NIPBL::NACC1 Fusion Hepatic Carcinoma.

Several reports describing a rare primary liver tumor with histologic features reminiscent of follicular thyroid neoplasms have been published under a variety of descriptive terms including thyroid-like, solid tubulocystic, and cholangioblastic cholangiocarcinoma. Although these tumors are considered to represent histologic variants, they lack classic features of cholangiocarcinoma and have unique characteristics, namely immunoreactivity for inhibin and NIPBL::NACC1 fusions. The purpose of this study is to present clinicopathologic and molecular data for a large series of these tumors to better understand their pathogenesis. We identified 11 hepatic tumors with these features. Immunohistochemical and NACC1 and NIPBL fluorescence in situ hybridization assays were performed on all cases. Four cases had available material for whole-genome sequencing (WGS) analysis. Most patients were adult women (mean age: 42 y) who presented with abdominal pain and large hepatic masses (mean size: 14 cm). Ten patients had no known liver disease. Of the patients with follow-up information, 3/9 (33%) pursued aggressive behavior. All tumors were composed of bland cuboidal cells with follicular and solid/trabecular growth patterns in various combinations, were immunoreactive for inhibin, showed albumin mRNA by in situ hybridization, and harbored the NIPBL::NACC1 fusion by fluorescence in situ hybridization. WGS corroborated the presence of the fusion in all 4 tested cases, high tumor mutational burden in 2 cases, and over 30 structural variants per case in 3 sequenced tumors. The cases lacked mutations typical of conventional intrahepatic cholangiocarcinoma. In this report, we describe the largest series of primary inhibin-positive hepatic neoplasms harboring a NIPBL::NACC1 fusion and the first WGS analysis of these tumors. We propose to name this neoplasm NIPBL:NACC1 fusion hepatic carcinoma.

Discovery of Selective Tertiary Amide Inhibitors of Cyclin-Dependent Kinase 2 (CDK2).

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.

CD8+ T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy.

Although immune checkpoint inhibitors (ICIs) are established as effective cancer therapies, overcoming therapeutic resistance remains a critical challenge. Here we identify interleukin 6 (IL-6) as a correlate of poor response to atezolizumab (anti-PD-L1) in large clinical trials of advanced kidney, breast, and bladder cancers. In pre-clinical models, combined blockade of PD-L1 and the IL-6 receptor (IL6R) causes synergistic regression of large established tumors and substantially improves anti-tumor CD8+ cytotoxic T lymphocyte (CTL) responses compared with anti-PD-L1 alone. Circulating CTLs from cancer patients with high plasma IL-6 display a repressed functional profile based on single-cell RNA sequencing, and IL-6-STAT3 signaling inhibits classical cytotoxic differentiation of CTLs in vitro. In tumor-bearing mice, CTL-specific IL6R deficiency is sufficient to improve anti-PD-L1 activity. Thus, based on both clinical and experimental evidence, agents targeting IL-6 signaling are plausible partners for combination with ICIs in cancer patients.

GLI1 -Rearranged Enteric Tumor : Expanding the Spectrum of Gastrointestinal Neoplasms With GLI1 Gene Fusions.

GLI1 encodes a transcription factor that targets cell cycle regulators affecting stem cell proliferation. GLI1 gene fusions were initially described in pericytomas with a t[7;12] translocation and more recently in gastric plexiform fibromyxomas and gastroblastomas. This study describes the clinicopathologic, immunohistochemical, and molecular features of three intestinal-based neoplasms harboring GLI1 gene fusions. We studied three unique mesenchymal small bowel tumors. Paraffin embedded tumor tissues from these cases and 62 additional tumor samples that included a plexiform fibromyxoma were sequenced using a targeted RNAseq method to detect fusion events. The study patients included two women and one man who were 52, 80, and 22 years of age at the time of diagnosis. The tumors involved the submucosa and muscularis propria of the duodenum, jejunum, and ileum. All 3 tumors contained a proliferation of monotonous oval or spindle cells with scattered, somewhat dilated vessels. Two cases showed epithelioid structures such as glands, tubules, or nests. Immunohistochemical analysis revealed cytokeratin expression in the epithelioid components of both tumors displaying these features, and variable numbers of mesenchymal cells. Diffuse CD56 positivity was seen in the mesenchymal component of 2 tumors and desmin and smooth muscle actin staining in the other tumor. Immunostains for S-100 protein, DOG-1, and CD117 were negative in all cases. GLI1 fusions with different partner genes were detected in all tumors, and in the plexiform fibromyxoma, used as a control. Validation by fluorescence in situ hybridization was performed. None of the tumors have recurred or metastasize after surgery. We describe novel GLI1 fusions in 3 mesenchymal neoplasms of the small intestine, including 2 with biphenotypic features. Thus far, all cases have pursued indolent clinical courses. We propose the term " GLI1 -rearranged enteric tumor" to encompass this group of unique neoplasms of the small intestine that harbor GLI1 gene fusions and expand the spectrum of gastrointestinal neoplasms with these alterations.

Features of fibrosis regression abound in "non-cirrhotic" patients with resected hepatocellular carcinoma.

Cirrhosis is a major risk factor for developing hepatocellular carcinoma (HCC). However, many surgically resected HCCs are presumably non-cirrhotic. The dynamic nature of chronic liver disease leads to periods of hepatic repair and fibrosis regression. We hypothesize that most resected HCCs, including those from non-cirrhotic patients, exhibit features of fibrosis regression in their background liver, suggesting previously more advanced liver disease. We reviewed the histology of 37 HCC resections performed between 2005-2020, including 30 from non-cirrhotic patients. The non-neoplastic liver was evaluated for features of liver disease and of the hepatic repair complex (HRC). CD34 immunohistochemistry was performed as a marker of sinusoidal capillarization. CD34 staining was evaluated manually and also by a digital image classifier algorithm. Overall, 28 cases (76%) had a high number of fibrosis regression and hepatic repair features (≥4 out of 8 features). Amongst the 30 non-cirrhotic patients, 21 (70%) showed a high number of repair features. Relative CD34 expression was increased in cases with a high number (≥4) of HRC features versus a low number (≤3) of features (p = 0.019). High HRC cases were more likely to exhibit nodular circumferential CD34 staining (p = 0.019). Our findings suggest that most resected HCC from non-cirrhotic patients display features of fibrosis regression in their background liver. Thus many, if not most, HCC patients who are "non-cirrhotic" may in fact have regressed cirrhosis. This finding reinforces that patients with regressed cirrhosis continue to be at high risk for HCC.

High interobserver variability and frequent overdiagnosis of dysplasia in fundic gland polyps can be improved by detecting atypia on the surface epithelium and an abrupt transition to non-neoplastic cells.

AIMS: Fundic gland polyps (FGPs) arise sporadically and in combination with familial adenomatous polyposis (FAP). Criteria for distinguishing low-grade dysplasia (LGD) from regenerative atypia in FGPs are not well established. The aims of study were to determine: (i) interobserver variability in diagnosing LGD in FGPs; (ii) bias in diagnosing LGD in FAP patients; and (iii) stringent criteria for LGD in FGPs. METHODS AND RESULTS: Five senior pathologists who were blinded to the clinical history reviewed 72 FAP-associated FGPs and 34 sporadic FGPs. Cases were classified as negative (score = 0) or positive (score = 1) for LGD. Each case was assigned a 'combined dysplasia score' (CDS) ranging from 0 to 5 to reflect all five opinions. Fleiss' kappa showed only moderate interobserver agreement (κ = 0.46). Forty-one FGPs were classified as negative for dysplasia by consensus (CDS = 0-1), including 10 (24%) originally diagnosed as LGD. In contrast, all 37 cases classified as LGD by consensus (CDS = 4-5) were originally diagnosed as LGD, indicating that overdiagnosis of dysplasia is more common than underdiagnosis (P = 0.0012). Cytological atypia in the surface epithelium and an abrupt transition between atypical and normal-appearing epithelium were the most sensitive (97% and 100%, respectively) and specific (100% and 98%, respectively) features of dysplasia (P < 0.0001 for both comparisons). Very good agreement was achieved when a diagnosis of dysplasia was based on the presence of both features (κ = 0.85). CONCLUSIONS: There is high interobserver variability and a tendency to overdiagnose LGD in FGPs. Strict criteria requiring both surface atypia and abrupt transition for LGD in FGPs result in low interobserver variability.

Controversies in appendiceal pathology: mucinous and goblet cell neoplasms.

Appendiceal goblet cell adenocarcinomas and mucinous neoplasms are uncommon compared with other epithelial tumours of the gastrointestinal tract. Both tumour types have been subjected to terminology that belies their biological risk and leads to confusion. Goblet cell adenocarcinomas display patchy staining for endocrine markers and, thus, were previously classified as goblet cell carcinoids. Unlike well-differentiated endocrine neoplasms, however, they often contain high-grade glandular elements and pursue an aggressive course akin to that of conventional adenocarcinoma. Although several authors have recently proposed grading schemes to predict behaviour among goblet cell neoplasms, most that contain high-grade components have already spread beyond the appendix at the time of diagnosis, whereas those confined to the appendix almost always have low-grade features; the added value of grading these tumours is limited. Contradictions also surround the nomenclature of mucinous neoplasms. The World Health Organization and others promote non-malignant terminology to describe metastatic mucinous neoplasms of the peritoneum and eliminate a benign category entirely, even though virtually all neoplasms confined to the appendix pose no recurrence risk following appendectomy. 'Low-grade appendiceal mucinous neoplasm' now encompasses a spectrum of benign tumours and malignant neoplasms in the appendix and peritoneum. Although using an umbrella term in this fashion simplifies the roles of pathologists, it provides essentially no actionable information beyond that which is already clinically apparent. Broad strokes nomenclature also ensures that many patients with no risk of recurrence will receive unnecessary surveillance while others will undergo inappropriate surgical procedures due to lapses in communication. Moreover, a surprising number of non-neoplastic mucinous lesions are misclassified as low-grade appendiceal mucinous neoplasms, which can result in unwarranted patient concern or even mismanagement. The purpose of this review is to critically evaluate the literature and describe an approach to appendiceal neoplasms that more clearly denotes their biologic risk.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Filaggrin expression via immunohistochemistry in basal cell carcinoma and squamous cell carcinoma.

BACKGROUND: Filaggrin is a protein integral to the structure and function of the epidermis. Filaggrin (FLG) loss-of-function (LOF) mutations are common and increase the risk of developing atopic dermatitis (AD) and ichthyosis vulgaris (IV). Epidemiologic data suggest a link between skin cancer and AD. We examined if FLG staining pattern can be used to characterize cutaneous squamous cell carcinomas (SCC), basal cell carcinomas (BCC), and reactive squamous epithelium. METHODS: Tissue microarrays (TMAs) were created from 196 cases of formalin-fixed paraffin-embedded (FFPE) SCC and 144 BCC cases. TMAs and sections of reactive squamous epithelium were stained with optimized anti-FLG antibody and evaluated for FLG expression (normal, abnormal, or negative). RESULTS: FLG was absent in poorly differentiated (PD) compared to well-differentiated (WD) SCC (P < .0001) and moderately-differentiated (MD) (P = .0231) SCC, and in MD compared to WD SCC (P = .0099). Abnormal staining was significantly increased in PD compared to WD cases (P = .0039) and in MD compared to WD cases (P = .0006). Most BCC did not exhibit FLG expression (P < .05). Reactive squamous epithelium demonstrated normal, but exaggerated FLG expression. CONCLUSIONS: Our findings demonstrate the differences in FLG expression patterns in types of keratinocyte carcinomas and their mimickers.

Venetoclax Increases Intratumoral Effector T Cells and Antitumor Efficacy in Combination with Immune Checkpoint Blockade.

The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti-PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. SIGNIFICANCE: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency-approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1.

Prevalence of histological features resembling autoimmune pancreatitis in neoplastic pancreas resections.

AIMS: Types 1 and 2 autoimmune pancreatitis (AIP) can mimic pancreatic neoplasia. Due to the small quantity of tissue in mass-targeted pancreas biopsies, inflammatory features may raise the differential of AIP. However, the frequency of AIP-like histology in neoplastic pancreas is not well characterised. Therefore, the specificity of inflammatory lesions on biopsy with respect to the diagnosis of AIP is uncertain. METHODS AND RESULTS: Neoplastic pancreas resections performed at our institution between 2008 and 2019 were retrospectively reviewed. Features of AIP types 1 and 2 were assessed in the non-neoplastic areas. If features of immunoglobulin (Ig)G4-associated AIP were seen, IgG4 immunohistochemistry was performed. We identified 163 neoplastic pancreas resections. Of these, 34 had one or more types of inflammatory lesions in non-neoplastic pancreatic tissue. Dense lymphoplasmacytic inflammation mimicking type 1 AIP was found in six cases with mild to moderately increased IgG4-positive plasma cells. Neutrophilic infiltrates in small intralobular ducts were found in 20 cases. Mild extralobular ductitis or duct microabscess was found in 10 specimens. Marked neutrophilic duct destruction that resembled granulocytic epithelial lesions was found in 12 cases. Some cases showed multiple features. CONCLUSION: Approximately 20% of neoplastic pancreas resections showed focal areas that could raise the differential of AIP. More cases showed neutrophilic predominant inflammation as seen in type 2 autoimmune pancreatitis, compared to dense lymphoplasmacytic infiltrates seen in type 1 AIP. Pathologists must be cautious when making a diagnosis of AIP on biopsy tissue based on histological findings alone.

Coaching by design: exploring a new approach to faculty development in a competency-based medical education curriculum.

As curricula move from a time-based system to a competency-based medical education system, faculty development will be required. Faculty will be asked to engage in the observation, assessment and feedback of tasks in the form of educational coaching. Faculty development in coaching is necessary, as the processes and tools for coaching learners toward competence are evolving with a novel assessment system. Here, we provide a scoping review of coaching in medical education. Techniques and content that could be included in the curricular design of faculty development programming for coaching (faculty as coach) are discussed based on current educational theory. A novel model of coaching for faculty (faculty as coachee) has been developed and is described by the authors. Its use is proposed for continuing professional development.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.

Correction: Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

Erratum: Clinical responses to ERK inhibition in BRAFV600E-mutant colorectal cancer predicted using a computational model.

[This corrects the article DOI: 10.1038/s41540-017-0016-1.].