Deregulation of L1 retrotransposon-encoded protein expression in oral cancer recurrence

Deregulation of L1 retrotransposon-encoded protein expression in oral cancer recurrence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deregulation of L1 retrotransposon-encoded protein expression in oral cancer recurrence Sujoy Kundu, Manali Ganguly, Koel Mukherjee, Gopal Sarkar, Shahab A Usmani, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6319447/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Oral squamous cell carcinoma (OSCC) often shows recurrence after surgery. To date, there is no significant study on OSCC that predicts recurrence after surgical removal of the cancer. Long INterpersed Element 1 (LINE-1 or L1) retrotransposons show very high activity in many cancers, suggesting a potential role in cancer onset and progression. We wished to assess the value of LINE-1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) as biomarkers of OSCC recurrence along with eight other established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44). Results: We collected 114 post-operative oral cancer patient samples, mostly from tobacco-addicted patients, and analysed the expression of both L1ORF1p and L1ORF2p and eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) by immunohistochemistry. We found 97% of samples (110 out of 114) showed significant expression of both the L1-encoded proteins. Among those 114 samples, 35 samples belonged to the recurrent group and showed strong association with L1ORF1p and L1ORF2p expression when compared with the non-recurrent group. Expression analysis of eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) by immunohistochemistry showed L1 proteins, along with CD105 and EGFR, can form a predictive panel for OSCC recurrence. Conclusions: The study revealed that the combined expression analysis of the four bio-markers (L1ORF1p, L1ORF2p, CD105 and EGFR) can distinguish recurrent from the non-recurrent OSCC samples. The findings have significant clinical relevance and applications in predicting oral cancer recurrence. retrotransposon LINE-1 oral squamous cell carcinoma (OSCC) cancer biomarker recurrent cancer non-recurrent cancer CD105 EGFR L1ORF1p and L1ORF2p. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background According to the Globocan report, oral cancer of the lip and oral cavity ranked 16th among all the reported cancers [ 1 ]. Almost 378,000 new cases and 178,000 deaths were reported from this particular cancer worldwide in 2020[ 1 ]. The global incidence and mortality rate of this particular cancer are more than double for men compared to women. Cancer of the tongue, buccal mucosa and lip mucosa is extremely common in India due to excessive use of betel quid and tobacco chewing [ 2 ]. Multiple genes required for normal growth and maintenance of cells show mutations in this particular cancer [ 3 ]. Although surgical removal of cancer tissue is effective, patients often report recurrence within 6 to 12 months after surgery [ 4 , 5 ]. Biomarker expression analysis through immunohistochemistry (IHC) is often used as the gold standard to predict the behaviour of cancer [ 6 , 7 ]. Here we have collected 114 patient samples with the history of excessive use of tobacco chewing and smoking habit. Here we collected 114 patient samples with a history of excessive tobacco chewing and/or smoking habit, and sought to determine if expression analysis of L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) in these oral cancer patients (n = 114) is efficacious in distinguishing recurrent from non-recurrent cancer. Long Interpersed Element (LINE1 or L1) is an autonomous non-LTR retrotransposon with around 500,000 copies occupying 17% of the human genome [ 8 – 11 ]. Although most copies are silent due to mutations, it was originally estimated 80 to 100 copies are potentially active in a human genome [ 12 ], although subsequent estimates have varied [ 13 , 14 ]. An active human L1 is 6.0 kb in length, and contains a 900 nt 5’-UTR with internal promoter, two open-reading frames (ORFs), designated ORF1p and ORF2p, separated by a small inter-ORF spacer sequence and followed by a ~ 200 bp 3’-UTR [ 15 ]. L1 ORF2p encodes a 150 kDa protein with reverse transcriptase (RT) [ 15 , 16 ] and endonuclease (EN) activities [ 17 ], whereas L1 ORF1p encodes a 40 kDa protein with demonstrated single-stranded nucleic acid binding and nucleic acid chaperone activities [ 18 ]. L1 retrotransposons are silent in normal somatic tissues due to multiple layers of restriction imposed by cellular factors [ 9 , 19 ]. Cancer tissues lose this restriction by some poorly defined, but possibly epigenetic mechanisms [ 20 ], and thus L1 becomes activated [ 21 , 22 ]. Previous studies showed that the expression of L1 ORF1p is significantly elevated and a potential biomarker in numbers of cancers [ 23 – 27 ]. By performing a pilot study with a small cohort of samples, we recently showed significant expression of both the L1-encoded proteins (ORF1p and ORF2p) in operated oral cancer samples [ 28 , 29 ]. A limited number of studies have investigated marker expression to distinguish recurrent from non-recurrent oral cancer [ 5 , 30 – 32 ]. Although L1 protein expression is ubiquitous in OSCC, expression status in recurrent and non-recurrent patients is unknown [ 23 – 29 ]. Identifying patients who are at risk of developing recurrence post-surgery is crucial in cancer treatment. In this study, we aimed to provide an improved solution for biomarker detection of OSCC recurrence. Therefore, we investigated the expression of L1ORF1p and L1ORF2p, along with eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44), in recurrent and non-recurrent patients. Although all these markers have previously been tested in other studies to assess their expression in oral cancer [ 6 , 7 ], a direct comparison of their expression between non-recurrent and recurrent OSCC was not explored. We found that 97% of oral cancer samples (110 out of 114) showed significant expression of both L1-encoded proteins. Importantly, we also found a strong association of L1 ORF1p and L1ORF2p expression with oral cancer recurrence. Our study suggests that IHC expression analysis of L1-encoded proteins, along with CD105 and EGFR, before surgery can predict behaviour of the disease. Methods Patient descriptions This study was approved by the Institutional ethics committee of All India Institute of Medical Sciences, Rishikesh and Indian Institute of Technology Roorkee. All methods were performed in accordance with relevant guidelines and regulations. Following full written informed consent from patients, post-operative cancer tissues were collected for preparation of formalin-fixed paraffin-embedded tissue blocks (FFPE). Doctors and pathologists verified the histopathology of OSCC in all patient samples. Patient follow-up data is provided in Supple table 2 . Histological grading and staging of cancers were performed as per the American Joint Committee on Cancer (AJCC) classification. Generation of L1ORF2 (RT1) antibodies in rabbit The cloning, expression and purification of hL1RT EH (RT1) protein was described earlier [ 29 ]. The purified protein was injected in rabbit to generate the antibody. The protocol used to generate the antibody was as follows: Day 0: Pre-immunization bleed (0.5 ml) Day 1: Immunization with 0.75 mg antigen in complete Freud’s adjuvant (Sigma, cat no. F5881), subcutaneous (SQ) 1 site Day 15: Boost with 0.5 mg antigen in incomplete Freud’s adjuvant (Sigma, cat no. F5506), SQ 1 site Day 30: Boost with 0.5 mg antigen in incomplete Freud’s adjuvant, SQ 1 site Day 35: Test-bleed (0.5 ml per rabbit) to check the titer and quality of the generated antibody by ELISA and Western blotting Day 45: Boost with 0.25 mg antigen in incomplete Freud’s adjuvant, SQ 1 site Day 60: Terminal blood collection (around 15 ml per rabbit from the central artery on the inner edge of the dorsal surface of the ear) Preparation of antigen affinity column: Sepharose CL-4B (Sigma, cat no. 61970-08-9) resin was used to conjugate the antigen (RT1). One ml of resin was mixed with freshly prepared CNBr solution (2 gm/ml, solvent acetonitrile) and incubated for 2 mins at room temp. Next, 800 µl of 5M NaOH was added to the slurry to adjust to pH 10–11 and the tube was incubated at 4C for 30 mins with gentle rotation. Following completion of the reaction, the bead slurry was washed with five column volumes (CVs) of ddH 2 O to remove any remaining unreacted CNBr. The beads were then ready to conjugate with the RT1 antigen. Around 10 mg of antigen dialyzed in coupling buffer (100 mM NaHCO 3 , 500 mM NaCl; pH 8.3) was incubated with 1 ml of CNBr activated Sepharose CL-4B resin at 4°C overnight with continuous gentle rotation. The next day, beads were washed with 5 CVs of coupling buffer, followed by incubation with 5 CVs of quenching buffer (0.1M Tris-Cl; pH 8.0) at room temperature for 2 hours with continuous gentle rotation to block residual binding sites. After quenching, beads were washed with three cycles of alternating pH buffers, i.e. 5 CVs of low pH (0.1 M sodium acetate; pH 4.0) and high pH (0.1 M Tris-Cl; pH 8.0) buffers one after the other. Finally, the antigen-bound resin was rinsed with 5 CVs of binding buffer (20 mM sodium phosphate buffer; pH 7.4). The RT1 antigen affinity resin was then ready to purify antigen-specific antibody from whole immune serum. Purification of antigen-specific antibody from immunized serum: Five hundred microliters of immunized rabbit whole serum was mixed with binding buffer (20 mM sodium phosphate buffer; pH 7.4) in a 1:1 ratio and incubated with 250 µl of antigen-coupled resin for 8–10 hours at 4°C with gentle rotation. Next, the resin was washed with 5 CVs of wash buffer (10 mM ammonium acetate; pH 4.6) followed by elution of the specific antibody with one CV of elution buffer (0.1 M citric acid). The elution step was repeated five times. Each elution step involves, i) incubation of the antibody-bound resin with elution buffer for two minutes on ice with intermittent gentle tapping, ii) centrifugation at 2000 rpm for two minutes at 4°C, iii) incubation for two minutes on ice to settle the beads completely, iv) collection of the supernatant, and v) immediate neutralization with 1 M Tris-Cl; pH 9.0 to adjust to pH 7–8. All the collected elution fractions were pooled and concentrated using a 0.5 ml 10-kDa Amicon concentrator (Millipore Sigma) to 2 CVs of resin (the final antibody concentration was 3 mg/ml). Preparation of antibody affinity resin: The purified ORF2p antibody was dialyzed in coupling buffer and then incubated with 500 µl of CNBr-activated Sepharose CL-4B beads at 4°C with continuous gentle rotation overnight. The rest of the procedure was the same as the preparation of antigen affinity column preparation. Tissue lysate preparation and immunoblotting: The tissue lysate was prepared by crushing OSCC tissues with a mortar-pestle in liquid N 2 and mixed with RIPA buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.5% NP-40, 1mM DTT, 1mM EGTA, 1mM NaF, 2mM Na 3 VO 4 , 1 mM PMSF, supplemented with 1X protease inhibitor cocktail) for 30 minutes with repeated vortexing after each 5 min. The lysate was then centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was transferred to a new 1.5 ml tube and stored at -70°C until further use. Around 60 µg of tissue lysate was resolved in a 10% SDS-PAGE gel (Mini-PROTEAN Tetra Cell, Bio-Rad) and transferred to PVDF membrane (Merck Immobilon, cat no. IPVH00010) by applying 100 V for 75 min using a BioRad Mini Trans-Blot Electrophoretic Transfer Cell (transfer buffer composition: 25 mM Tris-Cl, pH 7.6, 192 mM glycine, and 20% methanol). Following transfer to the membrane, the membrane was blocked with 1xPBST containing 5% w/v skimmed milk for 1 hour at room temperature. Then it was probed with primary antibody (purified polyclonal rabbit anti-ORF1(RRM) (1:5000), purified polyclonal rabbit anti-ORF2(RT1) (1:2000), anti-GAPDH (1:2000)) diluted in blocking buffer). The next day, the membrane was washed with 1xPBST 3 times for 10 minutes at room temperature and then incubated with secondary antibody (α-rabbit HRP, dilution 1:60,000; (Jackson ImmunoResearch, cat no. 111-035-003)) diluted in blocking buffer for an hour at room temperature. Following incubation, the membrane was washed 3x in 1xPBST for 10 minutes and then Western blots were developed with Chemiluminescence HRP Substrate (Takara, cat no. T7101A). The signal was detected by exposing the blot to Fuji X-ray film (Fujifilm). Immunoprecipitation (IP) of L1ORF2p: For immunoprecipitation of L1ORF2p, 200 µl of cancer tissue lysate (total protein ~ 300 µg) was incubated with 30 µl of Sepharose CL-4B beads coupled with L1ORF2p antibody (RT1) in a 1.5 ml micro-centrifuge tube overnight at 4°C with continuous gentle rotation. The following day, flowthrough was separated from the beads by centrifuging the beads at 2000 rpm for 3 minutes at 4°C. Next, the beads were washed with 5 CVs of wash buffer and then eluted with 2.5 CVs of elution buffer. In each elution step, beads were incubated with elution buffer for 5 min on ice with intermittent tapping. After incubation, beads were centrifuged at 2000 rpm for 2 min and then kept on ice for another 2 min to allow the beads to completely settle. Then the elution fraction was collected in a separate MCT tube, and pH was adjusted to 7–8 by adding neutralization buffer. This complete step was repeated 5 times. All the elution fractions were concentrated to 100 µl volume. From the concentrated sample, 30 µl was resolved in an 8% SDS-PAGE gel to perform immunoblotting for the detection of L1ORF2p in cancer samples. Immunohistochemistry Formalin fixed paraffin embedded (FFPE) tissue blocks and sections (4 µm thick) were prepared for all 114 post-operative OSCC samples. To perform histological examinations, hematoxylin and eosin staining was performed on tissue sections. Tissue sections were deparaffinised and rehydrated for antigen retrieval. After antigen retrieval, tissue sections were blocked by incubating in blocking solution followed by treatment with 3% hydrogen peroxide to quench endogenous peroxidase activity. Next, tissue sections were incubated with primary antibody at 4 o C overnight followed by incubation with secondary antibody (1:500 dilution, α-rabbit-HRP (Jackson ImmunoResearch, cat #111-035-003)) for an hour at room temperature. Signal was visualized by adding 3–3´-diaaminobenzidine tetrahydrochloride (DAB) solution to the slides and counterstaining with hematoxylin. All microscopic pictures were taken at 10X and 40X magnification. The antibodies against p53 (cat #PM101), PCNA (PR065), CD105 (MR1196), Ki67 (PM210), EGFR (PR040), CD44 (MM1121), and PDL1 (MR1247) were from PathnSitu Biotechnologies. MMP9 antibody (cat #BSB2138) was obtained from BIO SB Products and Technology for Molecular Pathology. In-house-generated Ag affinity column purified rabbit polyclonal antibodies against human L1ORF1p [ 27 , 32 ] and L1ORF2p [ 27 ] were used in this study. Image data analysis: The regions of interest (ROI) corresponding to histologically representative areas were identified for each image and confirmed by several authors and results were reviewed by a pathologist. For each tissue, images were taken from the same region for all the markers. The DAB-stained slide images were analysed using QuPath software (version 0.5.1) [ 34 ]. This program measures the number of stained cells and their intensity in four-grade semi-quantitative scale (3 = high, 2 = moderate, 1 = low and 0 = no stain) and calculates the immunohistochemical score or H-Score (H-Score = 3x% high + 2x% moderate + 1x% low). The image data analysis method is outlined in Supple Fig. 1 . Statistical analysis: Individual biomarker expression was analysed first with unpaired Welch’s t-test to identify significant differences in protein expression between non-recurrent and recurrent groups of samples. This was followed by two-tailed Pearson correlation test to assess correlation between biomarker expression and disease recurrence. The mean values of biomarker expression were used to compare data sets. The associations of expression of all the markers with clinical outcomes were determined using multivariate logistic regression analysis for the development and testing of the model. Kaplan-Meier survival analysis was used to construct diagrams of patient survival between sample cohorts and their relationship with each biomarker. The statistical analysis was performed using GraphPad Prism statistical software (version 9.5.1). Results The sample cohort comprises 114 cancer samples comprising 15 females and 99 males with a mean age of 47.4 years (Supple Table 1). The recurrent samples (n = 35) included in this study showed the incidence of recurrence within a year of surgical resection of the tumor and loco-regional spread (Supple Table 2). Almost all the patients were addicted to tobacco chewing and/or smoking, in addition to alcohol consumption in some cases. Most of the patients in this study belong to the moderately differentiated category (78%, including 18 non-recurrent and 16 recurrent), and only two patients from the recurrent group showed poorly differentiated grades (4%). The rest of the samples (18%) exhibited a well-differentiated nature (Supple Table 1). Expression analysis of L1 retrotransposon encoded proteins (L1ORF1p and L1ORF2p): In our previous study to check the expression of L1 encoded ORF1p we used an in-house antibody [ 28 , 29 , 33 ]. The antibody was raised against the RRM domain of L1ORF1p in rabbit and the immunized whole serum from the rabbit without further purification was used to detect L1ORF1p by Western blotting and IHC analysis. Here we purified the antibody using an antigen affinity column and the purity was checked by performing immunoblotting using operated cancer samples. The antigen column purified L1ORF1p antibody showed a discrete single band at around 40 kDa, the proposed molecular weight of L1ORF1p, in the patient cancer lysate ( Fig. 1 A ). The purified L1ORF1p antibody was used to check the expression of L1ORF1p in an operated oral cancer sample and adjacent normal tissue by performing immunohistochemistry. The operated cancer sample showed significant staining whereas adjacent normal tissue was completely negative and showed no staining with the L1ORF1p antibody ( Fig. 1 B). Next, IHC was conducted to assess the expression of L1ORF1p in 114 operated OCSS samples (Supple Table 1). We found 97% of samples (110 out of 114) with significant staining with anti-L1ORF1p. L1ORF1p staining intensity was measured using QuPath, analysis software which measures DAB staining semi-quantitatively [ 34 ] (Supple Fig. 1 ) . QuPath analysis determined four grades of overall L1ORF1p expressions in the cohort samples: high (43.8%, n = 50, H-scores = > 56), moderate (29.8%, n = 34, H-scores = 28–56), low (22.8%, n = 26, H-scores = 5–28) and no expression (3.6%, n = 4, H-scores = < 5) ( Fig. 1 C ) . L1ORF1p staining was readily detected mostly in the cytoplasm of tumor cells ( Fig. 1 B ); a few samples also showed L1ORF1p nuclear staining (Supple Fig. 2 ) . Many samples showed a diffuse pattern of staining all over the cancer tissues. Similarly, we interrogated L1ORF2p expression in the same large cohort (n = 114) of operated oral cancer samples. In a pilot study, we previously showed elevated expression of L1ORF2p in 39 operated oral cancer samples using an in-house L1ORF2p antibody [ 29 ]. This antibody was generated in mice using a 10-kDa fragment (hRT EH , L1ORF2p amino acids 479–558, accession number: AF148856) from the RT domain of ORF2p. Immunized whole serum from mouse without further purification was used to detect ORF2p by IHC and immunoblotting [ 29 ]. Here we raised an L1ORF2p antibody in rabbit, and the specific polyclonal antibody against L1ORF2p was purified by passing the whole serum through an antigen (Ag) affinity column. The purity of the column-purified L1ORF2p Ab was checked by immunoblotting using lysate prepared from the patient samples. As immunoblotting with the total lysate didn’t show any L1ORF2p signal, we next performed an immunoprecipitation experiment of L1ORF2p from the cancer tissue lysates. The Ag column-purified L1ORF2p antibody was cross-linked with sepharose beads and incubated with the cancer lysate, followed by washing and elution of bound L1ORF2p with citric acid elution buffer. The elution fractions were pooled, concentrated and analysed by immune blotting to determine the presence of L1ORF2p. A discrete band at around 150 kDa corresponding to the proposed molecular weight of L1ORF2p was detected in the two cancer samples analysed ( Fig. 1 A ) . Next, the newly made L1ORF2p rabbit antibody was used to detect by IHC L1ORF2p in an operated oral cancer sample along with adjacent normal mucosa. The L1ORF2p antibody showed distinct staining in the cancer samples and no stain in the normal mucosa tissue ( Fig. 1 B ). Next, the expression analysis of L1ORF2p was assayed by IHC for the 114 oral cancer samples (Supple Table 1). The results showed that all the samples positive for L1ORF1p (110 of 114, 97%) showed a significant expression of L1ORF2p, mostly in the cytoplasm; only a few samples showed a minor fraction of L1ORF2p in the nucleus (Supple Fig. 2 ). Expression analysis using QuPath showed high (40.3% n = 46, H-scores = > 84), moderate (43.9%, n = 50, H-scores = 42–84), low (12.3%, n = 14, H-scores = 12–42) and no (3.6%, n = 4, H scores = < 12) ORF2p expression ( Fig. 1 C ) . Thus, both the Ag column-purified L1ORF1p and L1ORF2p polyclonal rabbit antibodies are very effective in detecting L1 proteins in the operated oral cancer samples. We also found that L1ORF1p and L1ORF2p is ubiquitous: nearly all the oral cancer samples included in this study significantly expressed both the L1-encoded proteins. L1 protein expression is significantly elevated in recurrent versus non-recurrent oral cancer Although L1 expression is a hallmark for many cancers, its relation with recurrence outcome after surgery is unknown [ 23 – 29 ]. Here, we have investigated the expression of L1ORF1p and L1ORF2p between recurrent and non-recurrent cancer samples. Among 114 samples, 35 samples belonging to the recurrent categories showed loco-regional relapse of cancer within a year after surgery (Supple Table 2). Here, we have compared the expression of both the L1 encoded proteins between 35 non-recurrent and 35 recurrent samples by using QuPath software (version 0.2.0-m4). Significantly, higher expression was observed for both the L1 proteins when we compared the recurrent group with the non-recurrent ( Fig. 2 , Supple Fig. 3 ). The IHC images of 35 recurrent and non-recurrent samples are shown in supple Fig. 3 . L1ORF1p and L1ORF2p showed almost 2.5-fold and 1.8-fold increased expression, respectively, in the recurrent samples [(H-score: L1ORF1p non-rec = 31.6, rec = 80.2) and (H-score: L1ORF2p non-rec = 61.2, rec = 108.7) (p < 0.0001)]. L1ORF1p expression in an established oral cancer cell line The expression of L1ORF1p was analysed in an established oral squamous cell carcinoma cell line (AW13516) [ 35 ] and compared with other cancer cell lines (human embryonic kidney HEK293T, cervical cancer HeLa, and colon cancer HCT116 lines). We showed very high expression of L1ORF1p in AW13516 cells (Supple Fig. 4 ) when compared with other cancer cell lines. The expression of other cancer biomarkers, p53, PCNA and EGFR, was also analysed, and the results showed significant higher expression of p53 and EGFR in AW13516 (Supple Fig. 4 ) when compared to the other cell lines. Expression analysis of other biomarkers in the recurrent and non-recurrent oral cancer samples Along with the L1 retrotransposon encoded proteins, we also analysed eight established cancer biomarkers often used for IHC analysis of patient cancer samples. These biomarkers are p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44 [ 6 , 7 ]. The expression analysis of these eight markers was analysed by performing IHC in a group of 25 recurrent and 25 non-recurrent oral cancer patient samples ( Fig. 3 , Supple Fig. 5 –12, Supple table 3). The results showed a significant upregulation of CD105 [(H score: CD105, non-rec = 17.46, rec = 34.62) (p = 0.0473)] and downregulation of EGFR [(H score: EGFR, non-recurrent = 118.97, recurrent = 84.87) (p = 0.0296)] in the recurrent samples compared with non-recurrent samples ( Fig. 3 ). The other six markers (p53, PCNA, ki67, MMP8, EGFR, PDL1 and CD44) didn’t show any significant changes between non-recurrent and recurrent samples ( Fig. 3 ). Two-tailed Pearson test for recurrence assessment by investigating biomarker expression Next, we performed two-tailed Pearson test to find any association of L1 protein expression with recurrence outcome. The results show that expression levels of both L1 proteins are strongly correlated [(r value: L1ORF1p = 0.59, L1ORF2p = 0.50 (p < 0.0001)] ( Fig. 4 A). The same test was performed by including the expression values obtained from eight established biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) along with L1ORF1p and L1ORF2p. The result of the two-tailed Pearson test when including ten biomarkers showed that expressions of CD105, L1ORF1p and L1ORF2p are positively correlated [(r value: CD105 = 0.28 (p = 0.046), L1ORF1p = 0.53 (p < 0.0001), L1ORF2p = 0.39 (p = 0.0048)], whereas the expression of EGFR showed a negative association [(r-value: EGFR = -0.31 (p = 0.030)] ( Fig. 4 B, Supple table 4 ). The expression of p53, PCNA, Ki67, CD44, MMP9 and PDL1 didn’t show significant association with recurrence outcome (Fig. 4 B, Supple table 4). Generation of Receiver Operating Characteristic (ROC) curves as predictors of recurrence Multiple logistic regression analysis was used to generate a receiver operating curve (ROC) for the diagnostic ability of biomarker expression as predictors of recurrence. Considering the expression of L1ORF1p and L1ORF2p, we found that 80% of the recurrent samples (29 true positive and 7 false negative out of 35 recurrent samples included in this study) have a predicted value above 0.5, with an area under the curve (AUC) of 0.90 (95% confidence interval (CI) 0.8339 to 0.9702 ( Fig. 5 A, Supple Table 5) . Similarly, the analysis predicts 28 out of the 35 non-recurrent samples are true non-recurrent samples (Supple Table 5) . The same analysis was performed by including the eight other biomarkers with L1ORF1p and L1ORF2p, and the result showed that 92% of the samples (23 out of 25 samples) were correctly classified as recurrent with an AUC of 0.95 (95% CI 0.9037 to 1.0) ( Fig. 5 B, Supple Table 5) . In addition, the ROC curve was also generated using four biomarkers (CD105, EGFR, L1ORF1p and L1ORF2p) that showed significant expression changes between non-recurrent and recurrent samples ( Fig. 5 C ). Protein biomarker expression in relation to overall patient survival Next, we investigated if there was any correlation between individual marker expression and patient survivability by Kaplan Meier analysis. Comparing the survivability curves among all ten biomarkers, it is evident that the curves generated using L1ORF1p, L1ORF2p and EGFR are the most significant in predicting poor overall survivability (L1ORF1p, p = 0.0058; L1ORF2p, p = 0.0131; EGFR. p = 0.0296) ( Fig. 6 ). Discussion Cancer in the lip and oral cavity is very common in India due to excessive use of chewing tobacco [ 36 ]. Here we investigated the expression of L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) in a large cohort sample (n = 114) of patients with a history of excessive use of tobacco [Supple table 1]. We generated in-house antibodies against both the L1-encoded proteins. Previously we reported Western blotting and IHC analyses in a small cohort of oral cancer samples using our in-house L1ORF1p and L1ORF2p antibodies in the form of immune whole serum without further purification [ 28 , 29 , 33 ]. Here we purified both antibodies (anti-L1ORF1p and anti-L1ORF2p) to homogeneity by passage through their respective antigen affinity columns and checked specificity by immunoblotting before using them for IHC analysis of a large number of oral cancer samples. In our previous study, we reported an L1ORF2p antibody that was generated in mice [ 29 ]. Here the same antigen was injected into rabbit, which produced a very effective L1ORF2p antibody. Although numerous, including commercial, antibodies are available to detect endogenous L1ORF1p [ 23 – 25 , 29 ], there are few effective non-commercial antibodies that report detection of endogenous L1ORF2p [ 25 , 37 – 41 ]. We found our antigen column-purified antibodies against both the L1 proteins to be very effective for detecting L1 proteins in cancer samples. Previous reports from our and others' work demonstrated that almost 60% of head and neck cancer samples showed expression of L1-encoded proteins [ 23 , 29 ]. In this study, our sample cohort, collected mostly from tobacco-addicted patients, showed 97% expressing both L1-encoded proteins. Increased expression of L1ORF1p has been observed in many cancers [ 23 – 29 ]. A pioneer work from Rodic et al. showed that more than half of head & neck cancer samples expressed L1ORF1p [ 23 ]. In another study, Chen et al. [ 25 ] demonstrated ubiquitous expression of both L1 proteins in breast cancer cell lines and breast cancer tissues. The same study showed that breast cancers with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis and poor patient survival than those with cytoplasmic expression [ 25 ]. Harris et al. [ 24 ] earlier reported related findings for breast cancer. Hypomethylation of the L1 promoter and consequent high expression of L1ORF1p is common in high grade ovarian carcinoma, the most common and aggressive type of ovarian cancer [ 42 ]. The CpG sequences present in the L1 promoter are heavily methylated in normal tissues and restrict L1 transcription. In contrast, cancer tissues show severe hypomethylation of these CpGs and activated L1 transcription [ 28 , 42 , 43 ]. High prevalence of L1 proteins in OSCC could also be due to downregulation of Let-7 miRNA in patient samples [ 44 ]. Let-7 miRNA represses L1 retrotransposition by directly binding to L1 mRNA and impairing translation of L1 encoded proteins [ 45 ]. Although a few molecular-based studies have been described, IHC remains the gold standard for diagnosis and therapeutics of oral cancer [ 4 , 6 , 7 ]. In this study, we asked if expression analysis of L1 proteins could predict behaviour of the disease in non-recurrent and recurrent samples. Previous studies analyzed expression of p53, p63, pedoplanin and ki67 in oral leukoplakia and found that the expression of p53 is higher in recurrent compared to non-recurrent leukoplakia [ 46 ]. Another study on advanced stage laryngeal squamous cell carcinoma showed downregulation of MCM2, ki67 and EGFR [ 47 ]. In our sample cohort, 35 samples belonging to the recurrent group showed significantly higher expression of both L1 proteins when compared with non-recurrent samples. In parallel, we analysed eight established biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) and found that H-scores of L1 proteins are strongly associated with OSCC recurrence compared to other biomarkers. Among the other eight bio-markers, CD105 showed significant upregulation, while EGFR showed a downtrend trend in expression in recurrent samples. The remaining six biomarkers (P53, PCNA, Ki67, MMP9, CD44, and PDL1) showed no association with recurrent outcome. Micro-vessel density (MVD) is considered an independent indicator in a variety of human malignancies [ 48 ]. Higher MVD is correlated with progression of malignancy and shorter overall and relapse-free survival. Tumor vasculature is shaped by a number of angiogenesis markers, including CD105 (endoglin). CD105 is expressed on the surface of angiogenic endothelial cells and is induced by hypoxic conditions. In the case of laryngeal SCC, it was found that patients with higher values of CD105 have low disease-free survival (DFS) and significantly higher chances of developing recurrence than those without [ 48 ]. Our study showed a significant upregulation of CD105 expression in recurrent samples compared to nonrecurrent ones. One possible reason for high CD105 expression may be due to downregulation of MASPIN, a potent tumour suppressor that regulates tumour angiogenesis [ 49 ]. Several past studies have reported the pivotal role of EGFR in the pathogenesis of cancer including HNSCC but none of the surveys focused on EGFR expression in recurrent HNSCC samples [ 50 , 51 ]. Carcinogens induce expression of EGFR, which forms a complex with the heterodimer Ku70/80 and helps to repair DNA double-strand breaks by recruiting DNA protein kinase (DNA-PKcs) and the MRN protein complex [ 52 , 53 ]. Maiorano et al. [ 54 ] showed that oral cancer patients with downregulated expression of membranous EGFR are more likely to suffer recurrence and death. Our study showed a significant down-regulation of EGFR in recurrent oral cancer samples. One possible reason behind the downregulation of EGFR may be hypoxia that leads to the upregulation of prolyl hydroxylase 3 (PHD3), a protein interacting with hypoxia-inducible factor (HIF) to cause internalization of EGFR via endocytosis [ 55 ]. Our two-tailed Pearson test with four (CD105, EGFR, L1ORF1p, and L1ORF2p), out of ten bio-markers analysed in this study, showed significant changes in the recurrent samples when compared to non-recurrent. Parallelly, multiple logistic regression test showed that CD105, EGFR, and L1-encoded proteins (L1ORF1p and L1ORF2p) form a set and can efficiently identify disease recurrence in oral cancer patients. Thus, the expression analysis of L1 encoded proteins, along with CD105 and EGFR, allowed us to distinguish recurrent from non-recurrent samples. The findings of our study have significant clinical relevance and applications in predicting oral cancer recurrence. Implications for increased LINE-1 expression in OSCCs, and especially their recurrent tissues, extend beyond biomarker development. Activation of L1 in cancer has many consequences (insertion, deletion, duplication, etc.) that lead to structural variation of the genome [ 9 , 11 , 21 ] and accelerates genome plasticity [ 21 , 56 , 57 ]. Extensive increase in structural variation has been seen in many metastatic cancers, although the factors that increase structural variation are not entirely known [ 58 ]. It is possible that elevated expression of L1-encoded proteins could aggravate structural variation in the recurrent OSCC genome and contribute to cancer progression. Moreover, multiple studies have shown that elevated L1 activity may induce interferon production and an immune response, [ 59 – 61 ]. There is also increased understanding that immunological change is a hallmark of cancer and can influence tumor development and outcome [ 62 , 63 ]. We therefore believe expanded investigation of retrotransposon activity in the context of OCSS is warranted. Conclusion The global incidence of oral cancer is very common, with a significant number of new cases and deaths reported each year. In India oral cancer is the second most common cancer, primarily due to wide-spread consumption of chewing tobacco. Recurrence after surgical removal of cancer is often reported due to limited information that distinguishes recurrent from non-recurrent patients. Our work stems from previous findings that upregulation of L1 retrotransposon is a common factor in many cancers. Here, we investigated difference in expression of L1 retrotransposon-encoded proteins, along with eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44), between non-recurrent and recurrent patients. We found significant upregulation of both the L1-encoded proteins and CD105 and downregulation of EGFR in the recurrent samples when compared to non-recurrent samples. In summary, our results showed that combined expression of both L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p), CD105, and EGFR can be used as a prognostic marker to identify OSCC patients at high risk of developing recurrence. Abbreviations %: percentage; μg: microgram; µl: microliter; AUC: Area Under Curve; CV: column volume; CD105: Cluster of differentiation 105; CD44: Cluster of differentiation 44; DAB: Diaaminobenzidinetetrahydrochloride; DFS: Disease-free survival; DTT: Dithiothreitol; EGTA: Ethylene glycol tetra-Acetic Acid; EGFR: Epidermal growth factor receptor; EN: Endonuclease; FFPE: formalin fixed paraffin embedded; HIF: Hypoxia-inducible factor; IHC: Immunohistochemistry; kDa: Kilo Dalton; LINE-1/L1: Long Interspersed Element-1; miRNA: MicroRNA; MMP9: Matrix metalloprotease 9; MVD: Micro-vessel density; NaCl: Sodium Chloride; Na 3 VO 4 : sodium orthovanadate; NBF: Neutral Buffered Formalin; NR: Nonrecurrent; ORF: Open Reading Frame; OSCC: Oral Squamous Cell Carcinoma; PBS-T: Phosphate Buffered Saline-Tween 20; PCNA: Proliferating Cell Nuclear Antigen; PDL1: Programmed Cell Death Ligand 1; PMSF: phenylmethanesulfonyl fluoride; R: Recurrent; ROC: Receiver Operating Characteristic; RT: Reverse transcriptase. Declarations Ethics approval and consent to participate This study was approved by the Institutional ethics committee of All India Institute of Medical Sciences, Rishikesh (Letter No. AIIMS/IEC/22/191.Date:Feb18,2022) and Indian Institute of Technology Roorkee (Letter No. IITR/IEC/22/009. Date: Sept 10, 2022). All methods were performed in accordance with relevant guidelines and regulations. Consent for publication: No identifying individual person’s data are disclosed. Availability of data and materials: Plasmid constructs, ORF1 and ORF2 antibody used in this study will be provided to academic researcher upon request. Competing Interest: The authors declare that they have no competing interest. Funding: This work was supported by a grant to PKM from the Dept. of Science and Technology (DST), India (grant no. CRG/2021/002071), Dept. of Biotechnology (DBT), India (grant no. BT/PR41540/BRB/10/1960/2020) and Uttarakhand State Council for Science and Technology (UCOST) India (grant no. UCS&T/R&D 14/21-22/20409). Author Contributions: SK and PKM had complete access to all the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis. Concept and design: PKM. Acquisition, analysis and interpretation of data: SK and PKM. Sample acquisition & Histopathology: SAU, VKD, PD and AT. Antibody generation and immunohistochemistry: MG, KM, GS and SK. Drafting of the manuscript: SK and PKM. Critical revision of the manuscript for important intellectual content: JLG. Statistical analysis: SK. Obtained funding and Supervision: PKM. Acknowledgements: We thank Dr. Manoj Kumar (AIIMS Rishikesh, India) for helping with post-operative patient’s sample collection. We thank Dr. Amit Dutt and Mr. Rudransh Singh (Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Hospital, India) for providing AW13516 oral cancer cell line. References Sung H, et al. 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Competition between two HUSH complexes orchestrates the immune response to retroelement invasion. Mol Cell. 2024;84:2870–81. Sakowska J, Arcimowicz Ł, Jankowiak M, Papak I, Markiewicz A, Dziubek K, Kurkowiak M, Kote S, Kaźmierczak-Siedlecka K, Połom K, Marek-Trzonkowska N, Trzonkowski P. Autoimmunity and Cancer-Two Sides of the Same Coin. Front Immunol. 2022;13:793234. Wang M, Chen S, He X, Yuan Y, Wei X. Targeting inflammation as cancer therapy. J Hematol Oncol. 2024;17:13. Additional Declarations No competing interests reported. Supplementary Files SuppleFig112.pdf Supplemental figure legends Supplementary Figure 1: Workflow of QuPath analysis software to measure the DAB stain intensity. Supplementary Figure 2: Localization of L1ORF1p and L1ORF2p in cytoplasm and nucleus. Representative images at 40x magnification demonstrate localization and expression of L1ORF1p and L1ORF2p in post-operative OSCC samples. Black arrows denote nuclear staining. Supplementary Figure 3: IHC staining of both L1 proteins (L1ORF1p and L1ORF2p) in non-recurrent (n=35) and recurrent (n=35) post-operative OSCC samples. (A) L1ORF1p expression in non-recurrent samples, (B) L1ORF1p expression in recurrent samples, (C) L1ORF2p expression in non-recurrent samples, (D) L1ORF2p expression in recurrent samples. Supplementary Figure 4: Representative micrographs showing Immunocytochemistry of p53, PCNA, EGFR and L1ORF1p in HEK-293T, HeLa, HCT116, and AW13516 cell lines [35]. AW13516 (oral squamous cell carcinoma of the tongue) cell line staining (bottom panels) shows significantly greater expression of L1 ORF1p and EGFR compared to other cell lines. Supplementary Figure 5-12: IHC expression analysis of P53 (Fig. 5), PCNA (Fig. 6), CD105 (Fig. 7), Ki67 (Fig. 8), MMP9 (Fig. 9), EGFR (Fig. 10), CD44 (Fig. 11), and PDL1 (Fig. 12) in non-recurrent (n=25) (A) and recurrent (n=25) (B) OSCC samples. Suppletables.xls Supplemental table legends Supplementary Table 1: Clinicopathological details of all patients (n=114) used in the study. Patient samples (SI) were assigned serial numbers C1-C114. The table provides information about histological cancer grade (WDSCC: well differentiated squamous cell carcinoma; MDSCC: moderately differentiated squamous cell carcinoma; PDSCC: poorly differentiated squamous cell carcinoma) and their TNM (Tumor, Node, and Metastasis) staging. Supplementary Table 2- Immunostaining H-score data of L1 ORF1p and ORF2p (35 non-recurrent and 35 recurrent OSCC samples) and the survival information. Each patient's date of surgery (DOS) is mentioned in the table. The patient's survival was determined from the date of their last follow-up (FP) mentioned in the follow-up column. Patients expired during the study: their expiration date is mentioned in the follow-up column. Supplementary Table 3: Immunnostaining H-Score data of p53, PCNA, CD44, EGFR, Ki67, MMP9, CD105, PDL1, ORF1 and ORF2 (n=25). (A. Non-recurrent sample B. Recurrent sample) Supplementary Table 4- The Pearson correlation matrix shows an association between protein biomarker expression and OSCC recurrence. p-value of significance (p≤0.05) calculated from two-tailed analysis. (A) Association between L1ORF1p and L1ORF2p with OSCC recurrence (n=35); (B) Association between all 10 biomarkers with OSCC recurrence (n=25). Supplementary Table 5- Multivariate prediction model to check the statistical association of expression of biomarkers with recurrence outcome in OSCC patients. (A) involves L1ORF1p and L1ORF2p (n=70); (B) involves CD105, EGFR, L1ORF1p, and L1ORF2p (n=50); (C) involves all 10 protein biomarkers (n=50). The tables display each multivariate model with a predictive probability cut-off of 0.5. There is a predicted probability value for each, and based on the cut-off value, they are identified as either true positive/negative or false positive/negative (predicted 0/1). Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6319447","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444450138,"identity":"6fd0df00-0341-4880-8cda-15882e7681d6","order_by":0,"name":"Sujoy Kundu","email":"","orcid":"","institution":"Indian Institute of Technology Roorkee","correspondingAuthor":false,"prefix":"","firstName":"Sujoy","middleName":"","lastName":"Kundu","suffix":""},{"id":444450140,"identity":"de2e0abb-9502-4ec1-a282-7fcc81680fd3","order_by":1,"name":"Manali Ganguly","email":"","orcid":"","institution":"Indian Institute of Technology Roorkee","correspondingAuthor":false,"prefix":"","firstName":"Manali","middleName":"","lastName":"Ganguly","suffix":""},{"id":444450142,"identity":"15f11f35-9e46-404b-a217-46ef6e2d30ea","order_by":2,"name":"Koel Mukherjee","email":"","orcid":"","institution":"Indian Institute of Technology Roorkee","correspondingAuthor":false,"prefix":"","firstName":"Koel","middleName":"","lastName":"Mukherjee","suffix":""},{"id":444450145,"identity":"523a4d72-cceb-405b-8c32-5ad177221398","order_by":3,"name":"Gopal Sarkar","email":"","orcid":"","institution":"Indian Institute of Technology Roorkee","correspondingAuthor":false,"prefix":"","firstName":"Gopal","middleName":"","lastName":"Sarkar","suffix":""},{"id":444450146,"identity":"b73dcd94-94aa-48cb-9348-9de4c0e36ff8","order_by":4,"name":"Shahab A Usmani","email":"","orcid":"","institution":"All Indian Institute of Medical Sciences (AIIMS) Rishikesh","correspondingAuthor":false,"prefix":"","firstName":"Shahab","middleName":"A","lastName":"Usmani","suffix":""},{"id":444450147,"identity":"4f0dbdc1-a540-4e27-9d76-d1f1e22da1ee","order_by":5,"name":"Vandana Kumar Dhingra","email":"","orcid":"","institution":"All Indian Institute of Medical Sciences (AIIMS) Rishikesh","correspondingAuthor":false,"prefix":"","firstName":"Vandana","middleName":"Kumar","lastName":"Dhingra","suffix":""},{"id":444450148,"identity":"933e5913-64a7-4734-8ad2-c663f1f50b3d","order_by":6,"name":"Prashant Durgapal","email":"","orcid":"","institution":"All Indian Institute of Medical Sciences (AIIMS) Rishikesh","correspondingAuthor":false,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Durgapal","suffix":""},{"id":444450149,"identity":"9f6b7b5d-3a32-4bbd-8b9f-a3420f1e2e1c","order_by":7,"name":"Amit Tyagi","email":"","orcid":"","institution":"All Indian Institute of Medical Sciences (AIIMS) Rishikesh","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"","lastName":"Tyagi","suffix":""},{"id":444450150,"identity":"6cc06b67-e71e-4b7c-9f84-5a4e9d1c4044","order_by":8,"name":"John L. 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B) A representative sample showing the presence of L1ORF1p (left) and L1ORF2p (right) by immunohistochemistry in the operated cancer tissue (top). IHC was also performed in adjacent normal tissue (bottom) and no signal was detected with purified rabbit L1ORF1p and L1ORF2p antibodies. C) Level of expression of L1ORF1p and L1ORF2p in 114 cancer samples detected by IHC and measured using QuPath (H-scores are shown).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/344cf07c8465fdc8debc11b0.jpg"},{"id":82056437,"identity":"b6947a5d-8f1a-4507-b893-ece3e8072630","added_by":"auto","created_at":"2025-05-06 10:33:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":275114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of L1 proteins in the post-operative non-recurrent and recurrent oral cancer samples\u003c/strong\u003e. A) Representative micrographs showing the presence and intensities of L1ORF1p and L1ORF2p in the non-recurrent and recurrent oral cancer samples following DAB IHC staining. B) IHC was performed on 35 non-recurrent and 35 recurrent samples using L1ORF1p and L1ORF2p antibodies. The number of DAB stained cells and their intensities were measured semi-quantitatively using QuPath. A two-tailed unpaired Welch t-test was performed to see any significant difference in the expression between recurrent and non-recurrent samples and represented by box plot. The mean, standard deviation, and p values are shown for each plot (p ≤ 0.001).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/3bb7ff83366eea24fcdbdce8.jpg"},{"id":82055760,"identity":"8835a041-9336-47dc-96f7-1ac2166d730f","added_by":"auto","created_at":"2025-05-06 10:25:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of p53, PCNA, CD105, ki67, MMP9, EGFR, CD44 and PDL1 in the post-operative non-recurrent and recurrent oral cancer samples. \u0026nbsp;\u003c/strong\u003eImmunohistochemistry (IHC) of tissue sections stained with (A) anti-p53, (B) anti-PCNA, (C) anti-CD 105, (D) anti-ki67, (E) anti-MMP9, (F) anti-EGFR, (G) anti-CD44, and (H) anti-PDL1. IHC was performed on 25 non-recurrent and 25 recurrent samples. Representative micrographs of two recurrent and two non-recurrent samples are shown for each antibody staining (10X image with 40X inset). Immunostaining H-Score was measured by QuPath software to compare the expression difference between recurrent and non-recurrent samples. The box plots represent the average expression difference between recurrent and non-recurrent samples. A two-tailed unpaired Welch t-test was performed to determine any significant difference in expression between recurrent and non-recurrent samples. The mean, standard deviation, and p values are shown for each plot (p≤0.05 is considered statistically significant). The H-scores of CD105 and EGFR showed significant differences between non-recurrent and recurrent samples.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/63f743ec29baa66fdaf1ac78.jpg"},{"id":82053204,"identity":"fe982f2e-7941-4195-b38a-21ab758b1a7a","added_by":"auto","created_at":"2025-05-06 10:09:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePearson correlation analysis of protein biomarker expression to assess oral cancer recurrence\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003ePositive and negative correlations are displayed in blue and red, respectively. Color intensity is proportional to the correlation coefficients. Significance p-values (p ≤ 0.05 being statistically significant) were calculated from two-tailed analyses. (A) Association between L1ORF1p and L1ORF2p expression with oral cancer recurrence (n=70). (B) Association between expression of 10 biomarkers (p53, PCNA, CD105, Ki67, MMP9, PDL1, CD44, EGFR, L1ORF1p, and L1ORF2p) with oral cancer recurrence (n=50).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/4d381d93d748d27316ad99f4.jpg"},{"id":82053207,"identity":"de7443bc-64cb-4bc4-a78b-88fb01e04602","added_by":"auto","created_at":"2025-05-06 10:09:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReceiver operating characteristic (ROC) curves using multivariate models to predict OSCC recurrence\u003c/strong\u003e. (A) Area under curve (AUC) value is 0.9020 (p\u0026lt;0.0001)\u003cstrong\u003e \u003c/strong\u003ewhen L1ORF1p and L1ORF2p were used as biomarkers to construct the ROC curve (left). (B) Area under curve (AUC) value is 0.856 (p\u0026lt;0.0001)\u003cstrong\u003e \u003c/strong\u003ewhen CD105, EGFR, L1ORF1p, and L1ORF2p biomarkers are used to construct the ROC curve.\u003cstrong\u003e \u003c/strong\u003e(C) ROC curve generated with all 10 biomarkers showed an AUC value 0.9536 (p\u0026lt;0.0001). For each ROC curve, a predicted vs observed scatter plot is shown (right). The x-axis represents the actual binary outcome (0/1), and the y-axis represents the predicted outcome probability with a cut-off value 0.5. Blue dots (observed 0) and red (observed 1) represent non-recurrent and recurrent samples. Each dot on the plot has its own predicted probability value that determines whether a sample will be true positive/negative or false positive/negative; it determines the predictive power of the multivariate model.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/c86bd2f926c79f62d7db3358.jpg"},{"id":82053210,"identity":"466243fc-0af3-4536-9385-976e0ead4f82","added_by":"auto","created_at":"2025-05-06 10:09:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":106285,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier graphs showing cumulative probability of patients’ survival in connection with expression of (A) p53, (B) PCNA, (C) CD105, (D) Ki67, (E) MMP9, (F) EGFR, (G) CD44. (H) PDL1, (I) L1ORF1p, and (J) L1ORF2p. Patients expressing high L1ORF1p (p=0.0058) and L1ORF2p (p=0.0131) showed significantly poorer survivability. Patients with low EGFR (p=0.0296) also showed reduced survivability.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/ed759c84a411f6bb33a7b5e6.jpg"},{"id":95715375,"identity":"844fa79b-1469-409a-b280-290315d546c4","added_by":"auto","created_at":"2025-11-12 08:39:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2474466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/f3335758-e070-4695-ac6b-c0e9d985ce9d.pdf"},{"id":82053223,"identity":"18cbbf55-dd01-4712-99c0-1655f11b12ad","added_by":"auto","created_at":"2025-05-06 10:10:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20251077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental figure legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e: Workflow of QuPath analysis software to measure the DAB stain intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2:\u003c/strong\u003e Localization of L1ORF1p and L1ORF2p in cytoplasm and nucleus. Representative images at 40x magnification demonstrate localization and expression of L1ORF1p and L1ORF2p in post-operative OSCC samples. Black arrows denote nuclear staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3:\u003c/strong\u003e IHC staining of both L1 proteins (L1ORF1p and L1ORF2p) in non-recurrent (n=35) and recurrent (n=35) post-operative OSCC samples. (A) L1ORF1p expression in non-recurrent samples, (B) L1ORF1p expression in recurrent samples, (C) L1ORF2p expression in non-recurrent samples, (D) L1ORF2p expression in recurrent samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eRepresentative micrographs showing\u003cstrong\u003e \u003c/strong\u003eImmunocytochemistry of p53, PCNA, EGFR and L1ORF1p in HEK-293T, HeLa, HCT116, and AW13516 cell lines [35]. AW13516 (oral squamous cell carcinoma of the tongue) cell line staining (bottom panels) shows significantly greater expression of L1 ORF1p and EGFR compared to other cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5-12:\u003c/strong\u003e IHC expression analysis of P53 (Fig. 5), PCNA (Fig. 6), CD105 (Fig. 7), Ki67 (Fig. 8), MMP9 (Fig. 9), EGFR (Fig. 10), CD44 (Fig. 11), and PDL1 (Fig. 12) in non-recurrent (n=25) (A) and recurrent (n=25) (B) OSCC samples.\u003c/p\u003e","description":"","filename":"SuppleFig112.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/4d566a61b30203f702369999.pdf"},{"id":82053203,"identity":"aaa379f4-5314-42c1-b56e-ca9e417c5651","added_by":"auto","created_at":"2025-05-06 10:09:59","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":121344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental table legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 1: Clinicopathological details of all patients (n=114) used in the study\u003c/strong\u003e. Patient samples (SI) were assigned serial numbers C1-C114. The table provides information about histological cancer grade (WDSCC: well differentiated squamous cell carcinoma; MDSCC: moderately differentiated squamous cell carcinoma; PDSCC: poorly differentiated squamous cell carcinoma) and their TNM (Tumor, Node, and Metastasis) staging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2- Immunostaining H-score data of L1 ORF1p and ORF2p (35 non-recurrent and 35 recurrent OSCC samples) and the survival information\u003c/strong\u003e. Each patient's date of surgery (DOS) is mentioned in the table. The patient's survival was determined from the date of their last follow-up (FP) mentioned in the follow-up column. Patients expired during the study: their expiration date is mentioned in the follow-up column.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 3: Immunnostaining H-Score data of p53, PCNA, CD44, EGFR, Ki67, MMP9, CD105, PDL1, ORF1 and ORF2 (n=25).\u003c/strong\u003e (A. Non-recurrent sample B. Recurrent sample)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 4- The Pearson correlation matrix shows an association between protein biomarker expression and OSCC recurrence.\u003c/strong\u003e p-value of significance (p≤0.05) calculated from two-tailed analysis. (A) Association between L1ORF1p and L1ORF2p with OSCC recurrence (n=35); (B) Association between all 10 biomarkers with OSCC recurrence (n=25).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 5- Multivariate prediction model to check the statistical association of expression of biomarkers with recurrence outcome in OSCC patients.\u003c/strong\u003e (A) involves L1ORF1p and L1ORF2p (n=70); (B) involves CD105, EGFR, L1ORF1p, and L1ORF2p (n=50); (C) involves all 10 protein biomarkers (n=50). The tables display each multivariate model with a predictive probability cut-off of 0.5. \u0026nbsp;There is a predicted probability value for each, and based on the cut-off value, they are identified as either true positive/negative or false positive/negative (predicted 0/1).\u003c/p\u003e","description":"","filename":"Suppletables.xls","url":"https://assets-eu.researchsquare.com/files/rs-6319447/v1/072fa66b1a6c181039c5d442.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deregulation of L1 retrotransposon-encoded protein expression in oral cancer recurrence","fulltext":[{"header":"Background","content":"\u003cp\u003eAccording to the Globocan report, oral cancer of the lip and oral cavity ranked 16th among all the reported cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Almost 378,000 new cases and 178,000 deaths were reported from this particular cancer worldwide in 2020[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The global incidence and mortality rate of this particular cancer are more than double for men compared to women. Cancer of the tongue, buccal mucosa and lip mucosa is extremely common in India due to excessive use of betel quid and tobacco chewing [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Multiple genes required for normal growth and maintenance of cells show mutations in this particular cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although surgical removal of cancer tissue is effective, patients often report recurrence within 6 to 12 months after surgery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Biomarker expression analysis through immunohistochemistry (IHC) is often used as the gold standard to predict the behaviour of cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Here we have collected 114 patient samples with the history of excessive use of tobacco chewing and smoking habit. Here we collected 114 patient samples with a history of excessive tobacco chewing and/or smoking habit, and sought to determine if expression analysis of L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) in these oral cancer patients (n\u0026thinsp;=\u0026thinsp;114) is efficacious in distinguishing recurrent from non-recurrent cancer.\u003c/p\u003e \u003cp\u003eLong Interpersed Element (LINE1 or L1) is an autonomous non-LTR retrotransposon with around 500,000 copies occupying 17% of the human genome [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although most copies are silent due to mutations, it was originally estimated 80 to 100 copies are potentially active in a human genome [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], although subsequent estimates have varied [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. An active human L1 is 6.0 kb in length, and contains a 900 nt 5\u0026rsquo;-UTR with internal promoter, two open-reading frames (ORFs), designated ORF1p and ORF2p, separated by a small inter-ORF spacer sequence and followed by a\u0026thinsp;~\u0026thinsp;200 bp 3\u0026rsquo;-UTR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. L1 ORF2p encodes a 150 kDa protein with reverse transcriptase (RT) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and endonuclease (EN) activities [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], whereas L1 ORF1p encodes a 40 kDa protein with demonstrated single-stranded nucleic acid binding and nucleic acid chaperone activities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. L1 retrotransposons are silent in normal somatic tissues due to multiple layers of restriction imposed by cellular factors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Cancer tissues lose this restriction by some poorly defined, but possibly epigenetic mechanisms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and thus L1 becomes activated [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Previous studies showed that the expression of L1 ORF1p is significantly elevated and a potential biomarker in numbers of cancers [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. By performing a pilot study with a small cohort of samples, we recently showed significant expression of both the L1-encoded proteins (ORF1p and ORF2p) in operated oral cancer samples [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA limited number of studies have investigated marker expression to distinguish recurrent from non-recurrent oral cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although L1 protein expression is ubiquitous in OSCC, expression status in recurrent and non-recurrent patients is unknown [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Identifying patients who are at risk of developing recurrence post-surgery is crucial in cancer treatment. In this study, we aimed to provide an improved solution for biomarker detection of OSCC recurrence. Therefore, we investigated the expression of L1ORF1p and L1ORF2p, along with eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44), in recurrent and non-recurrent patients. Although all these markers have previously been tested in other studies to assess their expression in oral cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], a direct comparison of their expression between non-recurrent and recurrent OSCC was not explored. We found that 97% of oral cancer samples (110 out of 114) showed significant expression of both L1-encoded proteins. Importantly, we also found a strong association of L1 ORF1p and L1ORF2p expression with oral cancer recurrence. Our study suggests that IHC expression analysis of L1-encoded proteins, along with CD105 and EGFR, before surgery can predict behaviour of the disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient descriptions\u003c/h2\u003e \u003cp\u003e This study was approved by the Institutional ethics committee of All India Institute of Medical Sciences, Rishikesh and Indian Institute of Technology Roorkee. All methods were performed in accordance with relevant guidelines and regulations. Following full written informed consent from patients, post-operative cancer tissues were collected for preparation of formalin-fixed paraffin-embedded tissue blocks (FFPE). Doctors and pathologists verified the histopathology of OSCC in all patient samples. Patient follow-up data is provided in \u003cb\u003eSupple table 2\u003c/b\u003e. Histological grading and staging of cancers were performed as per the American Joint Committee on Cancer (AJCC) classification.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of L1ORF2 (RT1) antibodies in rabbit\u003c/h3\u003e\n\u003cp\u003eThe cloning, expression and purification of hL1RT\u003csub\u003eEH\u003c/sub\u003e (RT1) protein was described earlier [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The purified protein was injected in rabbit to generate the antibody. The protocol used to generate the antibody was as follows:\u003c/p\u003e \u003cp\u003eDay 0: Pre-immunization bleed (0.5 ml)\u003c/p\u003e \u003cp\u003eDay 1: Immunization with 0.75 mg antigen in complete Freud\u0026rsquo;s adjuvant (Sigma, cat no. F5881), subcutaneous (SQ) 1 site\u003c/p\u003e \u003cp\u003eDay 15: Boost with 0.5 mg antigen in incomplete Freud\u0026rsquo;s adjuvant (Sigma, cat no. F5506), SQ 1 site\u003c/p\u003e \u003cp\u003eDay 30: Boost with 0.5 mg antigen in incomplete Freud\u0026rsquo;s adjuvant, SQ 1 site\u003c/p\u003e \u003cp\u003eDay 35: Test-bleed (0.5 ml per rabbit) to check the titer and quality of the generated antibody by ELISA and Western blotting\u003c/p\u003e \u003cp\u003eDay 45: Boost with 0.25 mg antigen in incomplete Freud\u0026rsquo;s adjuvant, SQ 1 site\u003c/p\u003e \u003cp\u003eDay 60: Terminal blood collection (around 15 ml per rabbit from the central artery on the inner edge of the dorsal surface of the ear)\u003c/p\u003e\n\u003ch3\u003ePreparation of antigen affinity column:\u003c/h3\u003e\n\u003cp\u003eSepharose CL-4B (Sigma, cat no. 61970-08-9) resin was used to conjugate the antigen (RT1). One ml of resin was mixed with freshly prepared CNBr solution (2 gm/ml, solvent acetonitrile) and incubated for 2 mins at room temp. Next, 800 \u0026micro;l of 5M NaOH was added to the slurry to adjust to pH 10\u0026ndash;11 and the tube was incubated at 4C for 30 mins with gentle rotation. Following completion of the reaction, the bead slurry was washed with five column volumes (CVs) of ddH\u003csub\u003e2\u003c/sub\u003eO to remove any remaining unreacted CNBr. The beads were then ready to conjugate with the RT1 antigen. Around 10 mg of antigen dialyzed in coupling buffer (100 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 500 mM NaCl; pH 8.3) was incubated with 1 ml of CNBr activated Sepharose CL-4B resin at 4\u0026deg;C overnight with continuous gentle rotation. The next day, beads were washed with 5 CVs of coupling buffer, followed by incubation with 5 CVs of quenching buffer (0.1M Tris-Cl; pH 8.0) at room temperature for 2 hours with continuous gentle rotation to block residual binding sites. After quenching, beads were washed with three cycles of alternating pH buffers, i.e. 5 CVs of low pH (0.1 M sodium acetate; pH 4.0) and high pH (0.1 M Tris-Cl; pH 8.0) buffers one after the other. Finally, the antigen-bound resin was rinsed with 5 CVs of binding buffer (20 mM sodium phosphate buffer; pH 7.4). The RT1 antigen affinity resin was then ready to purify antigen-specific antibody from whole immune serum.\u003c/p\u003e\n\u003ch3\u003ePurification of antigen-specific antibody from immunized serum:\u003c/h3\u003e\n\u003cp\u003eFive hundred microliters of immunized rabbit whole serum was mixed with binding buffer (20 mM sodium phosphate buffer; pH 7.4) in a 1:1 ratio and incubated with 250 \u0026micro;l of antigen-coupled resin for 8\u0026ndash;10 hours at 4\u0026deg;C with gentle rotation. Next, the resin was washed with 5 CVs of wash buffer (10 mM ammonium acetate; pH 4.6) followed by elution of the specific antibody with one CV of elution buffer (0.1 M citric acid). The elution step was repeated five times. Each elution step involves, i) incubation of the antibody-bound resin with elution buffer for two minutes on ice with intermittent gentle tapping, ii) centrifugation at 2000 rpm for two minutes at 4\u0026deg;C, iii) incubation for two minutes on ice to settle the beads completely, iv) collection of the supernatant, and v) immediate neutralization with 1 M Tris-Cl; pH 9.0 to adjust to pH 7\u0026ndash;8. All the collected elution fractions were pooled and concentrated using a 0.5 ml 10-kDa Amicon concentrator (Millipore Sigma) to 2 CVs of resin (the final antibody concentration was 3 mg/ml).\u003c/p\u003e\n\u003ch3\u003ePreparation of antibody affinity resin:\u003c/h3\u003e\n\u003cp\u003eThe purified ORF2p antibody was dialyzed in coupling buffer and then incubated with 500 \u0026micro;l of CNBr-activated Sepharose CL-4B beads at 4\u0026deg;C with continuous gentle rotation overnight. The rest of the procedure was the same as the preparation of antigen affinity column preparation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTissue lysate preparation and immunoblotting:\u003c/h2\u003e \u003cp\u003eThe tissue lysate was prepared by crushing OSCC tissues with a mortar-pestle in liquid N\u003csub\u003e2\u003c/sub\u003e and mixed with RIPA buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.5% NP-40, 1mM DTT, 1mM EGTA, 1mM NaF, 2mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 mM PMSF, supplemented with 1X protease inhibitor cocktail) for 30 minutes with repeated vortexing after each 5 min. The lysate was then centrifuged at 10,000 rpm for 10 min at 4\u0026deg;C, and the supernatant was transferred to a new 1.5 ml tube and stored at -70\u0026deg;C until further use. Around 60 \u0026micro;g of tissue lysate was resolved in a 10% SDS-PAGE gel (Mini-PROTEAN Tetra Cell, Bio-Rad) and transferred to PVDF membrane (Merck Immobilon, cat no. IPVH00010) by applying 100 V for 75 min using a BioRad Mini Trans-Blot Electrophoretic Transfer Cell (transfer buffer composition: 25 mM Tris-Cl, pH 7.6, 192 mM glycine, and 20% methanol). Following transfer to the membrane, the membrane was blocked with 1xPBST containing 5% w/v skimmed milk for 1 hour at room temperature. Then it was probed with primary antibody (purified polyclonal rabbit anti-ORF1(RRM) (1:5000), purified polyclonal rabbit anti-ORF2(RT1) (1:2000), anti-GAPDH (1:2000)) diluted in blocking buffer). The next day, the membrane was washed with 1xPBST 3 times for 10 minutes at room temperature and then incubated with secondary antibody (α-rabbit HRP, dilution 1:60,000; (Jackson ImmunoResearch, cat no. 111-035-003)) diluted in blocking buffer for an hour at room temperature. Following incubation, the membrane was washed 3x in 1xPBST for 10 minutes and then Western blots were developed with Chemiluminescence HRP Substrate (Takara, cat no. T7101A). The signal was detected by exposing the blot to Fuji X-ray film (Fujifilm).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunoprecipitation (IP) of L1ORF2p:\u003c/h3\u003e\n\u003cp\u003eFor immunoprecipitation of L1ORF2p, 200 \u0026micro;l of cancer tissue lysate (total protein\u0026thinsp;~\u0026thinsp;300 \u0026micro;g) was incubated with 30 \u0026micro;l of Sepharose CL-4B beads coupled with L1ORF2p antibody (RT1) in a 1.5 ml micro-centrifuge tube overnight at 4\u0026deg;C with continuous gentle rotation. The following day, flowthrough was separated from the beads by centrifuging the beads at 2000 rpm for 3 minutes at 4\u0026deg;C. Next, the beads were washed with 5 CVs of wash buffer and then eluted with 2.5 CVs of elution buffer. In each elution step, beads were incubated with elution buffer for 5 min on ice with intermittent tapping. After incubation, beads were centrifuged at 2000 rpm for 2 min and then kept on ice for another 2 min to allow the beads to completely settle. Then the elution fraction was collected in a separate MCT tube, and pH was adjusted to 7\u0026ndash;8 by adding neutralization buffer. This complete step was repeated 5 times. All the elution fractions were concentrated to 100 \u0026micro;l volume. From the concentrated sample, 30 \u0026micro;l was resolved in an 8% SDS-PAGE gel to perform immunoblotting for the detection of L1ORF2p in cancer samples.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFormalin fixed paraffin embedded (FFPE) tissue blocks and sections (4 \u0026micro;m thick) were prepared for all 114 post-operative OSCC samples. To perform histological examinations, hematoxylin and eosin staining was performed on tissue sections. Tissue sections were deparaffinised and rehydrated for antigen retrieval. After antigen retrieval, tissue sections were blocked by incubating in blocking solution followed by treatment with 3% hydrogen peroxide to quench endogenous peroxidase activity. Next, tissue sections were incubated with primary antibody at 4\u003csup\u003eo\u003c/sup\u003eC overnight followed by incubation with secondary antibody (1:500 dilution, α-rabbit-HRP (Jackson ImmunoResearch, cat #111-035-003)) for an hour at room temperature. Signal was visualized by adding 3\u0026ndash;3\u0026acute;-diaaminobenzidine tetrahydrochloride (DAB) solution to the slides and counterstaining with hematoxylin. All microscopic pictures were taken at 10X and 40X magnification. The antibodies against p53 (cat #PM101), PCNA (PR065), CD105 (MR1196), Ki67 (PM210), EGFR (PR040), CD44 (MM1121), and PDL1 (MR1247) were from PathnSitu Biotechnologies. MMP9 antibody (cat #BSB2138) was obtained from BIO SB Products and Technology for Molecular Pathology. In-house-generated Ag affinity column purified rabbit polyclonal antibodies against human L1ORF1p [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and L1ORF2p [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] were used in this study.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImage data analysis:\u003c/h2\u003e \u003cp\u003eThe regions of interest (ROI) corresponding to histologically representative areas were identified for each image and confirmed by several authors and results were reviewed by a pathologist. For each tissue, images were taken from the same region for all the markers. The DAB-stained slide images were analysed using QuPath software (version 0.5.1) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This program measures the number of stained cells and their intensity in four-grade semi-quantitative scale (3\u0026thinsp;=\u0026thinsp;high, 2\u0026thinsp;=\u0026thinsp;moderate, 1\u0026thinsp;=\u0026thinsp;low and 0\u0026thinsp;=\u0026thinsp;no stain) and calculates the immunohistochemical score or H-Score (H-Score\u0026thinsp;=\u0026thinsp;3x% high\u0026thinsp;+\u0026thinsp;2x% moderate\u0026thinsp;+\u0026thinsp;1x% low). The image data analysis method is outlined in \u003cb\u003eSupple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eIndividual biomarker expression was analysed first with unpaired Welch\u0026rsquo;s t-test to identify significant differences in protein expression between non-recurrent and recurrent groups of samples. This was followed by two-tailed Pearson correlation test to assess correlation between biomarker expression and disease recurrence. The mean values of biomarker expression were used to compare data sets. The associations of expression of all the markers with clinical outcomes were determined using multivariate logistic regression analysis for the development and testing of the model. Kaplan-Meier survival analysis was used to construct diagrams of patient survival between sample cohorts and their relationship with each biomarker. The statistical analysis was performed using GraphPad Prism statistical software (version 9.5.1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe sample cohort comprises 114 cancer samples comprising 15 females and 99 males with a mean age of 47.4 years \u003cb\u003e(Supple Table\u0026nbsp;1).\u003c/b\u003e The recurrent samples (n\u0026thinsp;=\u0026thinsp;35) included in this study showed the incidence of recurrence within a year of surgical resection of the tumor and loco-regional spread \u003cb\u003e(Supple Table\u0026nbsp;2).\u003c/b\u003e Almost all the patients were addicted to tobacco chewing and/or smoking, in addition to alcohol consumption in some cases. Most of the patients in this study belong to the moderately differentiated category (78%, including 18 non-recurrent and 16 recurrent), and only two patients from the recurrent group showed poorly differentiated grades (4%). The rest of the samples (18%) exhibited a well-differentiated nature \u003cb\u003e(Supple Table\u0026nbsp;1).\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExpression analysis of L1 retrotransposon encoded proteins (L1ORF1p and L1ORF2p):\u003c/h2\u003e \u003cp\u003eIn our previous study to check the expression of L1 encoded ORF1p we used an in-house antibody [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The antibody was raised against the RRM domain of L1ORF1p in rabbit and the immunized whole serum from the rabbit without further purification was used to detect L1ORF1p by Western blotting and IHC analysis. Here we purified the antibody using an antigen affinity column and the purity was checked by performing immunoblotting using operated cancer samples. The antigen column purified L1ORF1p antibody showed a discrete single band at around 40 kDa, the proposed molecular weight of L1ORF1p, in the patient cancer lysate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e The purified L1ORF1p antibody was used to check the expression of L1ORF1p in an operated oral cancer sample and adjacent normal tissue by performing immunohistochemistry. The operated cancer sample showed significant staining whereas adjacent normal tissue was completely negative and showed no staining with the L1ORF1p antibody \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Next, IHC was conducted to assess the expression of L1ORF1p in 114 operated OCSS samples \u003cb\u003e(Supple Table\u0026nbsp;1).\u003c/b\u003e We found 97% of samples (110 out of 114) with significant staining with anti-L1ORF1p. L1ORF1p staining intensity was measured using QuPath, analysis software which measures DAB staining semi-quantitatively [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] \u003cb\u003e(Supple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. QuPath analysis determined four grades of overall L1ORF1p expressions in the cohort samples: high (43.8%, n\u0026thinsp;=\u0026thinsp;50, H-scores\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;56), moderate (29.8%, n\u0026thinsp;=\u0026thinsp;34, H-scores\u0026thinsp;=\u0026thinsp;28\u0026ndash;56), low (22.8%, n\u0026thinsp;=\u0026thinsp;26, H-scores\u0026thinsp;=\u0026thinsp;5\u0026ndash;28) and no expression (3.6%, n\u0026thinsp;=\u0026thinsp;4, H-scores\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;5) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. L1ORF1p staining was readily detected mostly in the cytoplasm of tumor cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e);\u003c/b\u003e a few samples also showed L1ORF1p nuclear staining \u003cb\u003e(Supple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Many samples showed a diffuse pattern of staining all over the cancer tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Similarly, we interrogated L1ORF2p expression in the same large cohort (n\u0026thinsp;=\u0026thinsp;114) of operated oral cancer samples. In a pilot study, we previously showed elevated expression of L1ORF2p in 39 operated oral cancer samples using an in-house L1ORF2p antibody [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This antibody was generated in mice using a 10-kDa fragment (hRT\u003csub\u003eEH\u003c/sub\u003e, L1ORF2p amino acids 479\u0026ndash;558, accession number: AF148856) from the RT domain of ORF2p. Immunized whole serum from mouse without further purification was used to detect ORF2p by IHC and immunoblotting [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Here we raised an L1ORF2p antibody in rabbit, and the specific polyclonal antibody against L1ORF2p was purified by passing the whole serum through an antigen (Ag) affinity column. The purity of the column-purified L1ORF2p Ab was checked by immunoblotting using lysate prepared from the patient samples. As immunoblotting with the total lysate didn\u0026rsquo;t show any L1ORF2p signal, we next performed an immunoprecipitation experiment of L1ORF2p from the cancer tissue lysates. The Ag column-purified L1ORF2p antibody was cross-linked with sepharose beads and incubated with the cancer lysate, followed by washing and elution of bound L1ORF2p with citric acid elution buffer. The elution fractions were pooled, concentrated and analysed by immune blotting to determine the presence of L1ORF2p. A discrete band at around 150 kDa corresponding to the proposed molecular weight of L1ORF2p was detected in the two cancer samples analysed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Next, the newly made L1ORF2p rabbit antibody was used to detect by IHC L1ORF2p in an operated oral cancer sample along with adjacent normal mucosa. The L1ORF2p antibody showed distinct staining in the cancer samples and no stain in the normal mucosa tissue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e Next, the expression analysis of L1ORF2p was assayed by IHC for the 114 oral cancer samples \u003cb\u003e(Supple Table\u0026nbsp;1).\u003c/b\u003e The results showed that all the samples positive for L1ORF1p (110 of 114, 97%) showed a significant expression of L1ORF2p, mostly in the cytoplasm; only a few samples showed a minor fraction of L1ORF2p in the nucleus \u003cb\u003e(Supple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Expression analysis using QuPath showed high (40.3% n\u0026thinsp;=\u0026thinsp;46, H-scores\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;84), moderate (43.9%, n\u0026thinsp;=\u0026thinsp;50, H-scores\u0026thinsp;=\u0026thinsp;42\u0026ndash;84), low (12.3%, n\u0026thinsp;=\u0026thinsp;14, H-scores\u0026thinsp;=\u0026thinsp;12\u0026ndash;42) and no (3.6%, n\u0026thinsp;=\u0026thinsp;4, H scores\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;12) ORF2p expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Thus, both the Ag column-purified L1ORF1p and L1ORF2p polyclonal rabbit antibodies are very effective in detecting L1 proteins in the operated oral cancer samples. We also found that L1ORF1p and L1ORF2p is ubiquitous: nearly all the oral cancer samples included in this study significantly expressed both the L1-encoded proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eL1 protein expression is significantly elevated in recurrent versus non-recurrent oral cancer\u003c/h2\u003e \u003cp\u003eAlthough L1 expression is a hallmark for many cancers, its relation with recurrence outcome after surgery is unknown [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Here, we have investigated the expression of L1ORF1p and L1ORF2p between recurrent and non-recurrent cancer samples. Among 114 samples, 35 samples belonging to the recurrent categories showed loco-regional relapse of cancer within a year after surgery \u003cb\u003e(Supple Table\u0026nbsp;2).\u003c/b\u003e Here, we have compared the expression of both the L1 encoded proteins between 35 non-recurrent and 35 recurrent samples by using QuPath software (version 0.2.0-m4). Significantly, higher expression was observed for both the L1 proteins when we compared the recurrent group with the non-recurrent \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The IHC images of 35 recurrent and non-recurrent samples are shown in supple Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. L1ORF1p and L1ORF2p showed almost 2.5-fold and 1.8-fold increased expression, respectively, in the recurrent samples [(H-score: L1ORF1p non-rec\u0026thinsp;=\u0026thinsp;31.6, rec\u0026thinsp;=\u0026thinsp;80.2) and (H-score: L1ORF2p non-rec\u0026thinsp;=\u0026thinsp;61.2, rec\u0026thinsp;=\u0026thinsp;108.7) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eL1ORF1p expression in an established oral cancer cell line\u003c/h2\u003e \u003cp\u003eThe expression of L1ORF1p was analysed in an established oral squamous cell carcinoma cell line (AW13516) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and compared with other cancer cell lines (human embryonic kidney HEK293T, cervical cancer HeLa, and colon cancer HCT116 lines). We showed very high expression of L1ORF1p in AW13516 cells \u003cb\u003e(Supple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e when compared with other cancer cell lines. The expression of other cancer biomarkers, p53, PCNA and EGFR, was also analysed, and the results showed significant higher expression of p53 and EGFR in AW13516 \u003cb\u003e(Supple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e when compared to the other cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExpression analysis of other biomarkers in the recurrent and non-recurrent oral cancer samples\u003c/h2\u003e \u003cp\u003eAlong with the L1 retrotransposon encoded proteins, we also analysed eight established cancer biomarkers often used for IHC analysis of patient cancer samples. These biomarkers are p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The expression analysis of these eight markers was analysed by performing IHC in a group of 25 recurrent and 25 non-recurrent oral cancer patient samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupple\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;12, \u003cb\u003eSupple table 3).\u003c/b\u003e The results showed a significant upregulation of CD105 [(H score: CD105, non-rec\u0026thinsp;=\u0026thinsp;17.46, rec\u0026thinsp;=\u0026thinsp;34.62) (p\u0026thinsp;=\u0026thinsp;0.0473)] and downregulation of EGFR [(H score: EGFR, non-recurrent\u0026thinsp;=\u0026thinsp;118.97, recurrent\u0026thinsp;=\u0026thinsp;84.87) (p\u0026thinsp;=\u0026thinsp;0.0296)] in the recurrent samples compared with non-recurrent samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The other six markers (p53, PCNA, ki67, MMP8, EGFR, PDL1 and CD44) didn\u0026rsquo;t show any significant changes between non-recurrent and recurrent samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTwo-tailed Pearson test for recurrence assessment by investigating biomarker expression\u003c/h2\u003e \u003cp\u003eNext, we performed two-tailed Pearson test to find any association of L1 protein expression with recurrence outcome. The results show that expression levels of both L1 proteins are strongly correlated [(r value: L1ORF1p\u0026thinsp;=\u0026thinsp;0.59, L1ORF2p\u0026thinsp;=\u0026thinsp;0.50 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The same test was performed by including the expression values obtained from eight established biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) along with L1ORF1p and L1ORF2p. The result of the two-tailed Pearson test when including ten biomarkers showed that expressions of CD105, L1ORF1p and L1ORF2p are positively correlated [(r value: CD105\u0026thinsp;=\u0026thinsp;0.28 (p\u0026thinsp;=\u0026thinsp;0.046), L1ORF1p\u0026thinsp;=\u0026thinsp;0.53 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), L1ORF2p\u0026thinsp;=\u0026thinsp;0.39 (p\u0026thinsp;=\u0026thinsp;0.0048)], whereas the expression of EGFR showed a negative association [(r-value: EGFR = -0.31 (p\u0026thinsp;=\u0026thinsp;0.030)] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cb\u003eSupple table 4\u003c/b\u003e). The expression of p53, PCNA, Ki67, CD44, MMP9 and PDL1 didn\u0026rsquo;t show significant association with recurrence outcome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cb\u003eSupple table 4).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of Receiver Operating Characteristic (ROC) curves as predictors of recurrence\u003c/h2\u003e \u003cp\u003eMultiple logistic regression analysis was used to generate a receiver operating curve (ROC) for the diagnostic ability of biomarker expression as predictors of recurrence. Considering the expression of L1ORF1p and L1ORF2p, we found that 80% of the recurrent samples (29 true positive and 7 false negative out of 35 recurrent samples included in this study) have a predicted value above 0.5, with an area under the curve (AUC) of 0.90 (95% confidence interval (CI) 0.8339 to 0.9702 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cb\u003eSupple Table\u0026nbsp;5)\u003c/b\u003e. Similarly, the analysis predicts 28 out of the 35 non-recurrent samples are true non-recurrent samples \u003cb\u003e(Supple Table\u0026nbsp;5)\u003c/b\u003e. The same analysis was performed by including the eight other biomarkers with L1ORF1p and L1ORF2p, and the result showed that 92% of the samples (23 out of 25 samples) were correctly classified as recurrent with an AUC of 0.95 (95% CI 0.9037 to 1.0) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eSupple Table\u0026nbsp;5)\u003c/b\u003e. In addition, the ROC curve was also generated using four biomarkers (CD105, EGFR, L1ORF1p and L1ORF2p) that showed significant expression changes between non-recurrent and recurrent samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProtein biomarker expression in relation to overall patient survival\u003c/h2\u003e \u003cp\u003eNext, we investigated if there was any correlation between individual marker expression and patient survivability by Kaplan Meier analysis. Comparing the survivability curves among all ten biomarkers, it is evident that the curves generated using L1ORF1p, L1ORF2p and EGFR are the most significant in predicting poor overall survivability (L1ORF1p, p\u0026thinsp;=\u0026thinsp;0.0058; L1ORF2p, p\u0026thinsp;=\u0026thinsp;0.0131; EGFR. p\u0026thinsp;=\u0026thinsp;0.0296) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer in the lip and oral cavity is very common in India due to excessive use of chewing tobacco [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here we investigated the expression of L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) in a large cohort sample (n\u0026thinsp;=\u0026thinsp;114) of patients with a history of excessive use of tobacco [Supple table 1]. We generated in-house antibodies against both the L1-encoded proteins. Previously we reported Western blotting and IHC analyses in a small cohort of oral cancer samples using our in-house L1ORF1p and L1ORF2p antibodies in the form of immune whole serum without further purification [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here we purified both antibodies (anti-L1ORF1p and anti-L1ORF2p) to homogeneity by passage through their respective antigen affinity columns and checked specificity by immunoblotting before using them for IHC analysis of a large number of oral cancer samples. In our previous study, we reported an L1ORF2p antibody that was generated in mice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Here the same antigen was injected into rabbit, which produced a very effective L1ORF2p antibody. Although numerous, including commercial, antibodies are available to detect endogenous L1ORF1p [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], there are few effective non-commercial antibodies that report detection of endogenous L1ORF2p [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We found our antigen column-purified antibodies against both the L1 proteins to be very effective for detecting L1 proteins in cancer samples. Previous reports from our and others' work demonstrated that almost 60% of head and neck cancer samples showed expression of L1-encoded proteins [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, our sample cohort, collected mostly from tobacco-addicted patients, showed 97% expressing both L1-encoded proteins.\u003c/p\u003e \u003cp\u003eIncreased expression of L1ORF1p has been observed in many cancers [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A pioneer work from Rodic et al. showed that more than half of head \u0026amp; neck cancer samples expressed L1ORF1p [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In another study, Chen et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] demonstrated ubiquitous expression of both L1 proteins in breast cancer cell lines and breast cancer tissues. The same study showed that breast cancers with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis and poor patient survival than those with cytoplasmic expression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Harris et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] earlier reported related findings for breast cancer. Hypomethylation of the L1 promoter and consequent high expression of L1ORF1p is common in high grade ovarian carcinoma, the most common and aggressive type of ovarian cancer [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The CpG sequences present in the L1 promoter are heavily methylated in normal tissues and restrict L1 transcription. In contrast, cancer tissues show severe hypomethylation of these CpGs and activated L1 transcription [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. High prevalence of L1 proteins in OSCC could also be due to downregulation of Let-7 miRNA in patient samples [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Let-7 miRNA represses L1 retrotransposition by directly binding to L1 mRNA and impairing translation of L1 encoded proteins [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough a few molecular-based studies have been described, IHC remains the gold standard for diagnosis and therapeutics of oral cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this study, we asked if expression analysis of L1 proteins could predict behaviour of the disease in non-recurrent and recurrent samples. Previous studies analyzed expression of p53, p63, pedoplanin and ki67 in oral leukoplakia and found that the expression of p53 is higher in recurrent compared to non-recurrent leukoplakia [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Another study on advanced stage laryngeal squamous cell carcinoma showed downregulation of MCM2, ki67 and EGFR [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our sample cohort, 35 samples belonging to the recurrent group showed significantly higher expression of both L1 proteins when compared with non-recurrent samples. In parallel, we analysed eight established biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) and found that H-scores of L1 proteins are strongly associated with OSCC recurrence compared to other biomarkers. Among the other eight bio-markers, CD105 showed significant upregulation, while EGFR showed a downtrend trend in expression in recurrent samples. The remaining six biomarkers (P53, PCNA, Ki67, MMP9, CD44, and PDL1) showed no association with recurrent outcome.\u003c/p\u003e \u003cp\u003eMicro-vessel density (MVD) is considered an independent indicator in a variety of human malignancies [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Higher MVD is correlated with progression of malignancy and shorter overall and relapse-free survival. Tumor vasculature is shaped by a number of angiogenesis markers, including CD105 (endoglin). CD105 is expressed on the surface of angiogenic endothelial cells and is induced by hypoxic conditions. In the case of laryngeal SCC, it was found that patients with higher values of CD105 have low disease-free survival (DFS) and significantly higher chances of developing recurrence than those without [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Our study showed a significant upregulation of CD105 expression in recurrent samples compared to nonrecurrent ones. One possible reason for high CD105 expression may be due to downregulation of MASPIN, a potent tumour suppressor that regulates tumour angiogenesis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral past studies have reported the pivotal role of EGFR in the pathogenesis of cancer including HNSCC but none of the surveys focused on EGFR expression in recurrent HNSCC samples [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Carcinogens induce expression of EGFR, which forms a complex with the heterodimer Ku70/80 and helps to repair DNA double-strand breaks by recruiting DNA protein kinase (DNA-PKcs) and the MRN protein complex [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Maiorano et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] showed that oral cancer patients with downregulated expression of membranous EGFR are more likely to suffer recurrence and death. Our study showed a significant down-regulation of EGFR in recurrent oral cancer samples. One possible reason behind the downregulation of EGFR may be hypoxia that leads to the upregulation of prolyl hydroxylase 3 (PHD3), a protein interacting with hypoxia-inducible factor (HIF) to cause internalization of EGFR via endocytosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur two-tailed Pearson test with four (CD105, EGFR, L1ORF1p, and L1ORF2p), out of ten bio-markers analysed in this study, showed significant changes in the recurrent samples when compared to non-recurrent. Parallelly, multiple logistic regression test showed that CD105, EGFR, and L1-encoded proteins (L1ORF1p and L1ORF2p) form a set and can efficiently identify disease recurrence in oral cancer patients. Thus, the expression analysis of L1 encoded proteins, along with CD105 and EGFR, allowed us to distinguish recurrent from non-recurrent samples. The findings of our study have significant clinical relevance and applications in predicting oral cancer recurrence.\u003c/p\u003e \u003cp\u003eImplications for increased LINE-1 expression in OSCCs, and especially their recurrent tissues, extend beyond biomarker development. Activation of L1 in cancer has many consequences (insertion, deletion, duplication, etc.) that lead to structural variation of the genome [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and accelerates genome plasticity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Extensive increase in structural variation has been seen in many metastatic cancers, although the factors that increase structural variation are not entirely known [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. It is possible that elevated expression of L1-encoded proteins could aggravate structural variation in the recurrent OSCC genome and contribute to cancer progression. Moreover, multiple studies have shown that elevated L1 activity may induce interferon production and an immune response, [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. There is also increased understanding that immunological change is a hallmark of cancer and can influence tumor development and outcome [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. We therefore believe expanded investigation of retrotransposon activity in the context of OCSS is warranted.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe global incidence of oral cancer is very common, with a significant number of new cases and deaths reported each year. In India oral cancer is the second most common cancer, primarily due to wide-spread consumption of chewing tobacco. Recurrence after surgical removal of cancer is often reported due to limited information that distinguishes recurrent from non-recurrent patients. Our work stems from previous findings that upregulation of L1 retrotransposon is a common factor in many cancers. Here, we investigated difference in expression of L1 retrotransposon-encoded proteins, along with eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44), between non-recurrent and recurrent patients. We found significant upregulation of both the L1-encoded proteins and CD105 and downregulation of EGFR in the recurrent samples when compared to non-recurrent samples. In summary, our results showed that combined expression of both L1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p), CD105, and EGFR can be used as a prognostic marker to identify OSCC patients at high risk of developing recurrence.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e%: percentage; \u0026mu;g: microgram; \u0026micro;l: microliter; AUC: Area Under Curve; CV: column volume; CD105: Cluster of differentiation 105; CD44: Cluster of differentiation 44; DAB: Diaaminobenzidinetetrahydrochloride; DFS: Disease-free survival; DTT: Dithiothreitol; EGTA: Ethylene glycol tetra-Acetic Acid; EGFR: Epidermal growth factor receptor; EN: Endonuclease; FFPE: formalin fixed paraffin embedded; HIF: Hypoxia-inducible factor; IHC: Immunohistochemistry; kDa: Kilo Dalton; LINE-1/L1: Long Interspersed Element-1; miRNA: MicroRNA; MMP9: Matrix metalloprotease 9; MVD: Micro-vessel density; NaCl: Sodium Chloride; \u0026nbsp;Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e: sodium orthovanadate; NBF: Neutral Buffered Formalin; NR: Nonrecurrent; ORF: Open Reading Frame; OSCC: Oral Squamous Cell Carcinoma; PBS-T: Phosphate Buffered Saline-Tween 20; PCNA: Proliferating Cell Nuclear Antigen; PDL1: Programmed Cell Death Ligand 1; PMSF: phenylmethanesulfonyl fluoride; R: Recurrent; ROC: Receiver Operating Characteristic; RT: Reverse transcriptase.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eapproval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional ethics committee of All India Institute of Medical Sciences, Rishikesh (Letter No. AIIMS/IEC/22/191.Date:Feb18,2022) and \u0026nbsp;Indian Institute of Technology Roorkee (Letter No. IITR/IEC/22/009. Date: Sept 10, 2022). All methods were performed in accordance with relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo identifying individual person’s data are disclosed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Plasmid constructs, ORF1 and ORF2 antibody used in this study will be provided to academic researcher upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by a grant to PKM from the Dept. of Science and Technology (DST), India (grant no. CRG/2021/002071), Dept. of Biotechnology (DBT), India (grant no. BT/PR41540/BRB/10/1960/2020) and Uttarakhand State Council for Science and Technology (UCOST) India (grant no. UCS\u0026amp;T/R\u0026amp;D 14/21-22/20409).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eSK and PKM had complete access to all the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis. Concept and design: PKM. Acquisition, analysis and interpretation of data: SK and PKM. Sample acquisition \u0026amp; Histopathology: SAU, VKD, PD and AT. Antibody generation and immunohistochemistry: MG, KM, GS and SK. Drafting of the manuscript: SK and PKM. Critical revision of the manuscript for important intellectual content: JLG. Statistical analysis: SK. Obtained funding and Supervision: PKM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We thank Dr. Manoj Kumar (AIIMS Rishikesh, India) for helping with post-operative patient’s sample collection. We thank Dr. Amit Dutt and Mr. Rudransh Singh (Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Hospital, India) \u0026nbsp;for providing AW13516 oral cancer cell line.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBagal S, et al. Head and neck cancer burden in India: an analysis from published data of 37 population-based cancer registries. 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J Hematol Oncol. 2024;17:13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"retrotransposon, LINE-1, oral squamous cell carcinoma (OSCC), cancer biomarker, recurrent cancer, non-recurrent cancer, CD105, EGFR, L1ORF1p and L1ORF2p.","lastPublishedDoi":"10.21203/rs.3.rs-6319447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6319447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eOral squamous cell carcinoma (OSCC) often shows recurrence after surgery. To date, there is no significant study on OSCC that predicts recurrence after surgical removal of the cancer. Long INterpersed Element 1 (LINE-1 or L1) retrotransposons show very high activity in many cancers, suggesting a potential role in cancer onset and progression. We wished to assess the value of LINE-1 retrotransposon-encoded proteins (L1ORF1p and L1ORF2p) as biomarkers of OSCC recurrence along with eight other established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe collected 114 post-operative oral cancer patient samples, mostly from tobacco-addicted patients, and analysed the expression of both L1ORF1p and L1ORF2p and eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) by immunohistochemistry. We found 97% of samples (110 out of 114) showed significant expression of both the L1-encoded proteins. Among those 114 samples, 35 samples belonged to the recurrent group and showed strong association with L1ORF1p and L1ORF2p expression when compared with the non-recurrent group. Expression analysis of eight established cancer biomarkers (p53, PCNA, CD105, ki67, MMP9, EGFR, PDL1, and CD44) by immunohistochemistry showed L1 proteins, along with CD105 and EGFR, can form a predictive panel for OSCC recurrence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe study revealed that the combined expression analysis of the four bio-markers (L1ORF1p, L1ORF2p, CD105 and EGFR) can distinguish recurrent from the non-recurrent OSCC samples. The findings have significant clinical relevance and applications in predicting oral cancer recurrence.\u003c/p\u003e","manuscriptTitle":"Deregulation of L1 retrotransposon-encoded protein expression in oral cancer recurrence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 10:09:54","doi":"10.21203/rs.3.rs-6319447/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"164d2a65-92c6-4848-9ab7-685994e8d7b1","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-12T08:39:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 10:09:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6319447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6319447","identity":"rs-6319447","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}