Artificial intelligence-based assessment of PD-L1 expression in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is an aggressive blood cancer known for its rapid progression and high incidence. The growing use of immunohistochemistry (IHC) has significantly contributed to the detailed cell characterization, thereby playing a crucial role in guiding treatment strategies for DLBCL. In this study, we developed an AI-based image analysis approach for assessing PD-L1 expression in DLBCL patients. PD-L1 expression represents as a major biomarker for screening patients who can benefit from targeted immunotherapy interventions. In particular, we performed large-scale cell annotations in IHC slides, encompassing over 5101 tissue regions and 146,439 live cells. Extensive experiments in primary and validation cohorts demonstrated the defined quantitative rule helped overcome the difficulty of identifying specific cell types. In assessing data obtained from fine needle biopsies, experiments revealed that there was a higher level of agreement in the quantitative results between Artificial Intelligence (AI) algorithms and pathologists, as well as among pathologists themselves, in comparison to the data obtained from surgical specimens. We highlight that the AI-enabled analytics enhance the objectivity and interpretability of PD-L1 quantification to improve the targeted immunotherapy development in DLBCL patients.

Anti-metabolic agent pegaspargase plus PD-1 antibody sintilimab for first-line treatment in advanced natural killer T cell lymphoma.

Natural killer T cell lymphoma (NKTCL) is highly aggressive, with advanced stage patients poorly responding to intensive chemotherapy. To explore effective and safe treatment for newly diagnosed advanced stage NKTCL, we conducted a phase II study of anti-metabolic agent pegaspargase plus PD-1 antibody sintilimab (NCT04096690). Twenty-two patients with a median age of 51 years (range, 24-74) were enrolled and treated with induction treatment of pegaspargase 2500 IU/m2 intramuscularly on day 1 and sintilimab 200 mg intravenously on day 2 for 6 cycles of 21 days, followed by maintenance treatment of sintilimab 200 mg for 28 cycles of 21 days. The complete response and overall response rate after induction treatment were 59% (95%CI, 43-79%) and 68% (95%CI, 47-84%), respectively. With a median follow-up of 30 months, the 2 year progression-free and overall survival rates were 68% (95%CI, 45-83%) and 86% (95%CI, 63-95%), respectively. The most frequently grade 3/4 adverse events were neutropenia (32%, n = 7) and hypofibrinogenemia (18%, n = 4), which were manageable and led to no discontinuation of treatment. Tumor proportion score of PD-L1, peripheral blood high-density lipoprotein cholesterol, and apolipoprotein A-I correlated with good response, while PD-1 on tumor infiltrating lymphocytes and peripheral Treg cells with poor response to pegaspargase plus sintilimab treatment. In conclusion, the chemo-free regimen pegaspargase plus sintilimab was effective and safe in newly diagnosed, advanced stage NKTCL. Dysregulated lipid profile and immunosuppressive signature contributed to treatment resistance, providing an alternative therapeutic approach dual targeting fatty acid metabolism and CTLA-4 in NKTCL.

Acquired cystic disease-associated renal cell carcinoma with PTCH1 mutation: a case report.

Acquired cystic disease-associated renal cell carcinoma (ACD-RCC) is an extremely rare kidney tumor seen mainly in patients with end-stage renal disease. Currently, there are few reports on this type of tumor. We describe the case of a 58-year-old man who had been receiving peritoneal dialysis for more than nine years due to chronic renal insufficiency and uremia. One year after undergoing left renal clear cell renal cell carcinoma resection, a space-occupying lesion was found in the right kidney for which he underwent right nephrectomy. The histopathology of this tumor showed solid or tubular cell arrangements, with some areas of cyst formation. Vacuoles of varying sizes were present in the cytoplasm, and varying amounts of calcium oxalate crystals were found in the tumor cells or interstitium. The pathological diagnosis was ACD-RCC. Next-generation sequencing detected mutations in the PTCH1, MTOR, FAT1, SOS1, RECQL4, and CDC73 genes in the right renal tumor. This is a rare case of a patient with ACD-RCC in the right kidney and clear cell renal cell carcinoma in the left kidney. The findings suggest that mutations in PTCH1 associated with ACD-RCC may have acted as oncogenic drivers for the development of ACKD-RCC, together with providing insight into mechanisms underlying ACD-RCC development, as well as diagnostic and treatment options.

Fumarate Hydratase-Deficient Renal Cell Carcinoma With Predominant Tubulocystic Features Mimics Tubulocystic Renal Cell Carcinoma.

CONTEXT.­: Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) rarely exhibits a predominant tubulocystic architecture with few other components. RCC with pure tubules and cysts lined by eosinophilic tumor cells with prominent nucleoli would raise the diagnosis of tubulocystic RCC. It is important to differentiate the 2 entities because they lead to different outcomes. OBJECTIVE.­: To address the concern, a multicenter study was implemented to explore useful clinicopathologic features in differentiation between tubulocystic FH-deficient RCC and tubulocystic RCC. DESIGN.­: Clinical factors included age, sex, tumor size, and outcome. Morphologic factors included cell morphology, presence or absence of a nontubulocystic component, and stromal findings. Immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing were performed to explore the protein expression and molecular profiles of the 2 entities. RESULTS.­: We evaluated 6 patients with tubulocystic RCC and 10 patients with tubulocystic FH-deficient RCC. Tubulocystic RCC exhibited a small size (<4.0 cm, pT1a), low Ki-67 index (<5%), retained FH, and negative 2SC expression. Tubulocystic FH-deficient RCC had a relatively large size and a high Ki-67 index. Perinucleolar haloes, loss of FH, and 2SC positivity were always observed. Pure tubulocystic architecture was not observed in FH-deficient RCC, because focal nontubulocystic components can always be seen. CONCLUSIONS.­: We emphasized multiple sectioning to identify a nontubulocystic architecture to exclude tubulocystic RCC. Moreover, tumor size, FH/2SC staining, and the Ki-67 index can differentiate tubulocystic FH-deficient RCC from tubulocystic RCC. The diagnosis of tubulocystic RCC was not recommended in renal mass biopsy because of the limited tissues sampled.

Prospective observational study on biomarkers of response in pancreatic ductal adenocarcinoma.

Adjuvant chemotherapy benefits patients with resected pancreatic ductal adenocarcinoma (PDAC), but the compromised physical state of post-operative patients can hinder compliance. Biomarkers that identify candidates for prompt adjuvant therapy are needed. In this prospective observational study, 1,171 patients with PDAC who underwent pancreatectomy were enrolled and extensively followed-up. Proteomic profiling of 191 patient samples unveiled clinically relevant functional protein modules. A proteomics-level prognostic risk model was established for PDAC, with its utility further validated using a publicly available external cohort. More importantly, through an interaction effect regression analysis leveraging both clinical and proteomic datasets, we discovered two biomarkers (NDUFB8 and CEMIP2), indicative of the overall sensitivity of patients with PDAC to adjuvant chemotherapy. The biomarkers were validated through immunohistochemistry on an internal cohort of 386 patients. Rigorous validation extended to two external multicentic cohorts-a French multicentric cohort (230 patients) and a cohort from two grade-A tertiary hospitals in China (466 patients)-enhancing the robustness and generalizability of our findings. Moreover, experimental validation through functional assays was conducted on PDAC cell lines and patient-derived organoids. In summary, our cohort-scale integration of clinical and proteomic data demonstrates the potential of proteomics-guided prognosis and biomarker-aided adjuvant chemotherapy for PDAC.

EBV-Associated Smooth Muscle Tumors With Autoimmune Hemolytic Anemia and Hepatitis B Infection: Report of a Previously Undescribed Neoplasm With Review.

Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is rare in adults. The presence of intratumoral T lymphocytes and primitive rounded cells characterized this neoplasm. We report a 24-year-old Chinese man who developed EBV-SMT in the right adrenal gland with hepatitis B infection and autoimmune hemolytic anemia without a history of HIV infection, primary immune deficiency, organ transplantation, or malignant tumor. This patient had an unknown immunodeficient state. EBV-SMTs are commonly located in the liver, lung, and gastrointestinal tract but rarely in the adrenal gland. We reviewed 10 reported literature on EBV-SMT in the adrenal gland. It is imperative to distinguish EBV-SMT from conventional somatic smooth muscle tumors. The discovery of EBV-SMT forces the clinician to conduct a thorough evaluation of immune function and immune status surveillance, and these patients are vulnerable to subsequent malignant tumors.

Long non-coding RNA HIF1A-AS2 promotes carcinogenesis by enhancing Gli1-mediated HIF1α expression in clear cell renal cell carcinoma.

BACKGROUND: The most common urologic tumor in humans with the highest incidence rate is clear cell renal cell carcinoma (ccRCC). Long non-coding RNAs (lncRNAs) act as regulatory factors in several tumors. Here, we studied ccRCC regulated by hypoxia-inducible factor 1α (HIF1α)-antisense RNA 2 (AS2) or HIF1A-AS2. METHODS: We performed wound-healing, transwell, and CCK-8 assays by decreasing or increasing the HIF1A-AS2 expression in RCC cell lines. Western blotting and qRT-PCR were used to identify the expression of downstream genes of the HIF1A-AS2 pathway. Gli1 and HIF1A-AS2 relationship was assessed using RIP and RNA pull-down assays. Lastly, transcriptome sequencing was performed on kidney cancer cells that had been knocked down to find possible regulatory mechanisms. RESULTS: Our results suggest that high expression of HIF1A-AS2 may promote RCC cell proliferation and Gli1 expression as a downstream factor. Furthermore, they have physical binding sites and together regulate HIF1α to encourage the development of ccRCC. HIF1A-AS2 lncRNA may offer a new molecular target for ccRCC treatment. CONCLUSION: lncRNA HIF1A-AS2 affects ccRCC development by regulating HIF1a expression through Gli1.

Mutated ASXL1 upregulates mTOR expression in renal cell carcinoma with fibromyomatous stroma.

Renal cell carcinoma with fibromyomatous stroma (RCC FMS), defined as an "emerging entity" in the 2016 WHO classification and recommended to be a novel entity by GUPS, is represented by tumor cells with clear to mildly eosinophilic cytoplasm displaying elongated and branching tubules and papillae. A fibromyomatous stroma could be observed in these tumors. These tumors are immunopositive for CK7 and featured by ELOC and/or TSC/mTOR gene mutations. In the 2022 WHO classification, ELOC mutated RCC is classified as a molecularly defined RCCs as an individual renal entity. However, there are limited descriptions of TSC/mTOR alterations in RCC FMS. Herein, we reported a case of 28-year-old woman with RCC FMS with intact ELOC and TSC/mTOR genes but ASXL1 mutation. The tumor cells were positive for mTOR expression. This case may indicate that altered mTOR expression, but not limited to mutated TSC/mTOR gene, that participates in the pathogenesis of RCC FMS.

The genetic landscape of histologically transformed marginal zone lymphomas.

BACKGROUND: Marginal zone lymphomas (MZLs) comprise a diverse group of indolent lymphoproliferative disorders; however, some patients develop histologic transformation (HT) with rapid progression to aggressive lymphoma. METHODS: Forty-three MZLs with HT (HT-MZLs), 535 MZLs, and 174 de novo diffuse large B-cell lymphomas (DLBCLs) without rearrangements of MYC, BCL2, and BCL6 were collected. Among these, 22 HT-MZLs, 39 MZLs, and 174 DLBCLs were subjected to 148-gene targeted exome sequencing. The clinicopathologic features of patients who had HT-MZL and their genetic alterations were compared with those of patients who had MZLs and DLBCLs. RESULTS: All 43 HT-MZLs corresponded to DLBCLs. No HT-MZLs harbored BCL2 and MYC and/or BCL6 rearrangements. Bone marrow involvement and higher levels of lactate dehydrogenase were significantly more common in HT-MZLs than in MZLs. Furthermore, upregulated BCL6, MUM1, C-MYC, and Ki-67 expression was observed more frequently in HT-MZLs than in MZLs. TBL1XR1 was the most frequently altered gene (63.6%) in HT-MZLs, followed by CCND3 (31.8%), CARD11, ID3, and TP53 (22.7%). A trend toward worse progression-free survival in patients with TBL1XR1 mutations was observed. Compared with MZLs and non-germinal center B-cell (GCB) type DLBCLs, significantly higher frequencies of TBL1XR1 and ID3 mutations were identified in HT-MZLs. PIM1 mutations frequently occurred in DLBCLs and were significantly associated with TBL1XR1 mutations but were mutated less in HT-MZLs that had TBL1XR1 mutations. CONCLUSIONS: The current findings reveal the clinicopathologic and genetic features of HT-MZLs, suggesting that these tumors might constitute a group distinct from MZL and de novo non-GCB type DLBCL. TBL1XR1 mutations may be considered a predictor of HT in MZL.

Clinicopathological and molecular analysis of microsatellite instability in prostate cancer: a multi-institutional study in China.

Background: Microsatellite instability (MSI), or mismatch repair-deficiency (dMMR), is rare in prostate cancers (PCas). The histological and molecular features of PCas with MSI/dMMR are incompletely described. Thus, we sought to identify the characteristics of PCas with MSI/dMMR. Methods and results: We analyzed 1,141 primary treatment-naive PCas by MMR-related protein immunohistochemistry (MLH1, PMS2, MSH2, and MSH6). We identified eight cases exhibiting MSI/dMMR (0.7%, 8/1141). Of these, six tumors had both MSH2 and MSH6 protein loss, one had both MLH1 and PMS2 protein loss, and one had only MSH6 loss. Histologically, MSI/dMMR-PCas frequently demonstrated high histological grade (Grade Group 4 or 5), ductal/intraductal histology (6/8 cases), pleomorphic giant-cell features (4/8 cases), and conspicuous tumor lymphocytic infiltration (8/8 cases). Polymerase chain reaction-based analysis of seven MSI/dMMR tumors revealed two MSI-H tumors with loss of both MSH2 and MSH6 proteins. Subsequently, the seven cases underwent next-generation sequencing (NGS) analysis with a highly validated targeted panel; four were MSI. All cases had a high tumor mutation burden (median: 45.3 mutations/Mb). Overall, the MSI/dMMR-PCas showed a high frequency of DNA damage-repair pathway gene changes, including five with pathogenic somatic or germline MMR gene mutations. Activating mutations in the MAPK pathway, PI3K pathway, and WNT/ß-catenin pathway were common. TMPRSS2::ERG rearrangement was identified in one case (1/7, 14.3%). Conclusions: Several pathological features are associated with MSI/dMMR in PCas. Identification of these features may help to select patients for genetic screening. As MSI/dMMR-PCas are enriched for actionable mutations, patients should be offered NGS to guide standard-of-care treatment.

GPCR signaling contributes to immune characteristics of microenvironment and process of EBV-induced lymphomagenesis.

Epstein-Barr virus (EBV) is the oncogenic driver of multiple cancers. However, the underlying mechanism of virus-cancer immunological interaction during disease pathogenesis remains largely elusive. Here we reported the first comprehensive proteogenomic characterization of natural killer/T-cell lymphoma (NKTCL), a representative disease model to study EBV-induced lymphomagenesis, incorporating genomic, transcriptomic, and in-depth proteomic data. Our multi-omics analysis of NKTCL revealed that EBV gene pattern correlated with immune-related oncogenic signaling. Single-cell transcriptome further delineated the tumor microenvironment as immune-inflamed, -deficient, and -desert phenotypes, in association with different setpoints of cancer-immunity cycle. EBV interacted with transcriptional factors to provoke GPCR interactome (GPCRome) reprogramming. Enhanced expression of chemokine receptor-1 (CCR1) on malignant and immunosuppressive cells modulated virus-cancer interaction on microenvironment. Therapeutic targeting CCR1 showed promising efficacy with EBV eradication, T-cell activation, and lymphoma cell killing in NKTCL organoid. Collectively, our study identified a previously unknown GPCR-mediated malignant progression and translated sensors of viral molecules into EBV-specific anti-cancer therapeutics.

TET (Ten-eleven translocation) family proteins: structure, biological functions and applications.

Ten-eleven translocation (TET) family proteins (TETs), specifically, TET1, TET2 and TET3, can modify DNA by oxidizing 5-methylcytosine (5mC) iteratively to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), and then two of these intermediates (5fC and 5caC) can be excised and return to unmethylated cytosines by thymine-DNA glycosylase (TDG)-mediated base excision repair. Because DNA methylation and demethylation play an important role in numerous biological processes, including zygote formation, embryogenesis, spatial learning and immune homeostasis, the regulation of TETs functions is complicated, and dysregulation of their functions is implicated in many diseases such as myeloid malignancies. In addition, recent studies have demonstrated that TET2 is able to catalyze the hydroxymethylation of RNA to perform post-transcriptional regulation. Notably, catalytic-independent functions of TETs in certain biological contexts have been identified, further highlighting their multifunctional roles. Interestingly, by reactivating the expression of selected target genes, accumulated evidences support the potential therapeutic use of TETs-based DNA methylation editing tools in disorders associated with epigenetic silencing. In this review, we summarize recent key findings in TETs functions, activity regulators at various levels, technological advances in the detection of 5hmC, the main TETs oxidative product, and TETs emerging applications in epigenetic editing. Furthermore, we discuss existing challenges and future directions in this field.

HKDC1 reprograms lipid metabolism to enhance gastric cancer metastasis and cisplatin resistance via forming a ribonucleoprotein complex.

As essential modulators of transcription and translation, RNA-binding proteins (RBPs) are frequently dysregulated in cancer. Bioinformatics study reveals that the RNA-binding protein hexokinase domain component 1 (HKDC1) is overexpressed in gastric cancer (GC). As HKDC1 plays a role in lipid homeostasis in the liver and glucose metabolism in certain cancers, the exact mechanism of action of HKDC1 in GC remains largely unknown. Upregulation of HKDC1 correlates with chemoresistance and poor prognosis in GC patients. HKDC1 enhances invasion, migration and resistance to cisplatin (CDDP) in GC cells in vitro and in vivo. Comprehensive transcriptomic sequencing and metabolomic analysis reveal that HKDC1 mediates abnormal lipid metabolism in GC cells. Herein, we identify a number of HKDC1-binding endogenous RNAs in GC cells, including protein kinase, DNA-activated, catalytic subunit (PRKDC) mRNA. We further validate that PRKDC is a crucial downstream effector of HKDC1 induced-GC tumorigenesis depends on lipid metabolism. Interestingly, G3BP1, a well-known oncoprotein, can be bound by HKDC1. HKDC1 cooperates with G3BP1 to enhance the stability of PRKDC transcript. Our results reveal a novel HKDC1/G3BP1-PRKDC regulatory axis that induces GC metastasis and chemoresistance via reprogramming lipid metabolism, which may provide an effective therapeutic strategy for a subset of GC with HKDC1 overexpression.

Schwann cells regulate tumor cells and cancer-associated fibroblasts in the pancreatic ductal adenocarcinoma microenvironment.

Neuropathy is a feature more frequently observed in pancreatic ductal adenocarcinoma (PDAC) than other tumors. Schwann cells, the most prevalent cell type in peripheral nerves, migrate toward tumor cells and associate with poor prognosis in PDAC. To unveil the effects of Schwann cells on the neuro-stroma niche, here we perform single-cell RNA-sequencing and microarray-based spatial transcriptome analysis of PDAC tissues. Results suggest that Schwann cells may drive tumor cells and cancer-associated fibroblasts (CAFs) to more malignant subtypes: basal-like and inflammatory CAFs (iCAFs), respectively. Moreover, in vitro and in vivo assays demonstrate that Schwann cells enhance the proliferation and migration of PDAC cells via Midkine signaling and promote the switch of CAFs to iCAFs via interleukin-1α. Culture of tumor cells and CAFs with Schwann cells conditioned medium accelerates PDAC progression. Thus, we reveal that Schwann cells induce malignant subtypes of tumor cells and CAFs in the PDAC milieu.

Early Screening of Colorectal Precancerous Lesions Based on Combined Measurement of Multiple Serum Tumor Markers Using Artificial Neural Network Analysis.

Many patients with colorectal cancer (CRC) are diagnosed in the advanced stage, resulting in delayed treatment and reduced survival time. It is urgent to develop accurate early screening methods for CRC. The purpose of this study is to develop an artificial intelligence (AI)-based artificial neural network (ANN) model using multiple protein tumor markers to assist in the early diagnosis of CRC and precancerous lesions. In this retrospective analysis, 148 cases with CRC and precancerous diseases were included. The concentrations of multiple protein tumor markers (CEA, CA19-9, CA 125, CYFRA 21-1, CA 72-4, CA 242) were measured by electrochemical luminescence immunoassays. By combining these markers with an ANN algorithm, a diagnosis model (CA6) was developed to distinguish between normal healthy and abnormal subjects, with an AUC of 0.97. The prediction score derived from the CA6 model also performed well in assisting in the diagnosis of precancerous lesions and early CRC (with AUCs of 0.97 and 0.93 and cut-off values of 0.39 and 0.34, respectively), which was better than that of individual protein tumor indicators. The CA6 model established by ANN provides a new and effective method for laboratory auxiliary diagnosis, which might be utilized for early colorectal lesion screening by incorporating more tumor markers with larger sample size.

Etoposide, dexamethasone, and pegaspargase with sandwiched radiotherapy in early-stage natural killer/T-cell lymphoma: A randomized phase III study.

Methotrexate, etoposide, dexamethasone, and pegaspargase (MESA) with sandwiched radiotherapy is known to be effective for early-stage extranodal natural killer/T-cell lymphoma, nasal type (NKTCL). We explored the efficacy and safety of reduced-intensity, non-intravenous etoposide, dexamethasone, and pegaspargase (ESA) with sandwiched radiotherapy. This multicenter, randomized, phase III trial enrolled patients aged between 14 and 70 years with newly diagnosed early-stage nasal NKTCL from 27 centers in China. Patients were randomly assigned (1:1) to receive ESA (pegaspargase 2,500 IU/m2 intramuscularly on day 1, etoposide 200 mg orally, and dexamethasone 40 mg orally on days 2-4) or MESA (methotrexate 1 g/m2 intravenously on day 1, etoposide 200 mg orally, and dexamethasone 40 mg orally on days 2-4, and pegaspargase 2,500 IU/m2 intramuscularly on day 5) regimen (four cycles), combined with sandwiched radiotherapy. The primary endpoint was overall response rate (ORR). The non-inferiority margin was -10.0%. From March 16, 2016, to July 17, 2020, 256 patients underwent randomization, and 248 (ESA [n = 125] or MESA [n = 123]) made up the modified intention-to-treat population. The ORR was 88.8% (95% confidence interval [CI], 81.9-93.7) for ESA with sandwiched radiotherapy and 86.2% (95% CI, 78.8-91.7) for MESA with sandwiched radiotherapy, with an absolute rate difference of 2.6% (95% CI, -5.6-10.9), meeting the non-inferiority criteria. Per-protocol and sensitivity analysis supported this result. Adverse events of grade 3 or higher occurred in 42 (33.6%) patients in the ESA arm and 81 (65.9%) in the MESA arm. ESA with sandwiched radiotherapy is an effective, low toxicity, non-intravenous regimen with an outpatient design, and can be considered as a first-line treatment option in newly diagnosed early-stage nasal NKTCL.

Exploration of clinicopathological features of rearranged renal cell carcinoma and TFE3, TFEB, and ALK staining performance in renal entities.

Rearranged renal cell carcinomas (RCC) are rare types of kidney cancer. The clinicopathological features of rearranged RCC require further validation. The pathological diagnosis usually depends on immunohistochemistry and molecular analysis. This study aimed to explore the expression features of anti-TFE3, TFEB, and ALK in different renal entities. In addition, we collected thirty-six TFE3-rearranged RCC, two TFEB-altered RCC, and one ALK-rearranged RCC to explore their clinicopathological features. We observed that TFE3 can sometimes be weakly expressed in non-TFE3-rearranged RCC. TFE3-rearranged RCC usually exhibited strong TFE3 expression. However, clear cell RCC and FH-deficient RCC also displayed strong TFE3 expression. TFEB also can be weakly expressed in clear cell RCC. However, ALK IHC showed a relatively high specificity and was negative for all non-ALK-rearranged RCC. The ALK-rearranged RCC was analyzed using next generation sequencing to explore gene alterations, and we identified a novel gene partner, SLIT1. ALK-rearranged RCC appears to have eosinophilic cytoplasm. Tumor cells with clear cytoplasm may exclude this diagnosis. Psammomatous bodies (22/38) and pattern multiplicity (35/38) were observed in more than half of the patients. In conclusion, weak TFE3 expression did not indicate TFE3 rearrangement. Strong TFE3 expression had a higher value for indicating TFE3-rearranged RCC, although other entities can also exhibit a strong pattern. Young age combined with morphological features (psammomatous calcification and pattern multiplicity) may indicate the diagnosis of rearranged RCC.

Exploration of Morphological Features of Clear Cell Renal Cell Carcinoma With PBRM1, SETD2, BAP1, or KDM5C Mutations.

The last decade has seen great advances in genomic profiling and prognosis-associated factors of clear cell renal cell carcinoma (RCC), the most common entity in kidney cancer. Following VHL, PBRM1, SETD2, BAP1, and KDM5C have been validated as the most common co-occurring gene mutations in clear cell RCC by multicenter studies. However, the morphological features of clear cell RCC with co-occurring gene mutations remain unclear. In this study, we presented 20 clear cell RCCs that underwent next-generation sequencing, of which 1 tumor was reclassified as ELOC-mutated RCC. PBRM1, SETD2, BAP1, and KDM5C were the most common mutations, following VHL. Morphologically, clear cell RCC with PBRM1 or KDM5C mutation usually displayed a low-grade pattern. Cystic changes and hyalinized stroma were often observed. The Ki67 index was <10%. These observations indicated good prognosis. However, mutated SETD2 may increase the malignancy of clear cell RCC with PBRM1 mutation. Two clear cell RCCs with mutated PBRM1 and SETD2 developed local or distant metastases. Clear cell RCC with BAP1 mutations always had high-grade patterns, and rhabdoid differentiation was also observed, indicating that BAP1 mutation was associated with poor outcomes. Papillary architecture was often a feature of BAP1 mutation, which is uncommon in clear cell RCC. PDL1 was positive in only one tumor with BAP1 mutation, and the positivity rate was limited to 5%. B7H3 was negative in all tumors. Morphologic findings in this small cohort may suggest why PBRM1 mutation does not correlate with decreased survival, whereas BAP1 mutation usually predicts poor outcomes.

Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment.

OBJECTIVE: Innate immunity plays important roles in pancreatic ductal adenocarcinoma (PDAC), as non-T-cell-enriched tumour. Neutrophils are major players in innate immune system. Here, we aimed to explore the heterogeneity and pro-tumour mechanisms of neutrophils in PDAC. DESIGN: We analysed single-cell transcriptomes of peripheral blood polymorphonuclear leucocytes (PMNs) and tumour-infiltrating immune cells from five patients with PDAC, and performed immunofluorescence/immunohistochemistry staining, multi-omics analysis and in vitro experiments to validate the discoveries of bioinformatics analysis. RESULTS: Exploration of the heterogeneity of tumour-associated neutrophils (TANs) revealed a terminally differentiated pro-tumour subpopulation (TAN-1) associated with poor prognosis, an inflammatory subpopulation (TAN-2), a population of transitional stage that have just migrated to tumour microenvironment (TAN-3) and a subpopulation preferentially expressing interferon-stimulated genes (TAN-4). Glycolysis signature was upregulated along neutrophil transition trajectory, and TAN-1 was featured with hyperactivated glycolytic activity. The glycolytic switch of TANs was validated by integrative multi-omics approach of transcriptomics, proteomics and metabolomics analysis. Activation of glycolytic activity by LDHA overexpression induced immunosuppression and pro-tumour functions in neutrophil-like differentiated HL-60 (dHL-60) cells. Mechanistic studies revealed BHLHE40, downstream to hypoxia and endoplasmic reticulum stress, was a key regulator in polarisation of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by chromatin immunoprecipitation assay. Pro-tumour and immunosuppression functions were observed in dHL-60 cells overexpressing BHLHE40. Importantly, immunohistochemistry analysis of PDAC tissues revealed the unfavourable prognostic value of BHLHE40+ neutrophils. CONCLUSION: The dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

Clinicopathological and molecular features of indolent natural killer-cell lymphoproliferative disorder of the gastrointestinal tract.

AIMS: Indolent natural killer (NK) cell lymphoproliferative disorder of the gastrointestinal (GI) tract (iNKLPD) is a rare, recently recognised neoplasm. Most of the reported tumours are confined to the GI tract, while a small subset of the tumours harbour JAK3 mutations. We collected four cases of iNKLPD with the goal of adding additional information to the current knowledge of this disease regarding the clinicopathological, immunohistochemical and molecular features. METHODS AND RESULTS: Similar features including medium- to large-sized lymphoid cells with variable amounts of pale or slightly eosinophilic cytoplasm, and no evidence of EBER, TCR rearrangement were found in four cases. JAK3 K563_C565del mutation was found in one of three cases that were subjected to targeted next-generation sequencing. Unique findings of our study include one iNKLPD encountered for the first time in nasopharynx, where lesions could be inadvertently diagnosed as extranodal NK/T cell lymphoma, and one iNKLPD located in the gallbladder extended deeply into muscular and adventitial layers. Exceptional CD8-positive expression was observed in one iNKLPD. In addition, positive staining of phospho-STAT5, phospho-STAT3 and phospho-p38 were found in our cases. None of the four patients received therapy for lymphoma, but all had a benign clinical outcome during a follow-up time of 20-99 months. CONCLUSIONS: We present four iNKLPDs with clinical, immunohistochemical and molecular features similar to the reported cases, as well as some unusual characters, which expand our knowledge on this disease, and further support the neoplastic nature of iNKLPDs.