Digital quantification of p16-positive foci in fibrotic interstitial lung disease is associated with a phenotype of idiopathic pulmonary fibrosis with reduced survival.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is associated with increased expression of cyclin-dependent kinase inhibitors such as p16 and p21, and subsequent induction of cell cycle arrest, cellular senescence, and pro-fibrotic gene expression. We sought to link p16-expression with a diagnosis of IPF or other fibrotic interstitial lung diseases (ILDs), radiographic pattern, senescent foci-specific gene expression, antifibrotic therapy response, and lung transplant (LTx)-free survival. METHODS: Eighty-six cases of fibrosing ILD were identified with surgical lung biopsy. Immunohistochemistry for p16 was performed on sections with the most active fibrosis. p16-positive foci (loose collection of p16-positive fibroblasts with overlying p16-positive epithelium) were identified on digital slides and quantified. Cases were scored as p16-low (≤ 2.1 foci per 100 mm2) or p16-high (> 2.1 foci per 100 mm2). Twenty-four areas including senescent foci, fibrotic and normal areas were characterized using in situ RNA expression analysis with digital spatial profiling (DSP) in selected cases. RESULTS: The presence of p16-positive foci was specific for the diagnosis of IPF, where 50% of cases expressed any level of p16 and 26% were p16-high. There was no relationship between radiographic pattern and p16 expression. However, there was increased expression of cyclin-dependent kinase inhibitors, collagens and matrix remodeling genes within p16-positive foci, and cases with high p16 expression had shorter LTx-free survival. On the other hand, antifibrotic therapy was significantly protective. DSP demonstrated that fibroblastic foci exhibit transcriptional features clearly distinct from that of normal-looking and even fibrotic areas. CONCLUSIONS: We demonstrated the potential clinical applicability of a standardized quantification of p16-positive fibroblastic foci. This method identifies an IPF phenotype associated with foci-specific upregulation of senescence-associated and matrix remodeling gene expression. While these patients have reduced LTx-free survival, good response to antifibrotic therapies was observed in those who were treated.

Fostering mentorship for clinician-investigator trainees: overview and recommendations.

PURPOSE: The Clinician Investigator Trainee Association of Canada/ Association des cliniciens-chercheurs en formation du Canada (CITAC/ACCFC) recently published the first survey to assess factors contributing to trainee satisfaction. One key finding is that increased level of mentorship strongly correlates with overall satisfaction; however, while 98% of respondents reported mentorship as important to success, more than 60% expressed some dissatisfaction with the mentorship received. To help address this discrepancy, we reviewed mentorship in academic medicine, focusing on clinician-investigator trainees, and distilled a set of recommendations for mentors, mentees and institutions. SOURCE: OVID and manual curation based on the search terms 'mentorship' AND 'education, medical and research' identified 198 articles. Two authours independently reviewed both titles and abstracts and narrowed them down to 75 articles, based on relevance to mentorship in academic medicine. Consensus resulted in the selection of 19 articles for detailed review. Principal findings and Conclusion: Mentorship is beneficial at each training stage and is associated with greater research productivity, career retention and promotion. Nevertheless, more rigorous studies are needed, especially regarding cost-effectiveness. Studies have identified the characteristics of good mentors, including the ability to ensure open communication, ability to maintain confidentiality and ability to ensure that there is no mentor-mentee competition. Similarly, the characteristics of good mentees have been identified as the ability to take ownership of a project and the ability to build a network or team of mentors. The literature has also identified the actions that institutions can take to facilitate mentorship, which include mentor training and recognizing mentorship through awards.

Scientific overview: CSCI-CITAC Annual General Meeting and Young Investigators' Forum 2014.

The Canadian Society of Clinician Investigators (CSCI) and Clinical Investigator Trainee Association of Canada/Association des cliniciens-chercheurs en formation du Canada (CITAC/ACCFC) annual general meeting (AGM) was held in Toronto during November 21-24, 2015 for the first time in conjunction with the University of Toronto Clinician-Investigator Program Research Day. The overall theme for this year's meeting was the role of mentorship in career development, with presentations from Dr. Chaim Bell (University of Toronto), Dr. Shurjeel Choudhri (Bayer Healthcare), Dr. Ken Croitoru (University of Toronto), Dr. Astrid Guttman (University of Toronto), Dr. Prabhat Jha (University of Toronto) and Dr. Sheila Singh (McMaster University). The keynote speakers of the 2014 AGM included Dr. Qutayba Hamid, who was presented with the Distinguished Scientist Award, Dr. Ravi Retnakaran, who was presented with the Joe Doupe Award, and Dr. Lorne Babiuk, who was the CSCI-RCPSC Henry Friesen Award winner. The highlight of the conference was, once again, the outstanding scientific presentations from the numerous clinician investigator (CI) trainees from across the country who presented at the Young Investigators' Forum. Their research topics spanned the diverse fields of science and medicine, ranging from basic science to cutting-edge translational research, and their work has been summarized in this review. Over 120 abstracts were presented at this year's meeting. This work was presented during two poster sessions, with the six most outstanding submitted abstracts presented in the form of oral presentations during the President's Forum.

Scientific overview: CSCI-CITAC Annual General Meeting and Young Investigator's Forum 2013.

The 2013 joint Canadian Society of Clinician Investigators (CSCI)-Clinical Investigator Trainee Association of Canada/Association des cliniciens-chercheurs en formation du Canada (CITAC/ACCFC) annual general meeting(AGM) was held in Ottawa, September 2013. The symposium focused on "Applications of the 'omics' to Clinical Practice", with presentations from Drs. William T. Gibson (University of British Columbia), Julie Ho (University of Manitoba) and David Hwang (University of Toronto), discussing topics of genome, proteome and the microbiome, respectively. Other highlights from the 2013 AGM include presentations by Dr. Salim Yusuf (McMaster University, 2013 CSCI-RCPSC Henry Friesen Award winner), Dr. Gary Lewis (University of Toronto, 2013 CSCI Distinguished Scientist Award winner) and Dr. Michael Taylor (University of Toronto, 2013 Joe Doupe Award winner). The CSCI/CITAC/Friends of CIHR Joint Symposium consisted of presentations from Drs. John Bell (University of Ottawa), Dan Drucker (University of Toronto) and Heather J. Dean (University of Manitoba). Finally, the meeting ended with the presentation "The Power of an Idea to Bring Ideas to Power" by Dr. Harvey V. Fineberg (President, U.S. Institute of Medicine), the winner of the 2013 Henry Friesen International Prize. Also presented at the conference was research by clinician investigator (CI) trainees from across Canada; ie., those enrolled in MD/MSc, MD/PhD or Clinician Investigator Program(CIP) programs. Canadian trainees' research extended beyond the pillar of biomedical research, covering the spectrum between basic and clinical research, with a focus on the causes of significant morbidity and mortality for Canadians, including cancers, infectious diseases and other maladies. It is this research that we have summarized in this review.

Merkel cell carcinoma mimicking acute leukemia.

Bone marrow aspirate showed diffuse infiltration by a population of monomorphic cells with scant cytoplasm, markedly increased nuclear-to-cytoplasmic ratio, and numerous indistinct nucleoli. Bone marrow biopsy confirmed extensive marrow infiltration by a malignant neoplasm with strong and diffuse expression of synaptophysin by immunohistochemistry, consistent with metastases from Merkel Cell carcinoma.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

Urticaria pigmentosa and systemic mastocytosis.

Key Clinical Message: Additional investigations for systemic involvement should be initiated once the diagnosis of cutaneous mastocytosis has been established in an adult patient. A serum tryptase can serve as a screening test for systemic mastocytosis, and persistent elevations should prompt further investigations, such as bone marrow studies. Abstract: Urticaria pigmentosa (UP) is the most common form of cutaneous mastocytosis, presenting as a wide variety of macroscopic appearances. Cutaneous mastocytosis in pediatric patients usually does not present with systemic involvement, but more than half of adult patients with cutaneous mastocytosis demonstrate systemic involvement. Currently, there is no guidance surrounding systemic testing in patients with UP. A 50-year-old Caucasian male was referred to the Clinical Immunology and Allergy clinic with a history of a rash. He initially presented to hospital 12 years prior with group A beta hemolytic streptococcus bacteremia treated with multiple different antibiotics. One week following discharge, he developed erythematous brown spots on his right leg which were flat, non-pruritic, and not painful. The rash later expanded to his trunk and extremities. A skin biopsy performed 2 years prior to referral to our clinic demonstrated urticaria pigmentosa. The CD117 immunohistochemical stain showed increased perivascular and interstitial mast cells in the superficial dermis. Darier's sign was negative on physical examination, and venom testing was also negative. Although he had no symptoms of systemic involvement, his serum tryptase was elevated at 47.6 ng/mL in the context of normal kidney and liver function. A skeletal survey was normal, and an abdominal ultrasound ruled out splenomegaly. Bone marrow biopsy demonstrated a mild increase in paratrabecular and perivascular atypical mast cells, in keeping with systemic mastocytosis. Adult patients with cutaneous mastocytosis have a high likelihood of having an underlying systemic mast cell disorder. Therefore, any patient presenting with characteristic skin findings should be investigated as having a cutaneous manifestation of systemic mastocytosis. This case demonstrates the utility of serum tryptase and its role in triggering additional investigations and guiding appropriate therapy.

Pulmonary carcinosarcoma with an aggressive heterologous angiosarcoma component: A case report.

Pulmonary carcinosarcomas are rare biphasic lung tumors comprised of malignant epithelial and malignant mesenchymal components. The most common heterologous sarcomatous elements are osteosarcoma, rhabdomyosarcoma, and chondrosarcoma; a heterologous angiosarcoma component in a pulmonary carcinosarcoma is exceedingly rare. We report a case of a pulmonary carcinosarcoma containing adenocarcinoma, squamous cell carcinoma, undifferentiated malignant spindle cell, and heterologous angiosarcoma components. The patient, a 64-year-old woman, had initially presented to medical attention with hemoptysis. Although the tumor was thought to be confined to the lung at resection (pT3N0), she developed multiple metastatic foci within 3 weeks of lobectomy and required the evacuation of an intraparenchymal left occipital hematoma secondary to a hemorrhagic intra-axial focus of metastatic carcinosarcoma. She died 6 weeks after her primary lung resection from rapidly progressive metastatic disease. We hope the description and discussion provided herein will further the medical community's understanding of this rare malignancy.

When donor T cells attack: The curious case of liver transplant-associated acute graft-versus-host-disease.

Graft versus host disease is a rare but deadly complication of solid organ transplant. Clinical features of graft-versus-host-disease are non-specific, which may lead to delayed diagnosis as more common conditions including infections or drug reactions are considered. We describe a 54-year-old male patient who underwent liver transplantation for alcohol use disorder-related cirrhosis and developed acute graft-versus-host disease. Initial clinical presentation included dermatitis, bone marrow failure and enteritis. Results of skin biopsy and cytogenetic studies were consistent with liver transplant-associated acute graft-versus-host disease. The importance of this case is to highlight to transplant physicians and surgeons the challenges of diagnosing graft-versus-host-disease. In our case, pre-existing partnerships among the liver and hematopoietic stem cell transplant teams, transfusion medicine specialists, critical care specialists and facilitated timely communication relevant to confirming graft-versus-host disease. We propose an algorithm to assist in the workup of suspected graft-versus-host disease. Because this condition is characterized by high mortality, a high index of suspicion is imperative for prompt diagnosis and optimal management of the donor-recipient immune interaction when patients present with classic clinical features.

EBV-positive T/NK-associated lymphoproliferative disorders of childhood: A complete autopsy report.

INTRODUCTION: Epstein-Barr Virus (EBV)-associated systemic T-cell lymphoproliferative disorder of childhood is a rare but severe manifestation of chronic EBV infection. Despite several case reports characterizing this rare hematological neoplasm, the literature describes extensive heterogeneity in the presentation of this disease. CASE PRESENTATION: Here we present a complete autopsy of a 16-year-old girl who ultimately succumbed to EBV-associated systemic T-cell lymphoproliferative disorder of childhood. Her clinical presentation demonstrated a non-specific pharyngitis with positive mono spot test, evolving into fulminant multi-organ failure, disseminated intravascular coagulopathy, sepsis, and ultimately death. CONCLUSIONS: Post-mortem findings included extensive hemorrhage, and infiltration of the liver, spleen, lymph nodes and bone marrow with neoplastic T-cells. There was extensive hemophagocytic lymphohistiocytosis (HLH) within these organs, suggesting overlap between the EBV-associated systemic T-cell lymphoproliferative disorder of childhood and EBV-associated HLH. We hope these findings provide a more comprehensive overview of several possible manifestations of EBV-associated systemic T-cell lymphoproliferative disorder of childhood.

Complete duodenal necrosis associated with non-traumatic duodenal hematoma requiring emergent pancreatico-duodenectomy.

INTRODUCTION: Duodenal necrosis is a rare complication of acute pancreatitis but can occur given the shared blood supply to the head of the pancreas and the duodenum. PRESENTATION OF CASE: A 55-year-old male presented with acute-on-chronic pancreatitis and a duodenal hematoma. The hematoma expanded to occlude the biliary tree and, shortly after, the duodenum necrosed and perforated. The patient required an emergent pancreaticoduodenectomy performed in two stages. DISCUSSION: Surgical management is complex and a difficult challenge for a general surgeon. Many advocate for wide drainage to create a controlled fistula using a malecot through the wall defect/separate duodenotomy/a retrograde jejunostomy tube. This case represents an extreme variation on this issue which was best managed by definitive resection given the extent of the necrosis. CONCLUSION: This case report demonstrates that duodenal hematoma and necrosis should be recognized as part of the spectrum of consequences of acute pancreatitis. General surgeons should have a surgical approach to this complication whether that be diversion or definitive resection.

Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.

Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently 'tagged' by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.

Differential in vivo zymography: a method for observing matrix metalloproteinase activity in the zebrafish embryo.

Investigations into the molecular mechanisms of, and cellular signaling pathways modulating ECM remodeling are especially challenging due to the complex post-translational regulation of the primary effectors of ECM catabolism - the matrix metalloproteinases (MMPs). Recently a variety of approaches to the detection of MMP activity have been developed, and the prospect of visualizing ECM remodeling activity in living tissues is now opening exciting avenues of research for matrix biologists. In particular the use of FRET-quenched MMP substrates, which generate a fluorescent signal upon hydrolysis, is becoming increasingly popular, especially because linkers with defined and/or restricted proteolytic sensitivity can be used to bind fluorophore-quencher pairs, making these probes useful in characterizing the activity of specific proteases. We have taken advantage of the transparency and amenability to reverse genetics of the zebrafish embryo, in combination with these fluorogenic MMP substrates, to develop a multiplex in vivo assay for MMP activity that we dub "differential in vivo zymography."

The zebrafish embryo: a powerful model system for investigating matrix remodeling.

Extracellular matrix (ECM) remodeling is a process that is crucial to the development of embryos, the growth and metastasis of tumors, and wound healing and homeostasis of tissues in adults. As such, it involves dozens of gene products that are regulated by mechanisms operating at transcriptional and multiple posttranslational levels. This complexity of regulation has made the development of a comprehensive understanding of the biology of ECM remodeling in vivo an unusually challenging task, yet such an understanding would be of profound value to our knowledge of and clinical approaches to the treatment of many cancers. The primary effectors of ECM remodeling are the matrix metalloproteinases (MMPs). Homologs of this gene family have been identified in every metazoan examined. We propose that the zebrafish embryo is an ideal system for the study of the regulation of MMP activity, and we present some progress we have made in the development of this organism as a platform for MMP research. We have identified 25 genes encoding MMPs in the zebrafish genome, and 5 genes encoding their endogenous inhibitors, the tissue inhibitors of MMPs. Based on a phylogenetic analysis, we have identified the most probable homologies of these sequences and found that there are two that are of equivocal identity. We have developed 17 antibodies specific to zebrafish MMPs and have begun characterizing the ontogeny of these molecules. Finally, we have developed two novel assays that allow the detection and characterization of active MMPs in vivo (differential in vivo zymography and activity-based protease profiling). In combination with the array of powerful biochemical, genomic, cell, and molecular biological techniques available to zebrafish researchers already, we feel that these new reagents and techniques make the zebrafish the best model system for the study of MMP regulation currently available.