HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

Progression patterns, resistant mechanisms and subsequent therapy for ALK-positive NSCLC in the era of second-generation ALK-TKIs.

BACKGROUND: In the era of second-generation ALK tyrosine kinase inhibitors (ALK-TKIs), there was a paucity of data regarding the progression patterns, resistant mechanisms, and subsequent therapeutic approaches for ALK-positive (ALK+) non-small cell lung cancer (NSCLC). METHODS: Patients with advanced ALK+ NSCLC were retrospectively selected from our center. Cohort 1 consisted of patients who experienced disease progression after receiving first-line alectinib treatment (n = 20), while Cohort 2 included patients who progressed following sequential treatment with crizotinib and second-generation ALK-TKIs (n = 53). Oligo-progression was defined as the occurrence of disease progression in no more than three lesions. Symptomatic progression was determined when patients developed new symptoms or experienced worsening of pre-existing symptoms during radiological progression. RESULTS: The incidence of central nervous system (CNS) progression and symptomatic CNS progression was significantly lower in Cohort 1 compared to patients treated with crizotinib, with rates of 15.0% vs. 56.6% (p = 0.002) and 5.0% vs. 32.1% (p = 0.016), respectively. A total of 60.3% (44/73) patients underwent repeated biopsy and next-generation sequencing subsequent to the second-generation ALK-TKI resistance, with secondary mutation in ALK kinase domain emerging as the predominant mechanism of resistance (56.8%). Local therapy was applied to 50% of oligo-progression cases. Subsequent ALK-TKIs demonstrated significantly prolonged progression-free survival (PFS) (8.6 m vs. 2.7 m, p = 0.021, HR = 0.43, 95%CI: 0.15-0.85) and long-term overall survival (OS) (NA vs. 11.9 m, p = 0.132, HR = 0.50, 95%CI: 0.18-1.25) in patients harboring ALK resistance mutations, compared to those without such mutations. For patients without ALK-resistant mutations following progression on second-generation ALK-TKIs, there was no statistically significant difference in survival outcomes between subsequent chemotherapy or alternative ALK-TKI treatments. CONCLUSIONS: First-line alectinib demonstrated superior efficacy in protecting the CNS compared to crizotinib. For patients with ALK-resistant mutations following the resistance to second-generation ALK-TKIs, appropriate sensitive ALK-TKI should be administered; for those without such mutations, the selection of chemotherapy or third-generation ALK-TKI should be based on the patient's overall physical health and personal preferences.

The Battle for Accuracy: Identifying the Most Effective Grading System for Lung Invasive Mucinous Adenocarcinoma.

BACKGROUND: The prognostic analysis of lung invasive mucinous adenocarcinoma (IMA) is deficient due to the lack of a universally recommended histological grading system, leading to unregulated treatment approaches. OBJECTIVE: We aimed to examine the clinical trajectory of IMA and assess the viability of utilizing the existing grading system for lung invasive non-mucinous adenocarcinoma in the context of IMA. METHODS: We retrospectively collected clinicopathological data from 265 IMA patients. Each case re-evaluated the tumor grade using the following three classification systems: the 4th Edition of the World Health Organization classification system, the International Association for the Study of Lung Cancer (IASLC) grading system, and a two-tier grading system. We performed a comparative analysis of these grading systems and identified the most effective grading system for IMA. RESULTS: The study comprised a total of 214 patients with pure IMA and 51 patients with mixed IMA. The 5-year overall survival (OS) rates for pure IMA and mixed IMA were 86.7% and 57.8%, respectively. All three grading systems proved to be effective prognostic classifiers for IMA. The value of area under the curve at 1-, 3-, and 5-year OS was highest for the IASLC grading system compared with the other grade systems and the clinical stage. The IASLC classification system was an independent prognostic predictor (p = 0.009, hazard ratio 2.243, 95% confidence interval 1.219-4.127). CONCLUSION: Mixed IMA is more aggressive than pure IMA, with an OS rate on par with that of high-grade pure IMA. The IASLC grading system can better indicate prognosis and is recommended for lung IMA.

Proficiency testing of diagnosis in histopathology and immunohistochemistry of breast pathology in China: results from a pilot work of National Single Disease Quality Control Program for breast cancer.

AIM: Pathologists are currently supposed to be aware of both domestic and international guidelines for breast cancer diagnosis, but it is unclear how successfully these guidelines have been integrated into routine clinical practice in China. Thus, this national proficiency testing (PT) scheme for breast pathology was set up to conduct a baseline assessment of the diagnostic capability of pathologists in China. METHODS: This national PT plan is designed and implemented according to the "Conformity assessment-General requirements for proficiency testing" (GB/T27043-2012/ISO/IEC 17043:2010). Five cases of breast cancer with six key items, including histologic type, grade, ER, PR, HER2, and Ki67, were selected for testing among 96 participants. The final PT results were published on the website of the National Quality Control Center for Cancer ( http://117.133.40.88:3927/cn/col22/362 ). RESULTS: Our study demonstrated that the median PT score was 89.5 (54-100). Two institutions with scores < 67 were deemed unacceptable. The accuracy of histologic type, ER, PR, HER2, and Ki67 was satisfactory (all > 86%). However, the histologic grade showed low accuracy (74.0%). The unacceptable results mainly included incorrect evaluation of histologic grade (36.7%), inaccurate evaluation of ER/PR/HER2/Ki67 (28.2%), incorrect identification of C-AD as IBC-NST (15.7%), inappropriate use of 1+/2+/3+ rather than staining percentage for ER/PR (6.1%), misclassification of ER/PR < 1% weak expression as positive staining (1.4%), and no evaluation of histologic grade in ILC, MC, and IMC (5.8%). CONCLUSIONS: our nationwide PT program exhibited a satisfactory baseline assessment of the diagnostic capability of pathologists in China. More importantly, we identify some areas for further improvement.

Gastric tubular adenocarcinoma with diffuse neutrophils infiltrating: characteristics and probable treatment strategy.

BACKGROUND: Gastric adenocarcinoma is a highly heterogeneous malignancy with varying prognoses. In clinicopathological practice, we noticed a special tubular adenocarcinoma with diffuse neutrophils infiltrating (TADNI). However, the proportion and characteristics of TADNI remain unclear. This study aimed to evaluate the features of TADNI and explore probable treatments. METHODS: We divided 289 tubular adenocarcinoma cases into the TADNI and non-TADNI (nTADNI) groups by histological neutrophil quantity and performed immunohistochemistry of treatment-associated markers (CXCR1, CXCR2, PD-L1, CD8, HER2 and VEGFR2). Then we evaluated the clinical and morphological features in these cases. We also compared the value of histological features and peripheral blood neutrophil test. In addition, multiomics bioinformatic analyses were performed using the public datasets. RESULTS: In our cohort, TADNI accounted for 10.4% of all tubular adenocarcinoma cases. These cases had worse prognoses (especially the neutrophils mainly outside the tubes) than nTADNI cases. The histological identification of TADNI had more prognostic value than peripheral blood neutrophils. CXCR1/CXCR2 expression was significantly high in TADNI group which indicated that CXCR1/CXCR2 inhibitors might be beneficial for TADNI patients. There were no significant differences in the expression of PD-L1, CD8, HER2 and VEGFR2. The analyses of TCGA data confirmed that TADNI cases had poorer prognoses and higher CXCR1/CXCR2 expression. Bioinformatic results also revealed molecular features (more hsa-mir-223 expression, fewer CD8-positive T cells and regulatory T cells, tighter communication between tumor cells' CXCR1/CXCR2 and neutrophils' CXCL5/CXCL8) of this type. CONCLUSIONS: TADNI is a special morphological subtype with poorer prognoses and unique molecular characteristics, which might benefit from CXCR1/CXCR2 inhibitors.

Low TP53 variant allele frequency as a biomarker for anti-programmed death (ligand) 1 monotherapy in lung adenocarcinoma.

BACKGROUND: TP53 mutation heterogeneity should be considered when using TP53 as a predictive biomarker for anti-programmed death (ligand) 1 (PD-(L)1) monotherapy in lung adenocarcinoma (LUAD). However, whether TP53 variant allele frequency (VAF) should also be considered remains unknown. METHODS: Patients with LUAD from both published research and the local cohort were included to discover and validate the relationship between TP53 VAF and the efficacy of PD-(L)1 inhibitors. The Cancer Genome Atlas (TCGA) LUAD data were included for genomic, transcriptomic, and tumor microenvironment analysis. RESULTS: Among 159 patients in the discovery cohort, low TP53 VAF patients (VAF ≤ 25%) experienced significantly longer progression-free survival (PFS) than both high TP53 VAF (5.4 vs. 3.3 months; p = .021) and TP53-wild-type patients (5.4 vs. 2.5 months; p = .011). Multivariate Cox regression revealed low TP53 VAF as an independent biomarker of better efficacy. Among 50 patients in the combined validation cohort, median PFS of low TP53 VAF patients was also significantly longer than that of high TP53 VAF patients (12.0 vs. 2.1 months; p = .037). Analyzed with 469 TCGA LUAD samples, low TP53 VAF is associated with significantly higher PD-L1 expression, enrichment of gene sets related to T-cell activation, T cell-mediated immunity, and interferon-γ signaling pathways, and independently associated with more tumor-infiltrating CD8+ T cells compared with both high TP53 VAF and TP53-wild type. CONCLUSIONS: TP53 VAF should also be considered when using TP53 as a predictive biomarker. Only low TP53 VAF is independently associated with better efficacy of anti-PD-(L)1 monotherapy, which may result from higher PD-L1 expression and more tumor-infiltrating CD8+ T cells.

The prognostic value of tumour-infiltrating lymphocytes, programmed cell death protein-1 and programmed cell death ligand-1 in Stage I-III triple-negative breast cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) represent a robust biological prognostic biomarker in triple-negative breast cancer (TNBC); however, the contribution of different subsets of immune cells is unclear. We investigated the prognostic value of immune markers, including stromal TILs (sTILs), CD8+T and FOPX3+T cells, PD-1 and PD-L1 in non-metastatic TNBC. METHODS: In total, 259 patients with Stage I-III TNBC were reviewed. The density of sTILs along with the presence of total (t), stromal (s), and intratumoral (i) CD8+T cells and FOPX3+T cells were evaluated by haematoxylin and eosin and immunohistochemical staining. Immunohistochemical staining of PD-1, PD-L1 was also conducted. RESULTS: All immune markers were positively correlated with each other (P < 0.05). In the multivariate analysis, sTILs (P = 0.046), tCD8+T cells (P = 0.024), iCD8+T cells (P = 0.050) and PD-1 (P = 0.039) were identified as independent prognostic factors for disease-free survival (DFS). Further analysis showed that tCD8+T cells (P = 0.026), iCD8+T cells (P = 0.017) and PD-1 (P = 0.037) increased the prognostic value for DFS beyond that of the classic clinicopathological factors and sTILs. CONCLUSIONS: In addition to sTILs, inclusion of tCD8+T, iCD8+T cells, or PD-1 may further refine the prognostic model for non-metastatic TNBC beyond that including classical factors alone.

Using immunovascular characteristics to predict very early recurrence and prognosis of resectable intrahepatic cholangiocarcinoma.

OBJECTIVE: To predict the very early recurrence (VER) of patients with intrahepatic cholangiocarcinoma (ICC) based on TLSs and MVI status, and further perform prognosis stratifications. METHODS: A total of 160, 51 ICC patients from two institutions between May 2012 and July 2022 were retrospectively included as training, external validation cohort. Clinical, radiological and pathological variables were evaluated and collected. Univariate and multivariate analysis were applied to select the significant factors related to VER of ICC. The factors selected were combined to perform stratification of overall survival (OS) using the Kaplan-Meier method with the log-rank test. RESULTS: Overall, 39 patients (24.4%) had VER, whereas 121 (75.6%) did not (non-VER group). In the training cohort, the median OS was 40.5 months (95% CIs: 33.2-47.7 months). The VER group showed significantly worse OS than the non-VER group (median OS: 14.8, 95% CI:11.6-18.0 months vs. 53.4, 34.3-72.6 months; p<0.001), and it was confirmed in the validation cohort (median OS: 22.1, 95% CI: 8.8-35.4 months vs. 40.1, 21.2-59.0 months; p = 0.003). According to the univariate analysis, four variables were significantly different between the VER group and non-VER group (TLSs status, p = 0.028; differentiation, p = 0.023; MVI status, p = 0.012; diameter, p = 0.028). According to the multivariate analysis, MVI-positive status was independently associated with a higher probability of VER (odds ratio [OR], 2.5; 95% CIs,1.16-5.18; p = 0.018), whereas intra-tumoral TLSs-positive status was associated with lower odds of VER (OR, 0.43; 95% CIs, 0.19-0.97; p = 0.041). Based on the TLSs and MVI status, patients of ICC were categorized into four groups: TLSs-positive and MVI-negative (TP/MN); TLSs-negative and MVI-negative (TN/MN); TLSs-positive and MVI-positive (TP/MP), TLSs-negative and MVI-positive groups (TN/MP). In the training cohort, the four groups could be correlated with OS significantly (p<0.001), and it was confirmed in the validation cohort (p<0.001). CONCLUSION: Intra-tumoral TLSs and MVI status are independent predictive factors of VER after surgery, based on which immunovascular stratifications are constructed and associated with OS significantly of resectable intrahepatic cholangiocarcinoma.

Association Between MRI Radiomics and Intratumoral Tertiary Lymphoid Structures in Intrahepatic Cholangiocarcinoma and Its Prognostic Significance.

BACKGROUND: Tertiary lymphoid structures (TLSs) have prognostic value in intrahepatic cholangiocarcinoma (ICC) patients. Noninvasive tool to preoperatively evaluate TLSs is still lacking. PURPOSE: To explore the association between TLSs status of ICC and preoperative MRI radiomics analysis. STUDY TYPE: Retrospective. SUBJECTS: One hundred and ninety-two patients with ICC, divided into training (T = 105), internal validation groups (V1 = 46), and external validation group (V2 = 41). SEQUENCE: Coronal and axial single-shot fast spin-echo T2-weighted, diffusion-weighted imaging, T1-weighted, and T1WI fat-suppressed spoiled gradient-recall echo LAVA sequence at 3.0 T. ASSESSMENT: The VOIs were drawn manually within the visible borders of the tumors using ITK-SNAP version 3.8.0 software in the axial T2WI, DWI, and portal vein phase sequences. Radiomics features were subjected to least absolute shrinkage and selection operator regression to select the associated features of TLSs and construct the radiomics model. Univariate and multivariate analyses were used to identify the clinical radiological variables associated with TLSs. The performances were evaluated by the area under the receiver operator characteristic curve (AUC). STATISTICAL TESTS: Logistic regression analysis, ROC and AUC, Hosmer-Lemeshow test, Kaplan-Meier method with the log-rank test, calibration curves, and decision curve analysis. P < 0.05 was considered statistically significant. RESULTS: The AUCs of arterial phase diffuse hyperenhancement were 0.59 (95% confidence interval [CI], 0.50-0.67), 0.52 (95% CI, 0.43-0.61), and 0.66 (95% CI, 0.52-0.80) in the T, V1, and V2 cohorts. The AUCs of Rad-score were 0.85 (95% CI, 0.77-0.92), 0.81 (95% CI, 0.67-0.94), and 0.84 (95% CI, 0.71-0.96) in the T, V1, and V2 cohorts, respectively. In cohort T, low-risk group showed significantly better median recurrence-free survival (RFS) than that of the high-risk group, which was also confirmed in cohort V1 and V2. DATA CONCLUSION: A preoperative MRI radiomics signature is associated with the intratumoral TLSs status of ICC patients and correlate significantly with RFS. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

Multi-dimensional characterization of immunological profiles in small cell lung cancer uncovers clinically relevant immune subtypes with distinct prognoses and therapeutic vulnerabilities.

Small-cell lung cancer (SCLC) is generally considered a 'homogenous' disease, with little documented inter-tumor heterogeneity in treatment guidance or prognosis evaluation. The precise identification of clinically relevant molecular subtypes remains incomplete and their translation into clinical practice is limited. In this retrospective cohort study, we comprehensively characterized the immune microenvironment in SCLC by integrating transcriptional and protein profiling of formalin-fixation-and-paraffin-embedded (FFPE) samples from 29 patients. We identified two distinct disease subtypes: immune-enriched (IE-subtype) and immune-deprived (ID-subtype), displaying heterogeneity in immunological, biological, and clinical features. The IE-subtype was characterized by abundant immune infiltrate and elevated levels of interferon-alpha/gamma (IFNα/IFNγ) and inflammatory response, while the ID-subtype featured a complete lack of immune infiltration and a more proliferative phenotype. These two immune subtypes are associated with clinical benefits in SCLC patients treated with adjuvant therapy, with the IE-subtype exhibiting a more favorable response leading to improved survival and reduced disease recurrence risk. Additionally, we identified and validated a personalized prognosticator of immunophenotyping, the CCL5/CXCL9 chemokine index (CCI), using machine learning. The CCI demonstrated superior predictive abilities for prognosis and clinical benefits in SCLC patients, validated in our institute immunohistochemistry cohort and multicenter bulk transcriptomic data cohorts. In conclusion, our study provides a comprehensive and multi-dimensional characterization of the immune architecture of SCLC using clinical FFPE samples and proposes a new immune subtyping conceptual framework enabling risk stratification and the appropriate selection of individualized therapy.

Single-cell RNA-sequencing data reveals the genetic source of extracellular vesicles in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is invasive cancer and the complex mechanisms underlying carcinogenesis remain unclear. Extracellular vesicles (EVs), secreted by most cell types, serve as a critical factor in tumorigenesis via intercellular communications. Our study aims to investigate the cellular origin of EVs in ESCC, and unveil the unknown molecular and cellular mechanisms underlying cell-cell communications. Six ESCC patients were enrolled and single-cell RNA sequencing (scRNA-seq) analyses were conducted to screen different cell subpopulations. The genetic origin of EVs was tracked using the supernatant from different cellular extracts. Nanoparticle tracking analysis (NTA), western blot analysis, and transmission electron microscopy (TEM) were performed for validation. Using scRNA-seq analysis, eleven cell subpopulations were identified in ESCC. Differences in gene expression in EVs between malignant and non-malignant esophageal tissues were found. Our findings demonstrated that epithelial cells releasing EVs were the most prevalent in malignant tissues, while endothelial cells and fibroblasts releasing EVs were predominant in non-malignant tissues. Furthermore, the high levels of gene expression in EVs released from these cells were correlated significantly with a worse prognosis. Our findings revealed the genetic origin of EVs in malignant and non-malignant esophageal tissues and provided a comprehensive overview of the associated cell-cell interactions in ESCC.

PSC subtyping based on TTF-1 and p40 expression reveals distinct molecular characteristics and therapeutic strategies.

Pulmonary sarcomatoid carcinoma (PSC) is a unique form of poorly differentiated nonsmall cell lung cancer (NSCLC) and is notorious for its highly malignant nature and dismal prognosis. To introduce effective treatment for PSC patients, precise subtyping of PSC is demanding. In our study, TTF-1 and P40 immunohistochemistry (IHC) staining were applied to 56 PSC patients with multiomics data. According to IHC results, we categorized these patients into three subgroups and profiled their molecular contexture using bioinformatic skills. IHC results classified these patients into three subgroups: TTF-1 positive subgroup (n = 27), P40 positive subgroup (n = 15) and double-negative subgroup (n = 14). Spindle cell samples accounted for 35.71% (5/14) of double-negative patients, higher than others (P = .034). The three subgroups were heterogeneous in the genomic alteration spectrum, showing significant differences in the RTK/RAS pathway (P = .004) and the cell cycle pathway (P = .030). The methylation profile of the double-negative subgroup was between the other two subgroups. In similarity analysis, the TTF-1 and p40 subgroups were closely related to LUAD and LUSC, respectively. The TTF-1 positive subgroup had the highest leukocyte fraction (LF) among several cancer types, and the tumor mutation burden (TMB) of the p40 positive subgroup ranked third in the TMB list, suggesting the applicability of immunotherapy for PSC. The study established a new subtyping method of PSC based on IHC results and reveals three subgroups with distinct molecular features, providing evidence for refined stratification in the treatment of PSC.

Integrated analysis of single-cell and bulk RNA-sequencing identifies a signature based on B cell marker genes to predict prognosis and immunotherapy response in lung adenocarcinoma.

As an essential component of the tumor microenvironment, B cells exist in all stages of tumor and exert important roles in anti-tumor immunity and shaping tumor development. We aimed to explore the expression profile of B cell marker genes and construct a prognostic signature based on these genes in Lung adenocarcinoma (LUAD). A total of 1268 LUAD patients from different cohorts were enrolled in this study. We performed an analysis of single-cell RNA-sequencing (scRNA-seq) data from Gene expression omnibus (GEO) database to identify B cell marker genes in LUAD. TCGA database was used to construct signature, and six cohorts from GEO database were used for validation. We also investigated the association between this signature and immunotherapy response. Based on 258 B cell marker genes identified by scRNA-seq analysis, a nine-gene signature was constructed for prognostic prediction in TCGA dataset, which classified patients into high-risk and low-risk groups according to overall survival. The multivariate analysis demonstrated that the signature was an independent prognostic factor. The signature's predictive power was verified in other six independent cohorts and different clinical subgroups. Analysis of immune profiles showed that high-risk groups presented discriminative immune-cell infiltrations and immune-suppressive states. More importantly, risk scores of the signature were closely correlated with PD-L1, tumor mutation burden, neoantigens, and tumor immune dysfunction and exclusion score. Our study proposed a novel prognostic signature based on B cell marker genes for LUAD patients. The signature could effectively indicate LUAD patients' survival and serve as a predictor for immunotherapy.

Immune-related histologic phenotype in pretreatment tumour biopsy predicts the efficacy of neoadjuvant anti-PD-1 treatment in squamous lung cancer.

BACKGROUND: Although neoadjuvant anti-PD-1 immunotherapies have shown good efficacy in non-small cell lung cancer (NSCLC) patients, there is still a lack of effective predictive markers. We aimed to develop a pretreatment histologic scoring system to predict the efficacy of neoadjuvant immunotherapy. METHODS: One hundred forty NSCLC cases were evaluated in this study. Initially, surgical specimens from 31 squamous cell lung cancer patients treated with neoadjuvant anti-PD-1 therapy and their eligible paired pretreatment biopsies were used for pathologic evaluation and developing the pretreatment scoring system, immune-related histologic phenotype assessment criteria (irHPC). Three trained pathologists independently scored the haematoxylin-eosin (HE) slides of the pretreatment tumour biopsies according to irHPC. The follow-up was from 07 March 2018 to 31 December 2021, mainly focusing on disease-free survival (DFS) and overall survival (OS). Second, 109 biopsies of lung squamous cell carcinoma were evaluated to explore the relationship between eosinophils and PD-L1 expression. RESULTS: Superior 2-year DFS rates and 2-year OS rates were observed in patients who achieved major pathologic response (MPR) (MPR vs. non-MPR: 92.9% vs. 78.6%; 100.0% vs. 93.3%). Whether necrosis was included in the calculation of the per cent of residual viable tumour (%RVT) or not had almost no effect on the consistency of pathologic assessment and the histological response grouping. The interpathologist variability in assessing %RVT with immune-activated phenotype was not statistically significant (P = 0.480). Four immune-related features of pretreatment biopsies were included for calculating the predictive score. The trained pathologist accurately predicted most cases according to irHPC. For interobserver reproducibility using "2 points" as the cutoff, the overall per cent agreement was 77.8%. The reliability between pathologists for a binary tumour evaluation showed "moderate" agreement (κ = 0.54). Patients with scores ≥ 2 points tended to have better 2-year DFS rates and 2-year OS rates than those with scores < 2 points (85.7% vs. 71.4%; 100.0% vs. 87.5%). CONCLUSIONS: The irHPC scoring system reflecting the preexisting immune response could be used to predict pathologic response to neoadjuvant immunotherapy, possibly further predicting the long-term prognosis, but larger trials are needed for verification.

Reliability analysis of exonic-breakpoint fusions identified by DNA sequencing for predicting the efficacy of targeted therapy in non-small cell lung cancer.

BACKGROUND: Diverse genomic breakpoints of fusions that localize to intronic, exonic, or intergenic regions have been identified by DNA next-generation sequencing (NGS), but the role of exonic breakpoints remains elusive. We investigated whether exonic-breakpoint fusions could predict matched targeted therapy efficacy in non-small cell lung cancer (NSCLC). METHODS: NSCLC samples were analyzed by DNA NGS, RNA NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization. RESULTS: Using DNA NGS, kinase fusions were identified in 685 of 7148 (9.6%) NSCLCs, with 74 harboring exonic-breakpoint fusions, mostly anaplastic lymphoma kinase (ALK) fusions. RNA NGS and IHC revealed that 11 of 55 (20%) exonic-breakpoint fusions generated no aberrant transcript/protein, possibly due to open reading frame disruption or different gene transcriptional orientations. Four cases of genomic-positive but RNA/protein-negative fusions were treated with matched targeted therapy, but progressive disease developed within 2 months. Nevertheless, 44 of 55 (80%) exonic-breakpoint fusions produced chimeric transcripts/proteins, possibly owing to various alternative splicing patterns, including exon skipping, alternative splice site selection, and intron retention. Most of these genomic- and RNA/protein-positive fusion cases showed a clinical response to matched targeted therapy. Particularly, there were no differences in objective response rate (P = 0.714) or median progression-free survival (P = 0.500) between intronic-breakpoint (n = 56) and exonic-breakpoint ALK fusion subtypes (n = 11) among ALK RNA/protein-validated patients who received first-line crizotinib. CONCLUSIONS: Exonic-breakpoint fusions may generate in-frame fusion transcripts/proteins or not, and thus are unreliable for predicting the efficacy of targeted therapy, which highlights the necessity of implementing RNA or protein assays for functional validation in exonic-breakpoint fusion cases.

Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions.

Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA' performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients' samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.

High PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor in urothelial carcinoma.

Aims: To explore the prognostic value of high PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (TIIC) in urothelial carcinoma (UC). Patients & methods: 162 UC specimens were evaluated for PD-L1 expression on TIIC and TC with the SP263 assay. High PD-L1 expression was defined as ≥25% staining. Results: High PD-L1 expression on TC in UC patients with stage T1-4 disease was associated with poor overall survival. However, high PD-L1 expression on TIIC in UC patients with stage T1-4 disease revealed favorable disease-free and overall survival; more significant differences were observed in patients with stages T2-4. Multivariate analysis revealed that high PD-L1 expression on TIIC was an independent prognostic predictor for better disease-free and overall survival. Conclusion: High PD-L1 expression on TIIC, but not on TC, is a favorable prognostic factor in UC.

Lay abstract Bladder cancer is the tenth most common form of cancer worldwide, and urothelial carcinoma is the most common type of bladder cancer. PD-L1 is a protein that can be expressed on the surface of many tissue types, including tumor cells (TC) and tumor-infiltrating immune cells (TIIC). PD-L1 can help the tumor evade the body's natural immune defense system. The expression of PD-L1 not only related to the response of immunotherapy but is also associated with the prognosis in bladder cancer. However, the prognostic significance of PD-L1 expression on TC and TIIC remains controversial. This study drew a conclusion that high PD-L1 expression on TIIC, but not on TC, is a favorable prognostic factor in urothelial carcinoma.

Distinct clinical phenotype and genetic testing strategy for Lynch syndrome in China based on a large colorectal cancer cohort.

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) predisposition syndrome. We performed a large-scale study to assess a screening strategy for identifying LS in Chinese CRC patients in routine clinical testing. A total of 4,195 eligible CRCs were universally screened. Then, 8.7% of CRCs were detected with dMMR. The incidence of LS was 2.7% (115 of 4,195) in this cohort; among patients over 70 years of age, only 0.3% (2 of 678) were diagnosed as LS. Then, 17.4% of LS cases showed large genomic deletions/duplications. LS probands developed CRCs predominantly at proximal colon location. The frequency of BRAF V600E mutation among Chinese CRCs was significantly lower than that among Western populations, and MLH1 promoter methylation significantly improved the efficiency of genetic screening for LS among MLH1-deficient patients. A comprehensive molecular testing strategy that includes detection of large genomic rearrangements is imperative for the diagnosis of LS. Among CRC patients aged 70 years or younger, a selective strategy for LS screening might be considered for routine clinical testing.

The efficiency of 18F-FDG PET-CT for predicting the major pathologic response to the neoadjuvant PD-1 blockade in resectable non-small cell lung cancer.

PURPOSE: Investigate whether 18F-FDG PET-CT has the potential to predict the major pathologic response (MPR) to neoadjuvant sintilimab in resectable NSCLC patients, and the potential of sifting patients who probably benefit from immunotherapy. METHODS: Treatment-naive patients with resectable NSCLC (stage IA-IIIB) received two cycles of sintilimab (200 mg, intravenously, day 1 and 22). Surgery was performed between day 29 and 43. PET-CT was obtained at baseline and prior to surgery. The following lean body mass-corrected metabolic parameters were calculated by PET VCAR: SULmax, SULpeak, MTV, TLG, ΔSULmax%, ΔSULpeak%, ΔMTV%, ΔTLG%. PET responses were classified using PERCIST. The above metabolic information on FDG-PET was correlated with the surgical pathology. (Registration Number: ChiCTR-OIC-17013726). RESULTS: Thirty-six patients received 2 doses of sintilimab, all of whom underwent PET-CT twice and had radical resection (35) or biopsy (1). MPR occurred in 13 of 36 resected tumors (36.1%, 13/36). The degree of pathological regression was positively correlated with SULmax (p = 0.036) of scan-1, and was negatively correlated with all metabolic parameters of scan-2, and the percentage changes of the metabolic parameters after neoadjuvant therapy (p < 0.05). According to PERCIST, 13 patients (36.1%, 13/36) showed partial metabolic response (PMR), 21 (58.3%, 21/36) had stable metabolic disease, and 2 (5.6%, 2/36) had progressive metabolic disease (PMD). There was a significant correlation between the pathological response and the PET responses which were classified using PERCIST. All (100.0%) the PMR (ΔSULpeak% < - 30.0%) tumors showed MPR. CONCLUSIONS: 18F-FDG PET-CT can predict MPR to neoadjuvant sintilimab in resectable non-small cell lung cancer.

DLBCL with amplification of JAK2/PD-L2 exhibits PMBCL-like CNA pattern and worse clinical outcome resembling those with MYD88 L265P mutation.

BACKGROUND: Recently, copy number alteration (CNA) of 9p24.1 were demonstrated in 10% of diffuse large b-cell lymphoma (DLBCL), with gene expression and mutation profiles that were similar to those of primary mediastinal large B-cell lymphoma (PMBCL). However, their CNA-based profile and clinical impact still remain unclear. METHODS: Multiplex ligation-dependent probe amplification were employed to investigate the prevalence of JAK2/PD-L2 amplification in DLBCL and their CNA-based pattern of driver genes. The clinical outcome and characteristics were also analyzed. RESULTS: Using unsupervised hierarchical clustering, a small group of DLBCL (10.5%, 8/76) was clustered together with PMBCL as Cluster_2, demonstrating amplification of JAK2 (100%,8/8) and PD-L2 (75.0%,6/8). This subgroups of DLBCL demonstrated significant higher expression of PD-L1 than those with MYD88 L265P mutation(p = 0.024). And they exhibited dismal OS and PFS as compared with DLBCL_others(p = 0.003 and 0.001, respectively), which is similar to DLBCL with MYD88 L265P mutation. CONCLUSIONS: DLBCL with amplification of JAK2/PD-L2 exhibits CNA pattern that is similar to PMBCL, and demonstrates unfavorable clinical outcome that resembles those with MYD88 L265P mutation. It is essential to identify this subgroup of DLBCL who may acquire more benefits from the JAK2 and PD-L1 signaling inhibition.