole slide digital imaging (WSI) review by members of the consensus conference. The digital images were then reviewed in two surveys separated by a washout period of at least 90 days. Five non-PVL histologic mimics were included as controls. Cases were re-evaluated during a consensus conference with 19 members reporting on the cases. The best inter-observer diagnostic agreement relative to PVL lesions were classified as "corrugated ortho(para)hyperkeratotic lesion, not reactive" and "SCC" (chi-square p = 0.015). There was less than moderate agreement (kappa < 0.60) for lesions in the "Bulky hyperkeratotic epithelial proliferation, not reactive" category. There was ≥ moderate agreement (> 0.41 kappa) for 35 of 48 cases. This expert consensus guideline has been developed with support and endorsement from the leadership of the American Academy of Oral and Maxillofacial Pathology and the North American Society of Head and Neck Pathologists to recommend the use of standardized histopathologic criteria and descriptive terminology to indicate three categories of lesions within PVL: (1) "corrugated ortho(para)hyperkeratotic lesion, not reactive;" (2) "bulky hyperkeratotic epithelial proliferation, not reactive;" and (3) "suspicious for," or "squamous cell carcinoma." Classification of PVL lesions based on a combination of clinical findings and these histologic descriptive categories is encouraged in order to standardize reporting, aid in future research and potentially guide clinical management.

Subperiosteal chondromyxoid fibroma: a rare case involving the humeral diaphysis.

Initially described, in 1948, as a tumor that could be mistaken with chondrosarcoma at histopathology, chondromyxoid fibroma is now a well-recognized entity. Surface-type chondromyxoid fibroma, however, remains an extremely rare occurrence. We present a case of a 55-year-old woman, who experienced right arm pain for 5 years. After unsuccessful treatment for presumed thoracic outlet syndrome, MRI revealed a large mass abutting the anteromedial cortex of the distal humeral diaphysis in a subperiosteal location. Further characterization was made with radiography, CT, and bone scan, which were followed by ultrasound-guided biopsy. Although histopathologic features were suggestive of chondromyxoid fibroma, the diagnosis remained somewhat uncertain initially due to the very unusual location involving the diaphysis of the humerus. Surgical resection was performed, and subsequent histopathologic analysis confirmed the diagnosis of chondromyxoid fibroma. Despite being a rare entity, surface-type chondromyxoid fibroma would need to be considered in the differential when dealing with expansile surface diaphyseal lesions.

An evidence-based guideline on the application of molecular testing in the diagnosis, prediction of prognosis, and selection of therapy in non-GIST soft tissue sarcomas.

AIMS: To make recommendations on the indications for molecular testing regarding the diagnosis, prediction of prognosis, and treatment selection in adult patients with s oft tissue sarcomas (STS) excluding gastrointestinal stromal tumour. MATERIALS AND METHODS: This guideline was developed by the Cancer Care Ontario's Program in Evidence-Based Care (PEBC) and the Sarcoma Disease Site Group (DSG). The medline, embase, and Cochrane Library databases, main guideline websites, abstracts of relevant annual meetings, and PROSPERO databases were searched (January 2005 to October 2016). Internal and external reviews were conducted, with final approval by the PEBC and the Sarcoma DSG. RESULTS: Based on the available evidence, we made three S trong Recommendations, 14 Recommendations, 9 Qualified Statements, and seven No Recommendations. The three Strong Recommendations include: i) MDM2 amplification by fluorescence in situ hybridization (FISH) is recommended as a sensitive and specific test to differentiate patients with atypical lipomatous tumour/well-differentiated liposarcoma, or dedifferentiated liposarcoma from lipoma or other STS in the differential diagnosis; ii) SS18 (SYT) break-apart by FISH or SS18-SSX (SYT-SSX) fusion by reverse transcription-polymerase chain reaction is recommended as a sensitive and specific test to differentiate patients with synovial sarcoma from other sarcomas; iii) CTNNB1 S45F mutation by polymerase chain reaction is recommended as a prognostic factor for poor recurrence-free survival in patients with desmoid tumours. CONCLUSION: This guideline may serve as a framework for the thoughtful implementation of molecular studies at cancer centres and other jurisdictions. Some of the recommendations may need to be updated when new evidence appears in the future.

Putting the patient at the centre of pathology: an innovative approach to patient education-MyPathologyReport.ca.

In many centres, patients now have access to their electronic medical record (EMR) and laboratory results, including pathology reports, are amongst the most frequently accessed pieces of information. The pathology report is an important but highly technical medical document that can be difficult for patient and clinicians alike to interpret. To improve communication and patient safety, pathologists are being called upon to play a more direct role in patient care. Novel approaches have been undertaken by pathologists to address this need, including the addition of patient-friendly summaries at the beginning of pathology reports and the development of patient education tools. MyPathologyReport.ca is a novel website exclusively providing pathology education to patients. It has been designed to help patients understand the language of pathology and to effectively navigate their pathology report. At present, the website includes over 150 diagnostic articles and over 125 pathology dictionary definitions. The diagnostic articles span all body sites and include a variety of malignant, benign and non-neoplastic conditions. Since its creation, this website has been visited over 14 000 times, with cancer-related diagnoses and definitions representing the most commonly accessed articles. This website has been embedded in patient accessible EMRs and shared through partnerships with patients, caregivers and their respective advocacy groups. Our next steps involve longitudinal assessment of MyPathologyReport.ca from non-medical community members, evaluation of patient satisfaction and understanding and further collaboration with hospitals and care-providers to increase patient access to this resource.

Paraneoplastic manifestations of salivary gland tumours: A case report and review.

Salivary gland cancers are an uncommon and heterogenous group of malignancies, accounting for approximately 3% of head and neck tumors. We describe a case of a patient who presented with paraneoplastic Cushing's syndrome secondary to metastatic salivary ductal carcinoma (SDC). Paraneoplastic ACTH secretion initially responded to chemotherapy with complete resolution of clinical symptoms. To our knowledge, this is the first described case of an ACTH-secreting SDC. We also review other paraneoplastic syndromes (PNS) that have been reported in association with salivary gland cancers.

Histologic Classification and Molecular Signature of Polymorphous Adenocarcinoma (PAC) and Cribriform Adenocarcinoma of Salivary Gland (CASG): An International Interobserver Study.

Polymorphous adenocarcinoma (PAC) shows histologic diversity with streaming and targetoid features whereas cribriform adenocarcinoma of salivary gland (CASG) demonstrates predominantly cribriform and solid patterns with glomeruloid structures and optically clear nuclei. Opinions diverge on whether CASG represents a separate entity or a variant of PAC. We aimed to assess the level of agreement among 25 expert Head and Neck pathologists in classifying these tumors. Digital slides of 48 cases were reviewed and classified as: PAC, CASG, tumors with ≥50% of papillary architecture (PAP), and tumors with indeterminate features (IND). The consensus diagnoses were correlated with a previously reported molecular alteration. The consensus diagnoses were PAC in 18/48, CASG in16/48, PAP in 3/48, and IND in 11/48. There was a fair interobserver agreement in classifying the tumors (κ=0.370). The full consensus was achieved in 3 (6%) cases, all of which were classified as PAC. A moderate agreement was reached for PAC (κ=0.504) and PAP (κ=0.561), and a fair agreement was reached for CASG (κ=0.390). IND had only slight diagnostic concordance (κ=0.091). PAC predominantly harbored PRKD1 hotspot mutation, whereas CASG was associated with fusion involving PRKD1, PRKD2, or PRKD3. However, such molecular events were not exclusive as 7% of PAC had fusion and 13% of CASG had mutation. In conclusion, a fair to moderate interobserver agreement can be achieved in classifying PAC and CASG. However, a subset (23%) showed indeterminate features and was difficult to place along the morphologic spectrum of PAC/CASG among expert pathologists. This may explain the controversy in classifying these tumors.

Early and Late Complications of Percutaneous Core Needle Biopsy of Retroperitoneal Tumors at Two Tertiary Sarcoma Centers.

BACKGROUND: Concern persists regarding percutaneous core needle biopsy (CNB) of a potentially malignant lesion of the retroperitoneum due to the perceived risk of immediate complications and adverse oncologic outcomes, including needle tract seeding (NTS). OBJECTIVE: The aim of this study was to evaluate the incidence of (1) early complications and (2) NTS following CNB of suspected retroperitoneal sarcoma (RPS). METHODS: Patients who underwent CNB of an RP mass with pre-biopsy suspicion of sarcoma were identified from a prospective database at two centers: (1) Princess Margaret Cancer Centre/Mount Sinai Hospital, Toronto (2009-2015); and (2) The Ottawa Hospital (1999-2015). Early complications, including bleeding, pain, infection, and organ injury, were recorded. Instances of NTS were identified from long-term follow-up of patients who underwent resection of primary RPS at these two centers after initial CNB (1996-2013). RESULTS: Of 358 percutaneous CNBs of suspected RPS performed over the study period, 7 (2.0%) resulted in minor bleeding with no transfusion, 3 (0.8%) resulted in significant pain, 1 (0.3%) resulted in unplanned admission to hospital for observation, and 1 (0.3%) resulted in a pneumothorax. There were no infections. In 203 patients who underwent resection of RPS following CNB, crude cumulative local recurrence was 24% at 5 years. At a median follow-up of 44 months, there was one case of NTS (approximately 0.5%). CONCLUSION: This large bi-institutional experience with CNB of an RP mass demonstrates that both the early complication rate and the incidence of NTS are very low. Physicians and patients can be reassured that the benefits of CNB in diagnosing sarcoma and determining its histologic subtype and grade far outweigh the risks.

PAX8-GLIS3 gene fusion is a pathognomonic genetic alteration of hyalinizing trabecular tumors of the thyroid.

The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8-GLIS1 or PAX8-GLIS3 gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8-GLIS3 fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n = 8), whole-exome sequencing (n = 3) or targeted massively parallel sequencing (n = 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8 (2q14.1) and GLIS3 (9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1-2 of PAX8 and exons 3-11 of GLIS3, GLIS3 is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8-GLIS3 gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1 rearrangements were identified. Conversely, no PAX8-GLIS3 gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8-GLIS3 fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8-GLIS3 fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.

Early squamous cell carcinoma of the oral tongue with histologically benign lymph nodes: A model predicting local control and vetting of the eighth edition of the American Joint Committee on Cancer pathologic T stage.

BACKGROUND: The eighth edition of the American Joint Committee on Cancer staging manual (AJCC8) added depth of invasion to the definition of pathologic T stage (pT). In the current study, the authors assess pT stage migration and the prognostic performance of the updated pT stage and compare it with other clinicopathologic variables in patients with early squamous cell carcinoma of the oral tongue (OTSCC; tumors measuring ≤4 cm) with histologically benign lymph nodes (pN0). METHODS: A multi-institutional cohort of patients with early OTSCC was restaged as per AJCC8. Primary endpoints were local recurrence (LR) and locoregional recurrence (LRR). Influential variables were identified and an LR/LRR prediction model was developed. RESULTS: There were a total of 494 patients, with 49 LR and 73 LRR. AJCC8 pT criteria resulted in upstaging of 37.9% of patients (187 of 494 patients), including 34.5% (64 of 185 patients) from pT2 to pT3, without improving the prognostication for LR or LRR. Both LR and LRR were found to be similar for patients with AJCC8 pT2 and pT3 disease. On multivariate analysis, LR was only found to be associated with distance to the closest margin (hazard ratio, 0.36; 95% CI, 0.20-0.64 [P = .0007]) and perineural invasion (hazard ratio, 1.92; 95% CI, 1.10-0.64 [P = .046]). Based on these 2 predictors, a final proportional hazards regression model (which may be used similar to a nomogram) was developed. The proposed model appeared to be superior to AJCC pT stage for estimating the probability of LR and LRR for individual patients with early OTSCC. CONCLUSIONS: AJCC8 pT criteria resulted in pT upstaging of patients with pN0 disease without improved LR or LRR prognostication. The proposed model based on distance to the closest margin and perineural invasion, status outperformed pT as a predictor of LR and LRR in patients with early OTSCC.

Metastasizing Pleomorphic Adenoma: Recurrent PLAG1/HMGA2 Rearrangements and Identification of a Novel HMGA2-TMTC2 Fusion.

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. On a molecular level PA is characterized by a translocation involving PLAG1 or HMGA2. PA is considered to be a benign tumor although it can undergo malignant transformation. Alternatively, cases of histologically benign PA "metastasizing" to lymph nodes or distant body sites are well documented. Several theories have been proposed to explain this behavior. However, there is a lack of molecular data available to assess the relationship of metastasizing PA (MPA) and their benign counterparts. In this study we describe 4 cases of MPAs and perform the first molecular study linking them to conventional PA. The index case was identified in the course of routine clinical practice, while the other cases were retrieved from the archives of the authors. Slides were reviewed to confirm the diagnosis of both the primary/recurrent tumor and the metastasis. Fluorescence in situ hybridization (FISH) was performed in all cases and RNA sequencing was performed on the index case. In all cases there was a history of recurrent PA involving the parotid. Lymph node metastases were identified in 2 cases; non-lymph node metastases were identified in 3 cases. All the metastases were histologically benign. RNA sequencing performed on the index case demonstrated a novel HMGA2-TMTC2 translocation, which was confirmed by separate FISH break-apart assays for both genes. FISH performed on the remaining cases demonstrated rearrangement of PLAG1 in all 3 cases. This study demonstrates that MPA harbors the same disease-defining molecular hallmark as their benign counterparts.

Measuring Depth of Invasion in Early Squamous Cell Carcinoma of the Oral Tongue: Positive Deep Margin, Extratumoral Perineural Invasion, and Other Challenges.

The 8th edition of American Joint Committee on Cancer (AJCC 8th) staging manual incorporated depth of invasion (DOI) into pT stage of oral cavity cancer. The aim of this study was to characterize several histological findings that may complicate measurement of DOI in early conventional squamous cell carcinomas (SCC) of the oral tongue: (1) lack of or minimal residual carcinoma following biopsy; (2) positive deep margin; (3) extratumoral perineural invasion (PNI); and (4) lymphatic or vascular invasion. Conventional SCC of the oral tongue (n = 407) with the largest dimension of ≤ 4 cm and with a negative elective cervical lymph node dissection (pN0) were reviewed. A clear plastic ruler was used to measure DOI by dropping a "plumb line" to the deepest point of the invasive tumor from the level of the basement membrane of the normal mucosa closest to the invasive tumor. Examples of identifying  reference point on the mucosal surface of oral tongue from which to measure the DOI are illustrated. In the experience of one contributing institution, the residual carcinoma was absent in 14.2% of glossectomies (34/239), while in 4.8% of cases (10/205) there was only minimal residual carcinoma. In 11.5% (21/183) of pT2 cases the deep margin was positive and thus DOI and pT may be underestimated. Of all cases with PNI, extratumoral PNI was identified in 23.1% (31/134) of cases, but represented the deepest point of invasion in only two cases. In one case, lymphatic invasion represented the deepest point of invasion and could have led to upstaging from pT1 to pT2. In conclusion, DOI measurement for SCC of the oral tongue may require re-examination of the diagnostic biopsy in up to 20% of cases due to the absence or only minimal residual carcinoma in glossectomy specimens. In 11.5% of apparently pT2 cases, DOI may be underestimated due to the positive deep margin. Rarely, extratumoral PNI or lymphatic invasion may be the deepest point of invasion. Overall, two issues (absent or minimal residual disease and positive deep margin) may confound DOI measurement in early SCCs of oral tongue.

Pannexin 1 inhibits rhabdomyosarcoma progression through a mechanism independent of its canonical channel function.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma of childhood thought to arise from impaired differentiation of skeletal muscle progenitors. We have recently identified Pannexin 1 (PANX1) channels as a novel regulator of skeletal myogenesis. In the present study, we determined that PANX1 transcript and protein levels are down-regulated in embryonal (eRMS) and alveolar RMS (aRMS) patient-derived cell lines and primary tumor specimens as compared to differentiated skeletal muscle myoblasts and tissue, respectively. While not sufficient to overcome the inability of RMS to reach terminal differentiation, ectopic expression of PANX1 in eRMS (Rh18) and aRMS (Rh30) cells significantly decreased their proliferative and migratory potential. Furthermore, ectopic PANX1 abolished 3D spheroid formation in eRMS and aRMS cells and induced regression of established spheroids through induction of apoptosis. Notably, PANX1 expression also significantly reduced the growth of human eRMS and aRMS tumor xenografts in vivo. Interestingly, PANX1 does not form active channels when expressed in eRMS (Rh18) and aRMS (Rh30) cells and the addition of PANX1 channel inhibitors did not alter or reverse the PANX1-mediated reduction of cell proliferation and migration. Moreover, expression of channel-defective PANX1 mutants not only disrupted eRMS and aRMS 3D spheroids, but also inhibited in vivo RMS tumor growth. Altogether our findings suggest that PANX1 alleviates RMS malignant properties in vitro and in vivo through a process that is independent of its canonical channel function.