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HPV16 is the most oncogenic type of human papillomaviruses (HPV). Integration of HPV into the human genome is an important mechanism of carcinogenesis but is absent in at least 30% of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors to characterize HPV16 carcinogenesis in the absence of integration. Large tandem arrays of full-length and unique truncated viral genomes integrated into multiple chromosomes were identified in two HPV16+ cell lines. The dispersion of characteristic viral variants to multiple integration sites indicates that viral deletions formed as extrachromosomal DNA (a phenomenon we term HPV superspreading). In addition, we identified an HPV16+ cell line with unintegrated (episomal) DNA that has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). Cytogenetic analysis of this cell line shows intense extrachromosomal HPV staining, including structures resembling double-minute chromosomes. WGS of HPV16+ cervical tumor samples from Latin America revealed that 11 of 20 tumors with only episomal HPV (EP) had intact monomer episomes. The remaining nine EP tumors had multimer and rearranged HPV genomes. The majority (80%) of HPV rearrangements and deletions disrupted the E1 and E2 genes, and EP tumors overexpressed the E6 and E7 viral oncogenes, a similar profile to tumors with HPV integration. Tumors with putative multimer HPV integrations display HPV multimers and concatemers of human and viral sequences. Our data uncovered a novel mechanism for HPV16 to cause cancer without integration through aberrant episomal replication, forming rearranged, mutated, and multimer episomes. Significance: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

The nonbiting midges are tiny insects with a wide distribution around West Lake, China. In this study, the seasonal dynamics of the local dominant midge species was investigated. Phylogenetic trees of this species also were analyzed to discover its evolutionary history. Ultraviolet (UV) light traps were used for surveillance around West Lake in the period from April 2017 to November 2018. External morphological identification was performed using a stereomicroscope. Two genes of adult female midges that encoded the cytochrome oxidase subunit I (COI) and internal transcribed spacer 1 (ITS-1) were sequenced and aligned online using GenBank nucleotide database and the Molecular Evolutionary Genetics Analysis (MEGA) software 10.1.7. According to the field surveillance in 2017–2018, Forcipomyia bikanni Chan and LeRoux (Diptera: Ceratopogonidae) is the representative dominant species of West Lake, with its activity peaking in August and October. The COI and ITS-1 gene sequences of F. bikanni (XH01-5) were 523 bp and 315 bp, respectively. The final phylogenetic tree of the COI implied that F. bikanni (XH01-5) has 97.9% significant similarity with conspecific F. bikanni (LC015045) from the adjacent Fujian Province, China (E-value ≤ 0.1%). Furthermore, the ITS-1 sequences with 98% likelihood showed the highest linkage between F. bikanni (XH01-5) and F. townsvillensis (Taylor) (HM775502-HM775504) with different branches. Thus, the final unrooted phylogenetic trees of COI and ITS-1 are essential tools for reconstructing the evolutionary histories of nonbiting midges.

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HPV16 is the most oncogenic type of human papillomaviruses (HPV). Integration of HPV into the human genome is an important mechanism of carcinogenesis but is absent in at least 30% of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors to characterize HPV16 carcinogenesis in the absence of integration. Large tandem arrays of full-length and unique truncated viral genomes integrated into multiple chromosomes were identified in two HPV16+ cell lines. The dispersion of characteristic viral variants to multiple integration sites indicates that viral deletions formed as extrachromosomal DNA (a phenomenon we term HPV superspreading). In addition, we identified an HPV16+ cell line with unintegrated (episomal) DNA that has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). Cytogenetic analysis of this cell line shows intense extrachromosomal HPV staining, including structures resembling double-minute chromosomes. WGS of HPV16+ cervical tumor samples from Latin America revealed that 11 of 20 tumors with only episomal HPV (EP) had intact monomer episomes. The remaining nine EP tumors had multimer and rearranged HPV genomes. The majority (80%) of HPV rearrangements and deletions disrupted the E1 and E2 genes, and EP tumors overexpressed the E6 and E7 viral oncogenes, a similar profile to tumors with HPV integration. Tumors with putative multimer HPV integrations display HPV multimers and concatemers of human and viral sequences. Our data uncovered a novel mechanism for HPV16 to cause cancer without integration through aberrant episomal replication, forming rearranged, mutated, and multimer episomes.Significance:Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this anti-tumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. While the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.

Supplementary Figures 1-11. Supplementary Figure 1. Sample and analyses diagram. Supplementary Figure 2. Aggregate mutation spectrum of Guatemalan cervical tumors. Supplementary Figure 3. Highly amplified regions of chromosome (Chr.) 2, 6 and 10 in tumor A4. Supplementary Figure 4. Chromosome breakpoints in cervical tumors. Supplementary Figure 5. PIK3CA mutation and pathology. Supplementary Figure 6. Co-occurring mutations in the PIK3CA and PTEN genes. Supplementary Figure 7. Sanger sequence concordance of mutant read percentage. Supplementary Figure 8. Mutation Fraction of PIK3CA mutations. Supplementary Figure 9. Age range associations with PIK3CA mutation and HPV type. Supplementary Figure 10. PIK3CA and HPV E6 and E7 mRNA expression. Supplementary Figure 11. Model of cervical cancer progression.

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Supplementary Tables 1-6. Supplemental Table 1. Primers used in Ion Torrent sequencing of HPV. Supplemental Table 2. Predicted somatic mutations in PIK3CA discovered by targeted sequencing of cervical tumors from Mexico, Venezuela, and Guatemala. Supplementary Table 3. Predicted somatic mutations in 5 candidate genes discovered by targeted sequencing of cervical tumors. Supplementary Table 4. Association of HPV type and PIK3CA mutation. Supplementary Table 5. PIK3CA mutations in histologic subtype in this and other published studies Supplementary Table 6. Fig.3A_B_C raw data

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence.Significance:This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

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Foxtail millet (Setaria italica L.) is one of the most economically valuable drought-resistant crops in arid and semi-arid regions as a nutrition health crop, which has garnered considerable research attention. We evaluated the effects of replacing chemical nitrogen fertilizers with organic fertilizers on two primary plant accessions of foxtail millet (Dungu and Jinfen no. 2). Nitrogen in a standard fertilizer was replaced with organic fertilizer at application levels of 0, 25, 50, 75, and 100%, with effects on crop yield, quality (appearance, taste, and nutritional value), and soil microbiome, assessed using field cultivation experiments. Our results indicate that partial replacement of conventional fertilizers with organic fertilizers improved both yield and quality. Specifically, the 75% replacement significantly improved the appearance (yellow pigment content and grain diameter) and taste (amylose content and soluble sugar content) of foxtail millet, while the 50% replacement significantly improved the taste (gel consistency) and nutritional qualities (crude protein content and seven amino acids’ content). The 50% replacement of organic fertilizer regulated amino acid content more significantly than starch content. Increased ratios of organic fertilizer significantly reduced the soil pH by 0.03–0.36 and increased the relative abundance of Chloroflexi as well as that of Basidiomycota and Cercozoa in the soil microbiome. Our findings provide a solid theoretical foundation for subsequent studies on fertilizer use for foxtail millet and contribute to developing functional nutritional foods in the foxtail millet industry.

This study is designed to determine the H 2 S origin in the Ordovician Ma5 Member of Daniudi gas field and study the corrosion behavior of N80, P110 and N80S tubing and casings in medium-sulfur content HE16, micro-sulfur HE3 and low-sulfur HE1 gas wells. Therefore, method of weight loss of coupon on site were used to determine the corrosion rate. Similarly, SEM, XRD and EDS analyses were employed to know the corrosion products while establishing the relationship with H 2 S formation conditions. Results of this study show that, as the production time increases, there was initial increase in the volume of H 2 S before it finally decreases. Among the different sulfur gas wells, the corrosion rate of the HE16 is the largest, followed by the low-sulfur HE3 and the micro-sulfur HE1 (smallest). Within a gas well there exist a proportional relationship where higher partial pressure of H 2 S leads to higher corrosion rate. Generally, there is a trend of initial increase followed by decrease in the corrosion temperature of the gas wells in order of decreasing sulfur content (HE16, HE3 and HE1 well). Thus, the average corrosion temperature changing-rate of N80, P110, N80S pipe in the medium sulfur content well area, low sulfur content and micro-sulfur wells are 15.13%, 31.01% and 49.45% respectively. Although, the wells have some coupon corrosion products in common, there is still some basic differences in the composition of the HE1 wells (FeCO 3 and CaCO 3), HE3 wells (FeCO 3, FeS and CaCO 3) and HE16 wells (FeCO 3, FeS 0.9 , FeS and CaCO 3). These corrosion products are clearly stratified in the HE16 and HE3 wells, while no stratification occur in the HE1 wells. The densely structured FeCO 3 is well-distributed within just as the loose and poorly-converted sulfides are distributed on the surface. Both uniform and local corrosion exist simultaneously in the study area. The galvanic corrosion between the product substrate as well as the pitting caused by the inorganic salt result in local corrosion. In the micro-sulfur gas well, only uniform corrosion occur to form FeCO 3 as the corrosion product. For future development of the M5 member of Daniudi gas field, N80 tubing and casing is recommended for the micro-sulfur gas well, P110 or N80S material tubing and casing for the low sulfur gas well and N80S oil jacket for the sulfur gas well. It is good to note that special attention should be paid to the influence of temperature changes on the corrosion rate of the tubing and casing in the low-sulfur and medium-sulfur well zones.

Myopia goes far beyond the inconvenience it brings. It is a prevailing and vision-threatening eye disease, especially in Asia. Aberrantly expressed miR-708a and miR-148 are critical for accurate diagnosis, good prognosis, and precise response prediction of myopia. In this paper, we aim to examine the potential contributions of miR-708a, miR-148a, and PAX6 to high myopia (HM). First, aqueous samples were taken from 25 exclusively HM eyes and 25 exclusively cataract eyes. For next-generation sequencing and bioinformatics analysis, RNA from sample 30one was used. Twenty more samples were used for RT-qPCR. 341 miRNAs in total were found in HM eyes; 249 mature miRNAs and 17 new miRNAs showed differential expression. The expression of hsa-miR-127-3p, hsa-let-7i-5p, and hsa-miR-98-5p was identified using RT-qPCR. MiR-708a and miR-148, which may be linked to the development of myopia and serve as possible biomarkers, are notably highly expressed in atrial tissues of HM patients. Our findings may help deepen the understanding of the mechanisms behind the high expression of miR-708a and miR-148 in atrial tissues of patients with HM.

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. Significance: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Extra-nodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTCL) is a highly aggressive lymphoma, in which the tumor suppressor gene PRDM1 is frequently lost or inactivated. We employed two different CRISPR/Cas9 approaches to generate PRDM1-/- primary NK cells to study the role of this gene in NK-cell homeostasis. PRDM1-/- NK cells showed a marked increase in cloning efficiency, higher proliferation rate and less apoptosis compared with their wild-type counterparts. Gene expression profiling demonstrated a marked enrichment in pathways associated with proliferation, cell cycle, MYC, MYB and TCR/NK signaling in PRDM1-/- NK cells, but pathways associated with normal cellular functions including cytotoxic functions were downregulated, suggesting that the loss of PRDM1 shifted NK cells toward proliferation and survival rather than the performance of their normal functions. We were also able to further modify a PRDM1-deleted clone to introduce heterozygous deletions of common tumor suppressor genes in ENKTCL such as TP53, DDX3X, and PTPN6. We established an in vitro model to elucidate the major pathways through which PRDM1 mediates its homeostatic control of NK cells. This approach can be applied to the study of other relevant genetic lesions and oncogenic collaborations in lymphoma pathogenesis.

To investigate the mechanism of the protective effects of blueberry anthocyanin extract (BAE) against oxidative stress and the roles of SIRT1 and NF-κB in the pathogenesis of diabetic cataracts. Ma...

Introduction: Recent pan-cancer whole-genome sequencing studies revealed the rich landscape of structural variants (SV), from simpler indels to complex events involving multiple breakpoints and sequence gain/loss. SVs may contribute to tumorigenesis through direct modification of coding sequence or deregulation from copy number alterations, enhancer hijacking, or topological domain modification. A substantial part of the variation in the human genome is not accessible to short reads due to mapping ambiguities. Recent benchmarking studies reported that the best short-read methods only have 30-70% SV sensitivity. Long-read sequencing (such as PacBio or Oxford Nanopore) can overcome the limitations of short reads, however the current methods were not designed for the analysis of rearranged cancer genomes with complex copy number profiles. Methods: We developed BGA (Breakpoint Graph Assembler), a method that combines the ideas from long-read assembly and breakpoint graph frameworks. BGA detects abnormally mapped reads and builds a breakpoint graph that characterizes the structure of derived cancer karyotypes. Complex events with multiple breakpoints form connectivity clusters and are classified based on the subgraph properties. BGA also takes advantage of phased haplotypes and can incorporate multiple related datasets (such as in tumor-normal comparison or multi-site tumor sampling). BGA is freely available at: https://github.com/KolmogorovLab/BGA. Results: We first analyzed three cancer cell lines and corresponding matching normal DNAs (HCC1954, H2009, and COLO829). In each cell line, we identified 8-56 somatic rearrangement clusters involving more than two breakpoints and at least 1kb of sequence. In H2009, we identified a chromoplexy event involving chr13 and chr1, consistent with previous FISH experiments. COLO829 showed the lowest number of somatic rearrangement clusters (n=8), including translocation and inversion events between chr3, chr10, and chr12 within the RARB, BICC1, and TRHDE genes. Homologous recombination deficient HCC1954 has the highest number of complex events including the chr17q arm which hosts ERBB2, chromoplexy between chr8, chr5, and chromothripsis in chr21. In addition, we analyzed three other HPV-infected cell lines (CaSki, SCC152, SNU1000). In each of them, we observed complex clusters of HPV-HPV and HPV-human breakpoints that formed cycles, suggesting extrachromosomal amplification. The HPV fragments had many interactions with chromosomal DNA in CaSki and SC152, but not in mostly episomal SNU1000 cells. We also observed karyotype-scale changes that did not involve HPV sequences, such as the simultaneous exchange of six chromosome arms of chr2, chr7, and chr17 in CaSki. Citation Format: Ayse Keskus, Tanveer Ahmad, Ataberk Donmez, Yi Xie, Isabel Rodriguez, Rose Milano, Nicole Rossi, Hong Lou, Laksh Malik, Kimberley Billingsley, Cornelis Blauwendraat, Michael Dean, Mikhail Kolmogorov. Long-read, assembly-based characterization of rearranged cancer karyotypes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4289.

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from the controls showed a reduced level of steady-state E7 protein, with 7/13 variants having deficient protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-normal E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2); and the R77S (CIN3) had activity only slightly reduced from wildtype E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay as compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration further demonstrating the importance of fully active E7 in clinical cancer development.

Simple Summary The coupling of metabolism with cellular status is critically important and highly evolutionarily conserved. However, how cells coordinate metabolism with transcription as they change their status is not clear. Utilizing multiomic and functional studies, we now demonstrate the dichotomous roles of the Kat3 coactivators CBP and p300 and, in particular, their extreme N-termini, in coordinating cellular metabolism with cell differentiation. Using multiple in vitro and in vivo systems, our study sheds new light on metabolic regulation in homeostasis and disease, including cancer. Abstract The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent β-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

SummaryIntegration of Human Papillomaviruses (HPV) is an important mechanism of carcinogenesis but is absent in a significant fraction of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors. In two HPV16+ cell lines, we identified large tandem arrays of full-length and truncated viral genomes integrated into multiple locations indicating formation as extrachromosomal DNA (HPV superspreading). An HPV16+ cell line with episomal DNA has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). WGS of HPV16+ cervical tumors revealed that 11/20 with only episomal HPV (EP) have intact monomer episomes. The remaining nine EP tumors have multimer and rearranged HPV genomes. Most HPV rearrangements disrupt the E1 and E2 genes, and EP tumors overexpress the E6 and E7 viral oncogenes. Tumors with both episomal and integrated HPV16 display multimer episomes and concatemers of human and viral sequences. One tumor has a recurrent deletion of an inhibitory site regulating E6 and E7 expression, and another has a recurrent duplication consistent with HPV superspreading. Therefore, HPV16 can cause cancer without integration through aberrant episomal replication, forming rearranged and multimer episomes.

Gene promoter hypermethylation is a vital step in tumorigenesis. This paper set out to explore the use of polymerase chain reaction - surface-enhanced Raman spectroscopy (PCR-SERS) for the detection of gene methylation levels, with a focus on cancer diagnosis. Methods: PCR with methylation independent primers were used on DNA samples to amplify target genes regardless of their methylation states. SERS was used on the obtained PCR products to generate spectra that contained peak changes belonging to CG and AT base pairs. Multiple linear regression (MLR) was then used to deconvolute the SERS spectra so that the CG/AT ratios of the sample could be obtained. These MLR results were used to calculate methylation levels of the target genes. For protocol verification, three sets of seven reference DNA solutions with known methylation levels (0%, 1%, 5%, 25%, 50%, 75%, and 100%) were analysed. Clinically, blood plasma samples were taken from 48 non-small-cell lung cancer (NSCLC) patients and 51 healthy controls. The methylation levels of the genes p16, MGMT, and RASSF1 were determined for each patient using this method. Results: Verification experiment on the mixtures with known methylation levels resulted in an error of less than 6% from the actual levels. When applied to our clinical samples, the frequency of methylation in at least one of the three target genes among the NSCLC patients was 87.5%, but this percentage decreased to 11.8% for the control group. The methylation levels of p16 were found to be significantly higher in NSCLC patients with more pack-years smoked (p=0.04), later cancer stages (p=0.03), and cancer types of squamous cell and large cell versus adenocarcinoma (p=0.03). Prediction accuracy of 88% was achieved from classification and regression trees (CART) based on methylation levels and states, respectively. Conclusion: This research showed that the PCR-SERS protocol could quantitatively measure the methylation levels of genes in plasma. The methylation levels of the genes p16, MGMT, and RASSF1 were higher in NSCLC patients than in controls.