Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Ultraviolet radiation shapes dendritic cell leukaemia transformation in the skin.

Tumours most often arise from progression of precursor clones within a single anatomical niche. In the bone marrow, clonal progenitors can undergo malignant transformation to acute leukaemia, or differentiate into immune cells that contribute to disease pathology in peripheral tissues1-4. Outside the marrow, these clones are potentially exposed to a variety of tissue-specific mutational processes, although the consequences of this are unclear. Here we investigate the development of blastic plasmacytoid dendritic cell neoplasm (BPDCN)-an unusual form of acute leukaemia that often presents with malignant cells isolated to the skin5. Using tumour phylogenomics and single-cell transcriptomics with genotyping, we find that BPDCN arises from clonal (premalignant) haematopoietic precursors in the bone marrow. We observe that BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet (UV) radiation. A reconstruction of tumour phylogenies reveals that UV damage can precede the acquisition of alterations associated with malignant transformation, implicating sun exposure of plasmacytoid dendritic cells or committed precursors during BPDCN pathogenesis. Functionally, we find that loss-of-function mutations in Tet2, the most common premalignant alteration in BPDCN, confer resistance to UV-induced cell death in plasmacytoid, but not conventional, dendritic cells, suggesting a context-dependent tumour-suppressive role for TET2. These findings demonstrate how tissue-specific environmental exposures at distant anatomical sites can shape the evolution of premalignant clones to disseminated cancer.

Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs.

Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.

Expanding the spectrum of tyrosine kinase fusions in calcified chondroid mesenchymal neoplasms: Identification of a novel PDGFRA::USP8 gene fusion.

Calcified chondroid mesenchymal neoplasms represent a distinct, and recently recognized, spectrum of tumors. To date most cases have been reported to be characterized by FN1 gene fusions involving multiple potential tyrosine kinase partners. Following incidental identification of a tumor morphologically corresponding to calcified chondroid mesenchymal neoplasm, but with a PDGFRA::USP8 gene fusion, we undertook a retrospective review to identify and characterize additional such cases. A total of four tumors were identified. Each was multilobulated and composed of polygonal-epithelioid-stellate cells with a background of chondroid matrix containing distinctive patterns of calcification. Targeted RNA sequencing revealed an identical PDGFRA (exon 22)::USP8 (exon 5) gene fusion in each case. Subsequent immunohistochemical staining confirmed the presence of PDGFRα overexpression. In summary, we report a series of four tumors within the morphologic spectrum of calcified chondroid mesenchymal neoplasms. In contrast to prior reports, these tumors harbored a novel PDGFRA::USP8 gene fusion, rather than FN1 rearrangement. Our findings expand the molecular diversity of these neoplasms, and suggest they are united through activation of protein kinases.

Twice-Daily Proton Pump Inhibitor Induces Higher Remission Rate in Eosinophilic Esophagitis Than Once-Daily Regimen Regardless of Total Daily Dose.

INTRODUCTION: The optimal proton pump inhibitor (PPI) regimen for eosinophilic esophagitis (EoE) is unclear. We compared histologic response rates of different dosing combinations. METHODS: A total of 305 patients with newly diagnosed EoE received standard (omeprazole 20 mg daily), once-daily moderate (40 mg daily), twice-daily moderate (20 mg twice daily), or high (40 mg twice daily) dose PPI for ≥8 weeks. RESULTS: Approximately 42.3% achieved histologic response to PPI, with higher rates for twice-daily (moderate 52.8%/high 54.3%) than once-daily (standard 11.8%/moderate 10%) dosing ( P < 0.0001). On multivariable analysis, twice-daily moderate (adjusted odds ratio 6.75, confidence interval 2.53-18.0, P = 0.0008) and high (adjusted odds ratio 12.8, confidence interval 4.69-34.8, P < 0.0001) doses independently predicted histologic response. DISCUSSION: Twice-daily PPI is associated with higher EoE histologic response rates than once-daily regimen.

CYP1A1 immunohistochemistry is highly specific for angiofibroma of soft tissue among morphological mimics.

AIMS: Angiofibroma of soft tissue (AFST) is a benign, morphologically distinctive tumour type that harbours recurrent AHRR::NCOA2 fusions in 60-70% of cases and shows a non-specific immunophenotype, expressing EMA in roughly half of cases. The AHRR::NCOA2 fusion results in increased expression of cytochrome P450 1A1 (CYP1A1); a recent study demonstrated CYP1A1 immunohistochemistry (IHC) to be moderately sensitive and highly specific for AFST. METHODS AND RESULTS: In this study, we sought to validate these findings in a larger independent cohort of 30 AFST, as well as 215 morphological mimics, including 30 solitary fibrous tumours, 29 myxoid liposarcomas, 28 low-to-intermediate grade myxofibrosarcomas (MFS), 20 atypical spindle cell lipomatous tumours (ASCLT), 20 cellular angiofibromas, 10 cases each of spindle cell lipoma, neurofibroma, malignant peripheral nerve sheath tumour, superficial angiomyxoma, cellular myxoma, soft tissue perineurioma and deep fibrous histiocytoma, and nine cases each of low-grade fibromyxoid sarcoma and mammary-type myofibroblastoma. We found CYP1A1 IHC to be 70% sensitive for AFST, with granular cytoplasmic staining in 21 of 30 tumours, and 98% specific, with staining in only five morphological mimics: two deep fibrous histiocytomas, one MFS, one cellular angiofibroma and one ASCLT. CONCLUSIONS: These findings confirm that CYP1A1 is 70% sensitive, consistent with the prevalence of AHRR::NCOA2 fusions that up-regulate this protein, and that it is highly specific among morphological mimics.

What Is the Value of Counting Mast Cells in Gastrointestinal Mucosal Biopsies?

Neoplastic and nonneoplastic mast cell disorders can cause diarrhea, nausea, and abdominal pain that result from heightened release of mast cell mediators. Systemic mastocytosis is characterized by neoplastic mast cell aggregates in the bone marrow and other sites, particularly the skin and gastrointestinal tract. In this situation, extramedullary mast cell aggregates display atypical morphology, with aberrant immunostaining for CD25 in addition to staining for other mast cell markers, such as mast cell tryptase and CD117. Morphologically normal mast cells have also been implicated in nonneoplastic conditions. For example, increased mast cell numbers have been reported in the mucosal biopsy samples from patients with irritable bowel syndrome and hereditary alpha-tryptasemia. Patients with mast cell activation syndrome presumably experience symptoms related to the aberrant elaboration of histamine and other mediators from normal-appearing mast cells present in normal numbers. Unfortunately, similarities in terminology among these biologically distinct clinical conditions have caused considerable diagnostic confusion among clinical colleagues, resulting in frequent requests for pathologists to quantify and characterize mast cells in normal gastrointestinal biopsy samples from patients with diarrheal symptoms. The purpose of this review is to summarize the available data related to mast cell assessment in the gastrointestinal tract and provide pathologists with practical information so that they can help their clinical colleagues manage patients with presumed mast cell disorders.

Anaplastic Kaposi Sarcoma: A Clinicopathologic and Molecular Genetic Analysis.

Kaposi sarcoma (KS) is a human herpesvirus 8 (HHV8)-associated vascular proliferation that most often involves the skin. Rarely, KS shows marked nuclear atypia or pleomorphism; such examples are known as "anaplastic" KS. This poorly characterized variant often pursues an aggressive course; little is known of its genetic landscape. This study evaluated the clinicopathologic and genomic features of anaplastic KS. We identified 9 anaplastic KS cases from 7 patients and 8 conventional KS cases, including a matched conventional KS and primary metastasis anaplastic KS pair from a single patient (anaplastic KS diagnosed 9 years after conventional KS). All patients with anaplastic KS were men, aged 51 to 82 years, who had locally aggressive tumors predominantly affecting the soft tissue and bone of the lower extremities (5/7 patients). Four patients were known to be HIV positive (all on antiretrovirals), 2 were HIV negative, and 1 was of unknown HIV status. The tumors showed angiosarcoma-like or pleomorphic spindle cell sarcoma morphology. Plasma cell-rich chronic inflammation and hemosiderin deposition were commonly present. Single-nucleotide polymorphism-based chromosomal microarray analysis showed the anaplastic KS cohort to demonstrate highly recurrent whole chromosome (chr) gains of chr 7, 11, 19, and 21, which primarily affected olfactory and G protein-coupled receptor signaling and losses of chr6_q and chrY. Compared with conventional KS, anaplastic KS cases showed significantly more total copy number alterations and more frequent gains of chr7 and chr11_q13.1 (MARK2, RELA, and ESRRA, including high copy number gain in 1 case). Pathway analysis demonstrated that these gains preferentially affected genes that facilitate cyclin-dependent cell signaling. Furthermore, anaplastic KS cases were phylogenetically distinct from conventional KS cases, including the patient-matched primary metastasis anaplastic KS pair and conventional KS. Our study is the first to demonstrate that a more complex genome and distinct copy number alterations distinguish anaplastic KS from conventional KS. Gains of chr7 and chr11_q13.1 appear central to biological transformation.

Expanding the Molecular Diversity of CIC-Rearranged Sarcomas With Novel and Very Rare Partners.

Capicua transcriptional repressor (CIC)-rearranged sarcoma represents a distinct pathologic entity and constitutes the second most prevalent category of undifferentiated round cell sarcomas (URCSs) after Ewing sarcoma. The 2 most common translocations are t(4;19) and t(10;19), resulting in CIC fusions with either DUX4 and DUX4L paralog, respectively; however, other rare variant fusions have also been reported. In this study, we expand the molecular spectrum of CIC-gene partners, reporting on 5 cases of URCSs showing CIC fusions with AXL, CITED1, SYK, and LEUTX by targeted RNA or DNA sequencing. There were 4 female patients and 1 male patient with a wide age range (12-70 years; median, 36 years). Four cases occurred in the deep soft tissues (lower extremity, 3; neck, 1) and 1 case in the central nervous system (midbrain/thalamus). All cases showed similar histologic findings within the spectrum of URCSs. Immunohistochemistry, showed variable positivity for ETV4 in 4 of the 4 cases and positive results for ERG in 3 of the 4 cases and for WT1 in 1 of the 4 cases. CD31 showed positivity in 2 of the 3 cases, including one coexpressing ERG. Unsupervised clustering of methylation profiles by T-distributed stochastic neighborhood embedding performed in 4 cases showed that all clustered tightly together and along the CIC sarcoma methylation class. RNA-sequencing data showed consistent upregulation of ETV1 and ETV4 mRNA in all cases examined, at similar levels to CIC::DUX4 URCSs. Our study expands the molecular diversity of CIC-rearranged URCSs to include novel and rare partners, providing morphologic, immunohistochemical, gene expression, and methylation evidence supporting their classification within the family of tumors harboring the more common DUX4/DUX4L partner genes.

Expanding the Clinicopathologic and Molecular Spectrum of Lipoblastoma-Like Tumor in a Series of 28 Cases.

Lipoblastoma-like tumor (LLT) is a rare adipocytic neoplasm with a predilection for the vulva. Since 2002, <30 cases have been reported, characterizing it as an indolent tumor that may sometimes recur locally. Diagnosis can be challenging due to its rarity and morphologic overlap with other adipocytic tumors. Thus far, there are no specific molecular or immunohistochemical features to aid in the diagnosis of LLT. Recent case reports have described LLT arising at other sites, including the spermatic cord and gluteal region, suggesting wider anatomical distribution. We present a large series of LLT to further characterize its clinicopathologic and molecular features. Twenty-eight cases of LLT were retrieved from departmental and consult archives (including 8 from a prior series). The cohort comprised 28 patients (8 males, 20 females) with a median age of 28 years (range: 1-80 years). There were 17 primary LLT of the vulva. Other anatomical sites included the scrotum (n = 3), spermatic cord (n = 2), inguinal region (n = 2), limbs (n = 2), pelvis (n = 1), and retroperitoneum (n = 1). Median tumor size was 6.0 cm (range: 1.8-30.0 cm). The tumors had a lobulated architecture and were typically composed of adipocytes, lipoblasts, and spindle cells in a myxoid stroma with prominent thin-walled vessels. Using immunohistochemistry, a subset showed loss of Rb expression (12/23 of samples). Follow-up in 15 patients (median: 56 months) revealed 8 patients with local recurrence and 1 patient with metastases to the lung/pleura and breasts. Targeted DNA sequencing revealed a simple genomic profile with limited copy number alterations and low mutational burden. No alterations in RB1 were identified. The metastatic LLT showed concurrent pathogenic PIK3CA and MTOR activating mutations, both in the primary and in the lung/pleural metastasis; the latter also harbored TERT promoter mutation. One tumor had a pathogenic TSC1 mutation, and one tumor showed 2-copy deletion of CDKN2A, CDKN2B, and MTAP. No biologically significant variants were identified in 8 tumors. No gene fusions were identified by RNA sequencing in 4 tumors successfully sequenced. This study expands the clinicopathologic spectrum of LLT, highlighting its wider anatomical distribution and potential for occasional metastasis. Molecularly, we identified activating mutations in the PI3K-MTOR signaling pathway in 2 tumors, which may contribute to exceptional aggressive behavior.

NR4A3 Expression Is Consistently Absent in Acinic Cell Carcinomas of the Breast: A Potential Nosologic Shift.

Acinic cell carcinoma (AciCC) is a tumor that is recognized in both the breast and salivary glands. Recently, the recurrent genomic rearrangement, t(4;9)(q13;q31) was identified in salivary AciCC that results in constitutive upregulation of the nuclear transcription factor NR4A3, which can be detected by immunohistochemistry. In this study, we sought to evaluate NR4A3 expression in breast AciCC using immunohistochemistry. Strong and diffuse nuclear staining was considered a positive result. Sixteen AciCCs were studied, including 8 pure AciCCs and 8 AciCCs admixed with other types (invasive carcinoma of no special type in 5 cases and metaplastic carcinoma in 3 cases). All 16 AciCCs showed negative results for NR4A3 expression. Four cases with available material were evaluated for rearrangements of the NR4A3 gene by fluorescence in situ hybridization and no rearrangements were observed. Whole-genome sequencing of 1 AciCC revealed a TP53 splice-site mutation, high levels of genomic instability, and genomic features of homologous recombination DNA repair defects; a structural variant analysis of this case did not reveal the presence of a t(4;9) rearrangement. We conclude that breast AciCCs consistently lack NR4A3 rearrangement or overexpression, unlike most of the salivary AciCCs, and that consistent with previous results, breast AciCCs are associated with genomic alterations more similar to those seen in triple-negative breast carcinomas than salivary gland AciCCs. These results suggest that unlike other salivary gland-like tumors that occur in the breast, the molecular underpinnings of the salivary gland and breast AciCCs are different and that the salivary gland and breast AciCCs likely represent distinct entities.

Extraenteric Malignant Gastrointestinal Neuroectodermal Tumor-A Clinicopathologic and Molecular Genetic Study of 11 Cases.

Malignant gastrointestinal neuroectodermal tumors (MGNETs), also known as "gastrointestinal clear cell sarcoma-like tumors", are very rare, aggressive sarcomas characterized by enteric location, distinctive pathologic features, and EWSR1/FUS::ATF1/CREB1 fusions. Despite identical genetics, the clinicopathologic features of MGNET are otherwise quite different from those of clear cell sarcoma of soft parts. Only exceptional extraenteric MGNET (E-MGNET) has been reported. We report a series of 11 E-MGNETs, the largest to date. Cases diagnosed with MGNET and occurring in nonintestinal locations were retrieved. A clinical follow-up was obtained. The tumors occurred in 3 men and 8 women (range, 14-70 years of age; median, 33 years) and involved the soft tissues of the neck (3), shoulder (1), buttock (2), orbit (1), tongue/parapharyngeal space (1), urinary bladder (1), and falciform ligament/liver (1). Tumors showed morphologic features of enteric MGNET (small, relatively uniform, round to ovoid cells with round, regular nuclei containing small nucleoli growing in multinodular and vaguely lobular patterns, with solid, pseudoalveolar, and pseudopapillary architecture). Immunohistochemical results were S100 protein (11/11), SOX10 (11/11), synaptophysin (3/10), CD56 (7/9), CD117 (3/9), DOG1 (0/4), ALK (4/8), chromogranin A (0/10), HMB-45 (0/11), Melan-A (0/11), tyrosinase (0/4), and MiTF (0/11). Next-generation sequencing results were EWSR1::ATF1 (7 cases), EWSR1::CREB1 (3 cases), and EWSR1::PBX1 (1 case). The EWSR1::PBX1-positive tumor was similar to other cases, including osteoclast-like giant cells, and negative for myoepithelial markers. A clinical follow-up (range, 10-70 months; median, 34 months) showed 4 patients dead of disease (10.5, 12, 25, and 64 months after diagnosis), 1 patient alive with extensive metastases (43 months after diagnosis), 1 patient alive with persistent local disease (11 months after diagnosis), and 4 alive without disease (10, 47, 53, and 70 months after diagnosis). One case is too recent for the follow-up. The clinicopathologic and molecular genetic features of rare E-MGNET are essentially identical to those occurring in intestinal locations. Otherwise, typical E-MGNET may harbor EWSR1::PBX1, a finding previously unreported in this tumor type. As in enteric locations, the behavior of E-MGNET is aggressive, with metastases and/or death from disease in at least 50% of patients. E-MGNET should be distinguished from clear cell sarcoma of soft parts and other tumors with similar fusions. ALK expression appears to be a common feature of tumors harboring EWSR1/FUS::ATF1/CREB1 fusion but is unlikely to predict the therapeutic response to ALK inhibition. Future advances in our understanding of these unusual tumors will hopefully lead to improved nomenclature.

Prognostic Significance of Percentage and Size of Dedifferentiation in Dedifferentiated Chondrosarcoma.

Dedifferentiated chondrosarcoma is rare, aggressive, and microscopically bimorphic. How pathologic features such as the amounts of dedifferentiation affect prognosis remains unclear. We evaluated the percentages and sizes of dedifferentiation in a consecutive institutional series of dedifferentiated chondrosarcomas from 1999 to 2021. The statistical analysis included cox proportional hazard models and log-rank tests. Of the 67 patients (26 women, 41 men; age, 39 to >89 [median 61] years; 2 with Ollier disease), 58 presented de novo; 9 were identified with conventional chondrosarcomas 0.6-13.2 years (median, 5.5 years) prior. Pathologic fracture and distant metastases were noted in 27 and 7 patients at presentation. The tumors involved the femur (n = 27), pelvis (n = 22), humerus (n = 7), tibia (n = 4), scapula/ribs (n = 4), spine (n = 2), and clivus (n = 1). In the 56 resections, the tumors ranged in size from 3.5 to 46.0 cm (median, 11.5 cm) and contained 1%-99.5% (median, 70%) dedifferentiated components that ranged in size from 0.6 to 24.0 cm (median, 7.3 cm). No correlation was noted between total size and percentage of dedifferentiation. The dedifferentiated components were typically fibrosarcomatous or osteosarcomatous, whereas the associated cartilaginous components were predominantly grade 1-2, rarely enchondromas or grade 3. The entire cohort's median overall survival and progression-free survival were 11.8 and 5.4 months, respectively. In the resected cohort, although the total size was not prognostic, the percentage of dedifferentiation ≥20% and size of dedifferentiation >3.0 cm each predicted worse overall survival (9.9 vs 72.5 months; HR, 3.76; 95% CI, 1.27-11.14; P = .02; 8.7 vs 58.9 months; HR, 3.03; 95% CI, 1.21-7.57; P = .02, respectively) and progression-free survival (5.3 vs 62.1 months; HR, 3.05; 95% CI, 1.13-8.28; P = .03; 5.3 vs 56.6 months; HR, 2.50; 95% CI, 1.06-5.88; P = .04, respectively). In conclusion, both the percentages and sizes of dedifferentiation were better prognostic predictors than total tumor sizes in dedifferentiated chondrosarcomas, highlighting the utility of their pathologic evaluations.

Distribution and Rate of Myxoid Liposarcoma Spine Metastases: Impact on Surveillance Imaging.

BACKGROUND: Myxoid liposarcoma (LPS) has a unique tendency to spread to extrapulmonary sites, including osseous sites such as the spine, and adjacent sites such as the paraspinous tissue. No clear consensus exists to guide the approach to imaging in these patients. OBJECTIVE: The aim of this study was to investigate the rate and distribution of spine metastases in patients with myxoid LPS and detection modality. METHODS: Records of all patients with myxoid LPS evaluated at our sarcoma center were retrospectively reviewed. Disease patterns and imaging modality utilization were analyzed. RESULTS: Between 2000 and 2020, 164 patients with myxoid LPS were identified. The majority (n = 148, 90%) presented with localized disease, with half (n = 82, 50%) of all patients developing metastases or recurrence during their disease course. With a median follow-up of 69.2 months, spine/paraspinous metastases developed in 38 patients (23%), of whom 35 (92%) already had synchronous, non-spine metastases. Spine disease was only visible on magnetic resonance imaging (MRI), as opposed to other imaging modalities, for over one-quarter of patients with spine metastases (n = 10). For patients with metastatic disease, spine metastases were associated with worse median overall survival (2.1 vs. 8.7 years, p < 0.001). CONCLUSION: Spine metastases occurred in nearly one-quarter of patients with myxoid LPS and represented an advanced disease state, as they primarily presented in the setting of synchronous, non-spine metastases, and were associated with worse overall survival. Routine surveillance with spine MRI in patients with localized disease likely provides no benefit but may be considered in those with known metastatic disease.

Loss of function SMAD4 nonstop mutations in human cancer.

BACKGROUND: SMAD4 is a tumour suppressor gene that is mutated in a variety of cancers. SMAD4 nonstop mutations, which convert stop codons to sense codons that extend transcription, have been identified in genomic databases but have not been characterised in human pathology samples. The frequency of SMAD4 nonstop mutations and the consequences of nonstop mutations on SMAD4 protein expression are unknown. METHODS: We retrospectively analysed our cancer sequencing database of 38,002 tumour specimens and evaluated the spectrum of SMAD4 mutations. SMAD4 protein expression was evaluated by immunohistochemistry in tumours with SMAD4 nonstop mutations. RESULTS: In total, 1956 SMAD4 mutations were identified in 1822 tumours. SMAD4 mutations were most common in tumours of the gastrointestinal tract and included nonsense variants (n = 344), frameshift indels (n = 258), splice site variants (n = 104), and missense variants at codon R361 (n = 245). In a subset of cases with immunohistochemistry, SMAD4 expression was lost in 23 of 25 tumours (92%) with protein truncating variants and in 7 of 27 tumours (26%) with missense variants. Four cases harboured SMAD4 nonstop mutations. SMAD4 nonstop mutations were identified in two pancreatic adenocarcinomas, one colonic adenocarcinoma, and one non-small cell lung carcinoma. Immunohistochemistry demonstrated loss of SMAD4 protein expression in each of the four tumours with SMAD4 nonstop mutations. CONCLUSION: SMAD4 nonstop mutations are associated with loss of SMAD4 protein expression in multiple tumour types. SMAD4 nonstop mutations should be clinically interpreted as pathogenic loss of function alterations when identified in cancer sequencing panels.

Diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification.

AIMS: Rhabdomyosarcomas currently are classified into one of four subtypes (alveolar, embryonal, spindle cell/sclerosing, or pleomorphic) according to their morphological, immunohistochemical, and molecular genetic features. The alveolar subtype is characterised by a recurrent translocation involving PAX3 or PAX7 and FOXO1; identification of this translocation is important for appropriate classification and prognostication. In this study, we aimed to explore the diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification. METHODS/RESULTS: A monoclonal antibody targeting a FOXO1 epitope retained in the fusion oncoprotein was used to study 105 rhabdomyosarcomas. FOXO1 was positive for expression by immunohistochemistry in all 25 alveolar rhabdomyosarcomas, with 84% showing diffuse expression in greater than 90% of neoplastic cells; the remainder of alveolar rhabdomyosarcomas displayed at least moderate staining in a minimum of 60% of lesional cells. Apart from three spindle cell rhabdomyosarcomas showing heterogeneous nuclear immunoreactivity in 40-80% of tumour cells, the 80 cases of embryonal, pleomorphic, and spindle cell/sclerosing rhabdomyosarcoma were negative for FOXO1 expression (96.3% specific) when using a threshold of nuclear staining in 20% of neoplastic cells to determine positivity. Variable cytoplasmic staining was present in a fraction of all rhabdomyosarcoma subtypes. Nonneoplastic lymphocytes, endothelial cells, and Schwann cells also showed variably intense nuclear anti-FOXO1 immunoreactivity. CONCLUSION: Taken together, our findings suggest that FOXO1 immunohistochemistry is a highly sensitive and relatively specific surrogate marker of the PAX3/7::FOXO1 fusion oncoprotein in rhabdomyosarcoma. Cytoplasmic immunoreactivity, expression in nonneoplastic tissues, and limited nuclear staining of nonalveolar rhabdomyosarcomas represent potential pitfalls in interpretation.

Dataset for reporting of gastrointestinal stromal tumours: recommendations from the International Collaboration on Cancer Reporting (ICCR).

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are among the most frequent sarcomas. Accurate diagnosis, classification, and reporting are critical for prognostication and patient management, including selection of appropriate targeted therapy. Here we report on international consensus-based datasets for the pathology reporting of biopsy and resection specimens of GIST. The datasets were produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of major international pathology and cancer organizations. An international expert panel consisting of pathologists, a surgical oncologist, and a medical oncologist produced a set of core and noncore data items for biopsy and resection specimens based on a critical review and discussion of current evidence. All professionals involved were subspecialized soft tissue tumour experts and affiliated with tertiary referral centres. Commentary was provided for each data item to explain its clinical relevance and the rationale for selection as a core or noncore element. Following international public consultation, the datasets, which include synoptic reporting guides, were finalized and ratified, and published on the ICCR website. These first international datasets for GIST are intended to promote high-quality, standardised pathology reporting. Their widespread adoption will improve consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will ultimately help to improve the management of patients with GIST. All the ICCR datasets, including these on GIST, are freely available worldwide on the ICCR website (www.iccr-cancer.org/datasets).

Assessment of the clinical and allergy profiles of PPI responsive and non-responsive eosinophilic esophagitis.

A subset of patients with eosinophilic esophagitis (EoE) respond to proton-pump inhibitor (PPI) therapy, however they cannot be distinguished prior to PPI trial and the mechanism of PPI response remains unclear. Improved understanding of the distinct patient phenotypes in PPI-responsive EoE (PPI-r-EoE), PPI-non-responsive EoE (PPI-nr-EoE) and erosive esophagitis (EE) may help guide management. The aim of this paper is to compare the clinical and allergy profiles of PPI-r-EoE versus PPI-nr-EoE and EE. This was a retrospective case-control study of EoE patients (>15 eos/hpf on esophageal biopsies) at a tertiary center. EE controls were identified from the pathology database. EoE patients were classified as PPI-r-EoE or PPI-nr-EoE based on histologic response to twice-daily PPI for ≥8 weeks. Patient demographics, comorbidities, symptoms, allergy history and endoscopic findings were recorded. Univariate analyses were performed using the Fisher-exact test or t-test. Multivariable analyses were performed using logistic regression. In all, 104 EoE (57 PPI-r-EoE/47 PPI-nr-EoE) and 80 EE subjects were included. On multivariable analyses, allergic conditions (aOR 20.1, P < 0.0001) and rings (aOR 108.3, P = 0.001) were independent predictors for PPI-r-EoE versus EE, whereas allergic conditions (aOR 4.8, P = 0.03), rings (aOR 27.5, P = 0.002) and furrows (aOR 17.1, P = 0.04) were independent predictors for PPI-nr-EoE versus EE. Esophageal rings was the only significant predictor found in PPI-nr-EoE versus PPI-r-EoE (OR 2.5, P = 0.03). Allergic conditions and esophageal rings are significantly more prevalent in PPI-r-EoE and PPI-nr-EoE compared with EE. PPI-r-EoE appears clinically similar to PPI-nr-EoE and significantly different from EE. Further studies are needed to delineate the underlying pathophysiology of PPI-r-EoE versus PPI-nr-EoE.

Concurrent inhibition of CDK2 adds to the anti-tumour activity of CDK4/6 inhibition in GIST.

BACKGROUND: Advanced gastrointestinal stromal tumour (GIST) is characterised by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximising response to therapeutic CDK4/6 inhibition. METHODS: Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples. The impact of inhibitors of CDK2, CDK4 and CDK2/4/6 was determined through cell proliferation and protein detection assays. CDK-inhibitor resistance mechanisms were characterised in GIST cell lines after long-term exposure. RESULTS: We identify recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation. Moreover, RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) are mechanisms of acquired CDK-inhibitor resistance in GIST. CONCLUSIONS: These studies establish the biological rationale for CDK2 and CDK4/6 co-inhibition as a therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors.