Comprehensive characterization of PKHD1 mutation in human colon cancer.

INTRODUCTION: The PKHD1 (Polycystic Kidney and Hepatic Disease 1) gene is essential for producing fibrocystin or polyductin, which is crucial in various cellular functions. Mutations in PKHD1 have been found to be involved in the development and progression of colorectal cancer (CRC). Along with APC, TP53, and KRAS, PKHD1 is one of the most frequently mutated genes in CRC. PKHD1 expression is governed by the Wnt/PCP pathway, often dysregulated in CRC. Targeting this pathway, crucial for CRC progression, could unveil potential therapeutic strategies for colon cancer treatment. METHODS: This study examined an in-house dataset of 3702 colon cancer samples, analyzing mutation landscapes, clinical features, tumor mutational burden (TMB), microsatellite instability (MSI), and chromosomal instability (CIN) score. For the survival analysis of PKHD1 patients, survival data of 436 colon adenocarcinoma samples were obtained from TCGA dataset. Additionally, 433 samples from TCGA with RNA-seq data were used for the assessment of immune cell infiltration and gene set enrichment analysis. RESULTS: Polycystic Kidney and Hepatic Disease 1 mutation was detected in 424 colon cancer patients from our in-house cohort and was associated with increased TMB, higher MSI, and lower CIN score. Importantly, within the TCGA dataset, PKHD1 mutations were identified as an independent prognostic factor, not merely correlated with established prognostic biomarkers, and were associated with poorer overall survival outcomes. In terms of immune response, these mutations correlated with increased enrichment scores for 12 immune cell types, including B cell plasma, macrophages, and naive CD4+ T cells. Additionally, interferon alpha and interferon-gamma gene sets were significantly down-regulated in patients with PKHD1 mutations (FDA q-value < 0.1). CONCLUSIONS: Overall, these findings suggest that PKHD1 may be a potential biomarker for the prognosis of colon cancer and provide some insight for personalized immunotherapy.

Enhanced Detection of Landmark Minimal Residual Disease in Lung Cancer Using Cell-free DNA Fragmentomics.

Currently, approximately 30%-55% of the patients with non-small cell lung cancer (NSCLC) develop recurrence due to minimal residual disease (MRD) after receiving surgical resection of the tumor. This study aims to develop an ultrasensitive and affordable fragmentomic assay for MRD detection in patients with NSCLC. A total of 87 patients with NSCLC, who received curative surgical resections (23 patients relapsed during follow-up), enrolled in this study. A total of 163 plasma samples, collected at 7 days and 6 months postsurgical, were used for both whole-genome sequencing (WGS) and targeted sequencing. WGS-based cell-free DNA (cfDNA) fragment profile was used to fit regularized Cox regression models, and leave-one-out cross-validation was further used to evaluate models' performance. The models showed excellent performances in detecting patients with a high risk of recurrence. At 7 days postsurgical, the high-risk patients detected by our model showed an increased risk of 4.6 times, while the risk increased to 8.3 times at 6 months postsurgical. These fragmentomics determined higher risk compared with the targeted sequencing-based circulating mutations both at 7 days and 6 months postsurgical. The overall sensitivity for detecting patients with recurrence reached 78.3% while using both fragmentomics and mutation results from 7 days and 6 months postsurgical, which increased from the 43.5% sensitivity by using only the circulating mutations. The fragmentomics showed great sensitivity in predicting patient recurrence compared with the traditional circulating mutation, especially after the surgery for early-stage NSCLC, therefore exhibiting great potential to guide adjuvant therapeutics. Significance: The circulating tumor DNA mutation-based approach shows limited performance in MRD detection, especially for landmark MRD detection at an early-stage cancer after surgery. Here, we describe a cfDNA fragmentomics-based method in MRD detection of resectable NSCLC using WGS, and the cfDNA fragmentomics showed a great sensitivity in predicting prognosis.

Letter to the Editor: An ultra-sensitive assay using cell-free DNA fragmentomics for multi-cancer early detection.

Early detection can benefit cancer patients with more effective treatments and better prognosis, but existing early screening tests are limited, especially for multi-cancer detection. This study investigated the most prevalent and lethal cancer types, including primary liver cancer (PLC), colorectal adenocarcinoma (CRC), and lung adenocarcinoma (LUAD). Leveraging the emerging cell-free DNA (cfDNA) fragmentomics, we developed a robust machine learning model for multi-cancer early detection. 1,214 participants, including 381 PLC, 298 CRC, 292 LUAD patients, and 243 healthy volunteers, were enrolled. The majority of patients (N = 971) were at early stages (stage 0, N = 34; stage I, N = 799). The participants were randomly divided into a training cohort and a test cohort in a 1:1 ratio while maintaining the ratio for the major histology subtypes. An ensemble stacked machine learning approach was developed using multiple plasma cfDNA fragmentomic features. The model was trained solely in the training cohort and then evaluated in the test cohort. Our model showed an Area Under the Curve (AUC) of 0.983 for differentiating cancer patients from healthy individuals. At 95.0% specificity, the sensitivity of detecting all cancer reached 95.5%, while 100%, 94.6%, and 90.4% for PLC, CRC, and LUAD, individually. The cancer origin model demonstrated an overall 93.1% accuracy for predicting cancer origin in the test cohort (97.4%, 94.3%, and 85.6% for PLC, CRC, and LUAD, respectively). Our model sensitivity is consistently high for early-stage and small-size tumors. Furthermore, its detection and origin classification power remained superior when reducing sequencing depth to 1× (cancer detection: ≥ 91.5% sensitivity at 95.0% specificity; cancer origin: ≥ 91.6% accuracy). In conclusion, we have incorporated plasma cfDNA fragmentomics into the ensemble stacked model and established an ultrasensitive assay for multi-cancer early detection, shedding light on developing cancer early screening in clinical practice.

Correlation of PD-L1 Expression with Clinicopathological and Genomic Features in Chinese Non-Small-Cell Lung Cancer.

Programmed cell death 1 ligand 1 (PD-L1) has been approved as predictive biomarker for non-small-cell lung cancer (NSCLC) patients treated with PD-(L)1 blockade therapy. The clinical/genomic features associated with PD-L1 are not well studied. Genomic profiling of tumor biopsies from 883 Chinese NSCLC patients was performed by targeted next-generation sequencing. Immunohistochemical analysis was conducted to evaluate PD-L1 expression levels using antibodies Dako 22C3 and 28-8, respectively. Our study showed distinct correlation between PD-L1 expression and clinical/genomic characteristics when using different PD-L1 antibodies and in different histological subtypes including adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively. PD-L1 high expression (22C3) was associated with male and lymph node metastasis only in ADC patients. Furthermore, mutations of TP53 and KRAS, KIF5B-RET fusion, copy number gains of PD-L1 and PD-L2, and arm-level amplifications of chr.12p were significantly associated with PD-L1 positive status in ADC patients. For SCC patients, the gain of EGFR and MDM2 and loss of PTPRD were negatively associated with PD-L1 expression. We also compared our results with other studies and found conflicting results presumably because of the multiplicity of antibody clones and platforms, the difference of cutoffs for assigning PD-L1 expression levels, and the variation in study populations. Our study can help to understand the utility and validity of PD-L1 as biomarker of response to immune checkpoint inhibitors.

Ultrasensitive and affordable assay for early detection of primary liver cancer using plasma cell-free DNA fragmentomics.

BACKGROUND AND AIMS: Early detection of primary liver cancer (PLC), including HCC, intrahepatic cholangiocarcinoma (ICC), and combined HCC-ICC (cHCC-ICC), is essential for patients' survival. This study aims to develop an accurate and affordable method for PLC early detection and differentiating ICC from HCC using plasma cell-free DNA (cfDNA) fragmentomic profiles. APPROACH AND RESULTS: Whole-genome sequencings (WGS) were performed using plasma cfDNA samples from 192 patients with PLC (159 HCC, 26 ICC, 7 cHCC-ICC) and 170 noncancer controls (including 53 liver cirrhosis [LC] or HBV-positive) enrolled in the training cohort. An ensembled stacked model for PLC detection was constructed using the training cohort. The model performance was assessed in an independent test cohort (189 patients with PLC [157 HCC, 26 ICC, 6 cHCC-ICC], 164 noncancer controls [including 51 LC/HBV]). Our model showed excellent performance for cancer detection in the test cohort (AUC: 0.995, 96.8% sensitivity at 98.8% specificity). It showed excellent sensitivities in detecting early-stage PLC (I: 95.9%, II: 97.9%), small tumors (≤3 cm: 98.2%), and HCC (96.2%) or ICC (100%). The AUC for distinguishing PLC from LC/HBV reached 0.985 (96.8% specificity at 96.1% specificity). Promisingly, our model maintained consistent performances during the downsampling process, even using 1X coverage data (AUC: 0.994, 93.7% sensitivity at 98.8% specificity). A separate model showed potential for distinguishing ICC from HCC (AUC: 0.776). CONCLUSIONS: Our model, outperforming previous reports at a lower cost by solely using low-coverage WGS data, exhibits excellent clinical potential for ultrasensitive and affordable detection of PLC and its subtypes.

Multi-dimensional fragmentomic assay for ultrasensitive early detection of colorectal advanced adenoma and adenocarcinoma.

Previous studies on liquid biopsy-based early detection of advanced colorectal adenoma (advCRA) or adenocarcinoma (CRC) were limited by low sensitivity. We performed a prospective study to establish an integrated model using fragmentomic profiles of plasma cell-free DNA (cfDNA) for accurately and cost-effectively detecting early-stage CRC and advCRA. The training cohort enrolled 310 participants, including 149 early-stage CRC patients, 46 advCRA patients and 115 healthy controls. Plasma cfDNA samples were prepared for whole-genome sequencing. An ensemble stacked model differentiating healthy controls from advCRA/early-stage CRC patients was trained using five machine learning models and five cfDNA fragmentomic features based on the training cohort. The model was subsequently validated using an independent test cohort (N = 311; including 149 early-stage CRC, 46 advCRA and 116 healthy controls). Our model showed an area under the curve (AUC) of 0.988 for differentiating advCRA/early-stage CRC patients from healthy individuals in an independent test cohort. The model performed even better for identifying early-stage CRC (AUC 0.990) compared to advCRA (AUC 0.982). At 94.8% specificity, the sensitivities for detecting advCRA and early-stage CRC reached 95.7% and 98.0% (0: 94.1%; I: 98.5%), respectively. Promisingly, the detection sensitivity has reached 100% and 97.6% in early-stage CRC patients with negative fecal occult or CEA blood test results, respectively. Finally, our model maintained promising performances (AUC: 0.982, 94.4% sensitivity at 94.8% specificity) even when sequencing depth was down-sampled to 1X. Our integrated predictive model demonstrated an unprecedented detection sensitivity for advCRA and early-stage CRC, shedding light on more accurate noninvasive CRC screening in clinical practice.

A comprehensive analysis of gorilla-specific LINE-1 retrotransposons.

BACKGROUND: Long interspersed element-1 (LINE-1 or L1) is the most abundant retrotransposons in the primate genome. They have approximately 520,000 copies and make up ~ 17% of the primate genome. Full-length L1s can mobilize to a new genomic location using their enzymatic machinery. Gorilla is the second closest species to humans after the chimpanzee, and human-gorilla split 7-12 million years ago. The gorilla genome provides an opportunity to explore primate origins and evolution. OBJECTIVE: L1s have contributed to genome diversity and variations during primate evolution. This study aimed to identify gorilla-specific L1s using a more recent version of the gorilla reference genome (Mar. 2016 GSMRT3/gorGor5). METHODS: We collected gorilla-specific L1 candidates through computational analysis and manual inspection. L1Xplorer was used to identify whether full-length gorilla-specific L1s were intact. In addition, to determine the level of sequence conservation between intact fulllength gorilla-specific L1s, two ORFs of intact L1s were aligned with the L1PA2 consensus sequence. RESULTS: 2002 gorilla-specific L1 candidates were identified through computational analysis. Among them, we manually inspected 1,883 gorilla-specific L1s, among which most of them belong to the L1PA2 subfamily and 12 were intact L1s that could influence genomic variations in the gorilla genome. Interestingly, the 12 intact full-length gorilla-specific L1s have 14 highly conserved nonsynonymous mutations, including 6 mutations and 8 mutations in ORF1 and ORF2, respectively. In comparison to the intact full-length chimpanzee-specific L1s and human-specific hot-L1s, two of these in ORF1 (L256F and E293G) were shown as gorilla-specific nonsynonymous mutations. CONCLUSION: The gorilla-specific L1s may have had significantly affected the gorilla genome to compose a genome different form that of other primates during primate evolution.

Identification of DNA methylation biomarkers for risk of liver metastasis in early-stage colorectal cancer.

BACKGROUND: Liver metastases can occur even in CRC patients who underwent curative surgery. While evidence suggested that adjuvant chemotherapy can help to reduce the occurrence of liver metastases for certain patients, it is not a recommended routine as the side effects outweigh the potential benefits, especially in Stage II CRC patients. This study aims to construct a model for predicting liver metastasis risk using differential methylation signals in primary CRC tumors, which can facilitate the decision for adjuvant chemotherapy. METHODS: Fifty-nine stage I/II and IV CRC patients were enrolled. Primary tumor, adjacent normal tissue, and metastatic tumor tissues were subject to targeted bisulfite sequencing for DNA methylation. The Least Absolute Shrinkage and Selection Operator (LASSO) algorithm was used to identify potential DMRs for predicting liver metastasis of CRC. RESULTS: We identified a total of 241,573 DMRs by comparing the DNA methylation profile of primary tumors of stage II patients who developed metastasis to those who were metastasis-free during the follow up period. 213 DMRs were associated with poor prognosis, among which 182 DMRS were found to be hypermethylated in the primary tumor of patients with metastases. Furthermore, by using the LASSO regression model, we identified 23 DMRs that contributed to a high probability of liver metastasis of CRC. The leave-one-out cross validation (LOOCV) was used to evaluate model predictive performance at an AUC of 0.701. In particular, 7 out of those 23 DMRs were found to be in the promoter region of genes that were previously reported prognostic biomarkers in diverse tumor types, including TNNI2, PAX8, GUF1, KLF4, EVI2B, CEP112, and long non-coding RNA AC011298. In addition, the model was also able to distinguish metastases of different sites (liver or lung) at an AUC of 0.933. CONCLUSION: We have identified DNA methylation biomarkers associated with the risk of cancer liver metastasis in early-stage CRC patients. A risk prediction model based on those epigenetic markers was proposed for outcome assessment.

Copy number loss in granzyme genes confers resistance to immune checkpoint inhibitor in nasopharyngeal carcinoma.

BACKGROUND: Anti-programmed death (PD)-1 therapy has recently been used in recurrent or metastatic (R/M) nasopharyngeal carcinoma (NPC). The long-term survival and its biomarkers responding to anti-PD-1 treatment in patients with R/M NPC remain unclear. METHODS: Patients with R/M NPC were enrolled between March 2016 and January 2018 from two phase I clinical trials. The median follow-up period was 24.7 months. Eligible patients progressed on standard chemotherapy had measurable disease by Response Evaluation Criteria in Solid Tumor V.1.1. Non-obligatory contemporaneous tumor samples were collected for whole-exome sequencing. The primary outcome was objective response rate (ORR). Duration of response (DOR), progression-free survival (PFS), and overall survival (OS) were secondary outcomes assessed in all patients. RESULTS: Among 124 evaluable patients, anti-PD-1 therapy achieved an ORR of 29.8% and a durable clinical benefit rate of 60.5%. The median OS (mOS) was 17.1 months (95% CI 14.2 to 24.7), median PFS (mPFS) was 3.8 months (95% CI 3.4 to 6.0), and median DOR was 9.5 months. Significant OS benefit from treatment was observed in patients without liver metastasis (23.8 vs 13.3 months, p=0.006). Copy number deletion in genes encoding granzyme B or granzyme H (GZMB/H) was associated with poor treatment outcome (mPFS altered vs wildtype: 1.7 vs 3.6 months, p=0.03; mOS altered vs wildtype: 10.1 vs 18 months, p=0.012). CONCLUSIONS: Anti-PD-1 treatment provided promising clinical benefit in pretreated patients with R/M NPC. Copy number loss in either GZMB or GZMH genes was associated with reduced survival.

L1 retrotransposons exploit RNA m6A modification as an evolutionary driving force.

L1 retrotransposons can pose a threat to genome integrity. The host has evolved to restrict L1 replication. However, mechanisms underlying L1 propagation out of the host surveillance remains unclear. Here, we propose an evolutionary survival strategy of L1, which exploits RNA m6A modification. We discover that m6A 'writer' METTL3 facilitates L1 retrotransposition, whereas m6A 'eraser' ALKBH5 suppresses it. The essential m6A cluster that is located on L1 5' UTR serves as a docking site for eukaryotic initiation factor 3 (eIF3), enhances translational efficiency and promotes the formation of L1 ribonucleoprotein. Furthermore, through the comparative analysis of human- and primate-specific L1 lineages, we find that the most functional m6A motif-containing L1s have been positively selected and became a distinctive feature of evolutionarily young L1s. Thus, our findings demonstrate that L1 retrotransposons hijack the RNA m6A modification system for their successful replication.

Alu master copies serve as the drivers of differential SINE transposition in recent primate genomes.

Alu elements, averaging ~300bp in length, are a family of primate-specific short intersperse nuclear elements (SINEs) with more than one million copies and contributing to ~11% of primate genomes. Despite mostly being shared among primates, our recent study revealed highly differential recent Alu transposition among the genomes of primates from Hominidae and Cercopithecidae families. To understand the underlying mechanism, we analyzed six primate genomes and revealed species- and lineage-specific Alu profile exclusively defined by AluY composition. Among all Alus from the 6 genomes, we identified 5401 Alu master copies with 99% being from the AluY subfamily. The numbers of Alu master copies are positively correlated to the number of AluY elements in the genomes with the baboon genome having the largest number of most recent Alu master copies at high activities, while the crab-eating macaque genome having a low number of Alu master copies with low activity. Furthermore, the expression level of Alu master copies is positively correlated with their transposition activity. Our results support the concept that Alu transposition in primate genomes is driven by a small number of master copies, the number and relative activity of which contribute to the differential Alu transposition in recent primate genomes.

Comparative Genomics Analysis Reveals High Levels of Differential Retrotransposition among Primates from the Hominidae and the Cercopithecidae Families.

Mobile elements (MEs), making ∼50% of primate genomes, are known to be responsible for generating inter- and intra-species genomic variations and play important roles in genome evolution and gene function. Using a bioinformatics comparative genomics approach, we performed analyses of species-specific MEs (SS-MEs) in eight primate genomes from the families of Hominidae and Cercopithecidae, focusing on retrotransposons. We identified a total of 230,855 SS-MEs, with which we performed normalization based on evolutionary distances, and we also analyzed the most recent SS-MEs in these genomes. Comparative analysis of SS-MEs reveals striking differences in ME transposition among these primate genomes. Interesting highlights of our results include: 1) the baboon genome has the highest number of SS-MEs with a strong bias for SINEs, while the crab-eating macaque genome has a sustained extremely low transposition for all ME classes, suggesting the existence of a genome-wide mechanism suppressing ME transposition; 2) while SS-SINEs represent the dominant class in general, the orangutan genome stands out by having SS-LINEs as the dominant class; 3) the human genome stands out among the eight genomes by having the largest number of recent highly active ME subfamilies, suggesting a greater impact of ME transposition on its recent evolution; and 4) at least 33% of the SS-MEs locate to genic regions, including protein coding regions, presenting significant potentials for impacting gene function. Our study, as the first of its kind, demonstrates that mobile elements evolve quite differently among these primates, suggesting differential ME transposition as an important mechanism in primate evolution.

A comprehensive analysis of the Baboon-specific full-length LINE-1 retrotransposons.

BACKGROUND: Long interspersed elements-1 (LINE-1s or L1s) and Alu elements are most successful retrotransposons that have generated genetic diversity and genomic fluidity in the primate genome. They account for ~ 27.7% of the primate genome. Interestingly, a previous study has shown that the retrotransposition rate of Alu elements is nine times higher in baboons than in humans. OBJECTIVE: The expansion of Alu copies could be dependent on the activity of L1-encoded proteins. Thus, we aimed to investigate full-length baboon-specific L1s and characterize structurally and functionally intact baboon-specific L1s (ORF1p/ORF2p and ORF2p only) that could induce trans-mobilization of Alu elements in the baboon genome. RESULTS: A total of 673 baboon-specific L1 candidates (> 4 kb) were identified through the comparative genomic analysis. Applying the baboon-specific correction value obtained from the experimental validation, it demonstrated that approximately 446 baboon-specific L1s (> 4 kb) were present in the baboon reference genome (papAnu2). In addition, we observed phylogenetic relationship of the baboon-specific L1s through the neighbor-joining method and they diverged from the L1PA6 consensus sequence. Finally, we identified 36 full-length baboon-specific L1s that were intact both ORF1p and ORF2p. CONCLUSION: The number of baboon-specific full-length L1s is fewer than the number of human-specific full-length L1s. Therefore, there is possibility that the "L1 master gene" or "L1 source gene" is more abundant in the baboon genome, or that in trans retrotransposition activity of baboon-specific L1s is relatively stronger than in the other genomes.

A comprehensive analysis of chimpanzee (Pan troglodytes)-specific LINE-1 retrotransposons.

Long interspersed element-1 (LINE-1 or L1) is one of the most abundant retrotransposons in the primate genomes and has contributed to their genome diversity and variations during the primate evolution. Among primate L1 subfamilies, L1Pt subfamilies include Pan troglodytes-specific L1s. L1Pt elements have been successfully expanded in the chimpanzee genome since the divergence of human and chimpanzee lineages. However, only a few full-length L1Pt copies were previously detected in the chimpanzee genome due to incomplete chimpanzee reference genome sequences at the time. In the present study, we aimed to identify chimpanzee-specific L1s using the most recent version of the chimpanzee reference genome (May 2016, panTro5). We identified a total of 3731 chimpanzee-specific L1s. This is much more than previously reported chimpanzee-specific L1 copies. Among these, 223 are full-length (>6 kb), and we annotated their subfamilies and examined their retrotransposition-competency. The result showed that there are two L1Pt subfamilies in the chimpanzee genome, and the nine structurally intact elements belong to L1Pt-1, L1Pt-2, and L1PA2 subfamilies. In addition, we found that the intact full-length L1 group showed significantly higher L1 expression level than the non-intact full-length L1 group using limited RNA-seq data. It is interesting to notice that the number of retrotransposition-competent elements is much less in the chimpanzee genome than that in the human genome. In conclusion, there is increasing evidence to indicate that chimpanzee-specific L1s have changed the chimpanzee genome, causing genomic difference from other primate genomes.

Mobile elements contribute to the uniqueness of human genome with 15,000 human-specific insertions and 14 Mbp sequence increase.

Mobile elements (MEs) collectively contribute to at least 50% of the human genome. Due to their past incremental accumulation and ongoing DNA transposition, MEs serve as a significant source for both inter- and intra-species genetic and phenotypic diversity during primate and human evolution. By making use of the most recent genome sequences for human and many other closely related primates and robust multi-way comparative genomic approach, we identified a total of 14,870 human-specific MEs (HS-MEs) with more than 8,000 being newly identified. Collectively, these HS-MEs contribute to a total of 14.2 Mbp net genome sequence increase. Several new observations were made based on these HS-MEs, including the finding of Y chromosome as a strikingly hot target for HS-MEs and a strong mutual preference for SINE-R/VNTR/Alu (SVAs). Furthermore, ∼8,000 of these HS-MEs were found to locate in the vicinity of ∼4,900 genes, and collectively they contribute to ∼84 kb sequences in the human reference transcriptome in association with over 300 genes, including protein-coding sequences for 40 genes. In conclusion, our results demonstrate that MEs made a significant contribution to the evolution of human genome by participating in gene function in a human-specific fashion.

Database documentation of retrotransposon insertion polymorphisms.

Retrotransposons constitute more than 40 percent of the human genome with L1, Alu, SVA, and HERVs known to remain active in transposition. Retrotransposition contribute to genetic diversity in the form of retrotransposon insertion polymorphism (RIP) that is defined as the presence or absence of a retrotransposon insertion among human populations at a specific genomic location. So far close to 5000 cases of RIPs have been identified with more than 50 cases associated with disease. A large number of new RIPs are being and to be identified from newly available personal genomes data, making RIPs an important source of genetic variations/mutations that deserve proper documentation. In this review, we discuss the special characteristics of RIPs and the challenges in their compiling and annotating, and we examine the current status of database documentation of RIPs and describe in details the design, data schema, and utilities of dbRIP, which is currently the only database dedicated to the documentation of retrotransposon insertion polymorphism. Some future perspectives and outstanding issues associated with documentation of RIPs are also presented.