Escaping but not the inactive X-linked protein complex coding genes may achieve X-chromosome dosage compensation and underlie X chromosome inactivation-related diseases.

X chromosome dosage compensation (XDC) refers to the process by which X-linked genes acquire expression equivalence between two sexes. Ohno proposed that XDC is achieved by two-fold upregulations of X-linked genes in both sexes and by silencing one X chromosome (X chromosome inactivation, XCI) in females. However, genes subject to two-fold upregulations as well as the underlying mechanism remain unclear. It's reported that gene dosage changes may only affect X-linked dosage-sensitive genes, such as protein complex coding genes (PCGs). Our results showed that in human PCGs are more likely to escape XCI and escaping PCGs (EsP) show two-fold higher expression than inactivated PCGs (InP) or other X-linked genes at RNA and protein levels in both sexes, which suggest that EsP may achieve upregulations and XDC. The higher expressions of EsP possibly result from the upregulations of the single active X chromosome (Xa), rather than escaping expressions from the inactive X chromosome (Xi). EsP genes have relatively high expression levels in humans and lower dN/dS ratios, suggesting that they are likely under stronger selection pressure over evolutionary time. Our study also suggests that SP1 transcription factor is significantly enriched in EsP and may be involved in the up-regulations of EsP on the active X. Finally, human EsP genes in this study are enriched in the toll-like receptor pathway, NF-kB pathway, apoptotic pathway, and abnormal mental, developmental and reproductive phenotypes. These findings suggest misregulations of EsP may be involved in autoimmune, reproductive, and neurological diseases, providing insight for the diagnosis and treatment of these diseases.

Targeted Next-Generation Sequencing Combined With Circulating-Free DNA Deciphers Spatial Heterogeneity of Resected Multifocal Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) has a high risk of recurrence after surgical resection, particularly among patients with multifocal HCC. Genomic heterogeneity contributes to the early recurrence. Few studies focus on targeted next-generation sequencing (tNGS) to depict mutational footprints of heterogeneous multifocal HCC. Methods: We conducted tNGS with an ultra-deep depth on 31 spatially distinct regions from 11 resected multifocal HCC samples. Matched preoperative peripheral circulating-free DNA (cfDNA) were simultaneously collected. Genomic alterations were identified and compared to depict the heterogeneity of multifocal HCC. Results: Widespread intertumoral heterogeneity of driver mutations was observed in different subfoci of multifocal HCC. The identified somatic mutations were defined as truncal drivers or branchy drivers according to the phylogenetic reconstruction. TP53 and TERT were the most commonly altered truncal drivers in multifocal HCC, while the most frequently mutated branchy driver was TSC2. HCC patients with a higher level of intertumoral heterogeneity, defined by the ratio of truncal drivers less than 50%, had a shorter RFS after surgical resection (HR=0.17, p=0.028). Genome profiling of cfDNA could effectively capture tumor-derived driver mutations, suggesting cfDNA was a non-invasive strategy to gain insights of genomic alterations in patients with resected multifocal HCC. Conclusions: Truncal mutations and the level of genomic heterogeneity could be identified by tNGS panel in patients with resected multifocal HCC. cfDNA could serve as a non-invasive and real-time auxiliary method to decipher the intertumoral heterogeneity and identify oncodrivers of multifocal HCC.

Integration of comprehensive genomic profiling, tumor mutational burden, and PD-L1 expression to identify novel biomarkers of immunotherapy in non-small cell lung cancer.

OBJECTIVES: This study aimed to explore the novel biomarkers for immune checkpoint inhibitor (ICI) responses in non-small cell lung cancer (NSCLC) by integrating genomic profiling, tumor mutational burden (TMB), and expression of programmed death receptor 1 ligand (PD-L1). MATERIALS AND METHODS: Tumor and blood samples from 637 Chinese patients with NSCLC were collected for targeted panel sequencing. Genomic alterations, including single nucleotide variations, insertions/deletions, copy number variations, and gene rearrangements, were assessed and TMB was computed. TMB-high (TMB-H) was defined as ≥10 mutations/Mb. PD-L1 positivity was defined as ≥1% tumor cells with membranous staining. Genomic data and ICI outcomes of 240 patients with NSCLC were derived from cBioPortal. RESULTS: EGFR-sensitizing mutations, ALK, RET, and ROS1 rearrangements were associated with lower TMB and PD-L1+/TMB-H proportions, whereas KRAS, ALK, RET, and ROS1 substitutions/indels correlated with higher TMB and PD-L1+/TMB-H proportions than wild-type genotypes. Histone-lysine N-methyltransferase 2 (KMT2) family members (KMT2A, KMT2C, and KMT2D) were frequently mutated in NSCLC tumors, and these mutations were associated with higher TMB and PD-L1 expression, as well as higher PD-L1+/TMB-H proportions. Specifically, patients with KMT2C mutations had higher TMB and PD-L1+/TMB-H proportions than wild-type patients. The median progression-free survival (PFS) was 5.47 months (95% CI 2.5-NA) in patients with KMT2C mutations versus 3.17 months (95% CI 2.6-4.27) in wild-type patients (p = 0.058). Furthermore, in patients with NSCLC who underwent ICI treatment, patients with TP53/KMT2C co-mutations had significantly longer PFS and greater durable clinical benefit (HR: 0.48, 95% CI: 0.24-0.94, p = 0.033). TP53 mutation combined with KMT2C or KRAS mutation was a better biomarker with expanded population benefit from ICIs therapy and increased the predictive power (HR: 0.46, 95% CI: 0.26-0.81, p = 0.007). CONCLUSION: We found that tumors with different alterations in actionable target genes had variable expression of PD-L1 and TMB in NSCLC. TP53/KMT2C co-mutation might serve as a predictive biomarker for ICI responses in NSCLC. IMPLICATIONS FOR PRACTICE: Cancer immunotherapies, especially immune checkpoint inhibitors (ICIs), have revolutionized the treatment of non-small cell lung cancer (NSCLC); however, only a proportion of patients derive durable responses to this treatment. Biomarkers with greater accuracy are highly needed. In total, 637 Chinese patients with NSCLC were analyzed using next-generation sequencing and IHC to characterize the unique features of genomic alterations and TMB and PD-L1 expression. Our study demonstrated that KMT2C/TP53 co-mutation might be an accurate, cost-effective, and reliable biomarker to predict responses to PD-1 blockade therapy in NSCLC patients and that adding KRAS to the biomarker combination creates a more robust parameter to identify the best responders to ICI therapy.

Identification of "regulation of RhoA activity panel" as a prognostic and predictive biomarker for gastric cancer.

RhoA is a member of the RHO family GTPases and is associated with essential functions in gastric cancer. In this study, we identified a gastric cancer biomarker, termed the "regulation of RhoA activity panel" (RRAP). Patients with gastric cancer from The Cancer Genome Atlas database were divided into training (N=160) and validation (N=155) cohorts. A cohort of 109 Chinese gastric cancer patients was utilized as an independent validation. Patients with mutated RRAP showed significantly better overall survival than patients with wild type RRAP. We also analyzed the association between RRAP and the migration capacity, immune-related signatures, and the tumor microenvironment. RRAP-mutant tumors had a significantly lower degree of lymph node metastasis and lower activities of migration-related pathways. These tumors also showed significantly increased immune cell infiltration and cytotoxic activity. Furthermore, two independent patient cohorts who received immune checkpoint blockade therapy were assessed for RRAP mutant status. As expected, for both immunotherapy cohorts, higher response rates to immune checkpoint blockade therapy were observed in patients with RRAP-mutant tumors than in patients with wild type RRAP tumors. Overall, this study indicates that the RRAP gene set is a potential biomarker for gastric cancer prognosis and therapeutic selection.

Intrahepatic Cholangiocarcinoma: Genomic Heterogeneity Between Eastern and Western Patients.

PURPOSE: Intrahepatic cholangiocarcinoma (IHCCA), a global health problem, is increasing in incidence and has differing etiologies worldwide. Next-generation sequencing (NGS) is rapidly being incorporated into the clinical management of biliary cancers. IHCCA is enriched with actionable mutations, and there are several promising targeted therapies under development. NGS data from Asia, where IHCCA is most prevalent, are limited. METHODS: Comprehensive genomic profiling of formalin-fixed paraffin-embedded tumor tissue from 164 Asian and 283 Western patients with IHCCA was performed using NGS. We measured the distribution of DNA repair genetic aberrations (GAs) in IHCCA, along with actionable mutations. Also, we evaluated the association between DNA repair GAs and tumor mutation burden (TMB). Based on the TMB status, patients were distinguished into 3 levels: low (< 6 mut/Mb), intermediate (6-10 mut/Mb), and high (TMB-H; ≥ 10 mut/Mb). RESULTS: Seventy-two percent of Asian patients had ≥ 1 actionable GA, with a significantly higher frequency in KMT2C , BRCA1/2, and DDR2 compared with Western patients (P = .02, .003, and .003, respectively); 60.9% of Western patients had ≥ 1 actionable GA and higher frequency of CDKN2A/B and IDH1/2 GAs (P = .0004 and < .001, respectively). GAs in nuclear factor kappa B pathway regulators and DNA repair genes occurred more frequently in Asian patients (P = .006 and .001, respectively). There was a higher frequency of TMB-H in Asian compared with the Western cohort (12.2% v 5.9%; P = .07). CONCLUSION: A higher burden of DNA repair mutations and frequency of patients with TMB-H in the Asian IHCCA cohort compared with the Western patients suggests a potential role for DNA repair and immune checkpoint inhibitors in the Asian population. Future clinical trials should account for this genetic heterogeneity.

Pembrolizumab combined with lenvatinib as non-first-line therapy in patients with refractory biliary tract carcinoma.

BACKGROUND: A therapeutic strategy involving combined treatment with lenvatinib plus pembrolizumab (LEP) has demonstrated a relatively high antitumor response in several solid tumors; however, the efficacy and safety of LEP in patients with refractory bile tract carcinoma (BTC) remains unknown. METHODS: This is a single-arm study for a preliminary assessment of the efficacy and tolerability of LEP in patients who experienced progression from prior systemic treatments. Pre-treatment tumor tissues were collected to retrospectively evaluate the expression status of PDL1. RESULTS: Thirty-two patients received second-line and above treatment with LEP. Overall, the objective response rate (ORR) was 25%, the disease control rate (DCR) was 78.1%, and the clinical benefit rate (CBR) was 40.5%. The median progression-free survival (PFS) was 4.9 months (95% CI: 4.7-5.2 months), and the median overall survival (OS) was 11.0 months (95% CI: 9.6-12.3 months). For tolerability, no grade 5 serious adverse events (AEs) were reported. All patients had any-grade AEs, and 59.3% of the patients experienced grade 3 AEs, while only 1 patient experienced a grade 4 AE of stomach bleeding. Fatigue was the most common AE, followed by hypertension and elevated aminotransferase levels. Retrospective analysis for PDL1 expression revealed that PDL1 positive tumor cells were associated with improved clinical benefits and survival outcomes. CONCLUSIONS: LEP is a promising alternative as a non-first-line therapeutic regimen for patients with refractory BTC. Furthermore, well-designed prospective clinical trials with a control arm are still needed to obtain more evidences to confirm the efficacy and safety of this particular regimen as well as the role of PDL1 expression.

Chromatin-based reprogramming of a courtship regulator by concurrent pheromone perception and hormone signaling.

To increase fitness, animals use both internal and external states to coordinate reproductive behaviors. The molecular mechanisms underlying this coordination remain unknown. Here, we focused on pheromone-sensing Drosophila Or47b neurons, which exhibit age- and social experience-dependent increase in pheromone responses and courtship advantage in males. FruitlessM (FruM), a master regulator of male courtship behaviors, drives the effects of social experience and age on Or47b neuron responses and function. We show that simultaneous exposure to social experience and age-specific juvenile hormone (JH) induces chromatin-based reprogramming of fruM expression in Or47b neurons. Group housing and JH signaling increase fruM expression in Or47b neurons and active chromatin marks at fruM promoter. Conversely, social isolation or loss of JH signaling decreases fruM expression and increases repressive marks around fruM promoter. Our results suggest that fruM promoter integrates coincident hormone and pheromone signals driving chromatin-based changes in expression and ultimately neuronal and behavioral plasticity.

NFE2L2/KEAP1 Mutations Correlate with Higher Tumor Mutational Burden Value/PD-L1 Expression and Potentiate Improved Clinical Outcome with Immunotherapy.

BACKGROUND: Abnormalities in the KEPA1-NRF2 pathway have a role in cancer progression, metastasis, and resistance to chemo- and radiotherapies. Persistent activation of NRF2 associates with poor prognosis across different cancer types. However, the beneficial therapeutic strategy to harness this pathway in cancer remains unclear. This study aimed to investigate the clinical outcome with immunotherapy in NFE2L2/KEAP1 mutant population. MATERIALS AND METHODS: We investigated the correlation between NFE2L2/KEAP1 mutations and tumor mutational burden (TMB) and programmed death-ligand 1 (PD-L1) expression status to identify the therapeutic vulnerability. For this purpose, relevance analysis with TMB value was performed in 9,040 patients with cancer, and relevance analysis with PD-L1 expression was performed in 3,457 patients. The Memorial Sloan Kettering Cancer Center (MSKCC) database and real-world evidence were used to assess the immunotherapy response in NFE2L2/KEAP1 mutant subsets. RESULTS: NFE2L2/KEAP1 mutations occurred in various cancers, and the highest mutation incidences occurred in lung squamous cell carcinoma (LUSC) at 19.16% (NFE2L2) and 10.31% (KEAP1). We confirmed that higher TMB value and PD-L1 expression were associated with NFE2L2/KEAP1 mutations compared with wild-type, especially in non-small lung cancer. MSKCC database analysis showed the improved survival of patients with NFE2L2/KEAP1 mutant with immunotherapy compared with other treatments (median overall survival 22.52 VS 12.89, p = .0034). Real-world evidence further confirmed the efficacy of immunotherapy in the mutant population. CONCLUSION: Our study revealed that patients with NFE2L2/KEAP1 mutant could achieve improved outcomes from immunotherapy than the other treatments. These findings may broaden the application of immune checkpoint blockade to patients harboring NFE2L2/KEAP1 mutations. IMPLICATIONS FOR PRACTICE: NFE2L2/KEAP1 alterations occur frequently in multiple cancer types and are associated with poor prognosis; however, the efficacious strategy to inhibit this pathway in cancer is poorly understood. This study was designed to analyze the mutational characteristics of NFE2L2/KEAP1 alterations in 9,243 Chinese patients. The highest mutation incidences occurred in lung squamous cell carcinoma at 19.16% (NFE2L2) and 10.31% (KEAP1). Relevance analysis showed the NFE2L2/KEAP1 mutant subsets were associated with higher tumor mutational burden value and programmed death-ligand 1 expression. Clinical data further confirmed NFE2L2/KEAP1 mutations correlate with improved outcome with immunotherapy. These findings suggest the clinical application of immunotherapy in the NFE2L2/KEAP1 mutant population.

Mutation Variants and Co-Mutations as Genomic Modifiers of Response to Afatinib in HER2-Mutant Lung Adenocarcinoma.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-mutant lung cancer remains an orphan of specific targeted therapy. The variable responses to anti-HER2 therapies in these patients prompt us to examine impact of HER2 variants and co-mutations on responses to anti-HER2 treatments in lung cancer. PATIENTS AND METHODS: Patients with stage IV/recurrent HER2-mutant lung cancers identified through next-generation sequencings were recruited from seven hospitals. The study comprised a cohort A to establish the patterns of HER2 variants and co-mutations in lung cancer and a cohort B to assess associations between HER2 variants, co-mutations, and clinical outcomes. RESULTS: The study included 118 patients (cohort A, n = 86; cohort B, n = 32). Thirty-one HER2 variants and 35 co-mutations were detected. Predominant variants were A775_G776insYVMA (49/118, 42%), G778_P780dup (11/118, 9%), and G776delinsVC (9/118, 8%). TP53 was the most common co-mutation (61/118, 52%). In cohort B, objective response rates with afatinib were 0% (0/14, 95% confidence interval [CI], 0%-26.8%), 40% (4/10, 14.7%-72.6%), and 13% (1/8, 0.7%-53.3%) in group 1 (A775_G776insYVMA, n = 14), group 2 (G778_P780dup, G776delinsVC, n = 10), and group 3 (missense mutation, n = 8), respectively (p = .018). Median progression-free survival in group 1 (1.2 months; 95% CI, 0-2.4) was shorter than those in group 2 (7.6 months, 4.9-10.4; hazard ratio [HR], 0.009; 95% CI, 0.001-0.079; p < .001) and group 3 (3.6 months, 2.6-4.5; HR, 0.184; 95% CI, 0.062-0.552; p = .003). TP53 co-mutations (6.317; 95% CI, 2.180-18.302; p = .001) and PI3K/AKT/mTOR pathway activations (19.422; 95% CI, 4.098-92.039; p < .001) conferred additional resistance to afatinib. CONCLUSION: G778_P780dup and G776delinsVC derived the greatest benefits from afatinib among HER2 variants. Co-mutation patterns were additional response modifiers. Refining patient population based on patterns of HER2 variants and co-mutations may help improve the efficacy of anti-HER2 treatment in lung cancer. IMPLICATIONS FOR PRACTICE: Human epidermal growth factor receptor 2 (HER2)-mutant lung cancers are a group of heterogenous diseases with up to 31 different variants and 35 concomitant genomic aberrations. Different HER2 variants exhibit divergent sensitivities to anti-HER2 treatments. Certain variants, G778_P780dup and G776delinsVC, derive sustained clinical benefits from afatinib, whereas the predominant variant, A775_G776insYVMA, is resistant to most anti-HER2 treatments. TP53 is the most common co-mutation in HER2-mutant lung cancers. Co-mutations in TP53 and the PI3K/AKT/mTOR pathway confer additional resistance to anti-HER2 treatments in lung cancer. The present data suggest that different HER2 mutations in lung cancer, like its sibling epidermal growth factor receptor, should be analyzed independently in future studies.

Precision oncology for gallbladder cancer: insights from genetic alterations and clinical practice.

BACKGROUND: Gallbladder cancer (GBC) is an uncommon but highly fatal malignancy, with limited adjuvant therapy. The present study aims to explore the actionable alterations and precision oncology for GBC patients. METHODS: Patients with pathologically confirmed GBC who progressed after first-line systemic treatment were enrolled. Genomic alterations were captured by ultra-deep targeted next-generation sequencing (tNGS). The actionabilities of alterations and the therapeutic regimens were evaluated by a multidisciplinary tumor board (MDTB). RESULTS: Sixty patients with GBC were enrolled and analyzed. tNGS was successfully achieved in all patients. The median tumor mutation burden for GBC patients was 5.4 (range: 0.8-36.74) mutations/Mb, and the most common mutations were in TP53 (73%), CDKN2A (25%) and PIK3CA (20%). The most frequently copy-number altered genes were CDKN2A deletion (11.7%) and ERBB2 amplification (13.3%). 23% of the patients displayed gene fusion; 17 fusion events were identified, and 14 of the 17 fusion events co-occurred with mutations in driver genes. In total, 46 of the 60 (76%) patients were identified as possessing at least one actionable target to proceed precision oncology. CONCLUSIONS: The present study revealed the mutational profile for the clinical practice of precision oncology in GBC patients.

Genomic based analyses reveal unique mutational profiling and identify prognostic biomarker for overall survival in Chinese small-cell lung cancer.

OBJECTIVE: As an aggressive subtype of lung cancer, small-cell lung cancer (SCLC) presents a poor prognosis. Although molecular and clinical characteristics have been established for SCLC, limited investigation has been performed for predicting survival of SCLC patients. METHODS: Genomic alterations were profiled in Chinese SCLC patients (N = 37) using targeted sequencing. Clonal mutation burden (CMB) integrated the number of mutations with the clonal structure of the tumor. Specific pathways involving DNA damage repair (DDR) and cell cycle as well as CMB were studied as potential biomarkers for prognosis of SCLC. RESULTS: TP53 and RB1 gene mutations were the most common alterations (91.9% and 83.8%, respectively), followed by LRP1B, FAM135B, SPTA1, KMT2D, FAT1, and NOTCH3. Survival analysis revealed that mutation status of the DDR pathway was associated with worse OS in our cohort. Importantly, patients with higher CMB exhibited worse OS in our cohort and this observation was successfully validated in the cBioportal cohort. Moreover, multivariate analysis demonstrated CMB as a promising independent prognostic factor for OS in Chinese SCLC patients. Interestingly, patients with loss of function of RB1, validated by immunohistochemistry staining, appeared to have worse OS. CONCLUSIONS: The mutational profiling of Chinese SCLC patients signified an ethnicity dependent component. CMB was firstly found to be associated with OS of Chinese SCLC patients and could be regarded as a prognostic marker for SCLC.

Comprehensive molecular profiling of extrahepatic cholangiocarcinoma in Chinese population and potential targets for clinical practice.

BACKGROUND: Cholangiocarcinoma (CCA) is a diverse group of malignancies arising from the intra- or extrahepatic biliary epithelium and characterized by its late diagnosis and fatal outcome. Extrahepatic cholangiocarcinoma (ECC) accounts for 90% of CCA. However, little is known about the comprehensive genomic alterations of ECC in Chinese population for providing clinical managements especially targeted therapy. METHODS: Comprehensive genomic profiling (CGP) was performed with next generation sequencing panel on paraffin-embedded tumor from a cohort of 80 Chinese ECC patients. RESULTS: The most frequently altered genes were TP53 (68%), KRAS (46%), SMAD4 (22%), ARID1A (20%) and CDKN2A (19%). Mutual exclusivity was observed between multiple genes including ARID1A:TP53, KRAS:LRP1B and NF2:TP53. Genetic alterations with potential therapeutic implications were identified in 43% of patients. The top three actionable alterations include CDKN2A (n=11), BRAF (n=5) and ERBB2 (n=4). Potentially actionable alterations were mainly enriched in the G1-S transition, homologous recombination repair, MAPK/ERK pathway. CONCLUSIONS: This is the largest data set of ECC cases providing a comprehensive view on genetic alterations in Chinese population which differs significantly from a US cohort, and indicates the potential clinical implications for targeted therapies.

Patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species.

Organisms have evolved strikingly parallel phenotypes in response to similar selection pressures suggesting that there may be shared constraints limiting the possible evolutionary trajectories. For example, the behavioral adaptation of specialist Drosophila species to specific host plants can exhibit parallel changes in their adult olfactory neuroanatomy. We investigated the genetic basis of these parallel changes by comparing gene expression during the development of the olfactory system of two specialist Drosophila species to that of four other generalist species. Our results suggest that the parallelism observed in the adult olfactory neuroanatomy of ecological specialists extends more broadly to their developmental antennal expression profiles, and to the transcription factor combinations specifying olfactory receptor neuron (ORN) fates. Additionally, comparing general patterns of variation for the antennal transcriptional profiles in the adult and developing olfactory system of the six species suggest the possibility that specific, non-random components of the developmental programs underlying the Drosophila olfactory system harbor a disproportionate amount of interspecies variation. Further examination of these developmental components may be able to inform a deeper understanding of how traits evolve.