Activation-neutral gene editing of tonsillar CD4 T cells for functional studies in human ex vivo tonsil cultures.

The molecular and immunological properties of tissue-resident resting CD4 T cells are understudied due to the lack of suitable gene editing methods. Here, we describe the ex vivo culture and gene editing methodology ediTONSIL for CD4 T cells from human tonsils. Optimized CRISPR-Cas9 RNP nucleofection results in knockout efficacies of over 90% without requiring exogenous activation. Editing can be performed on multiple cell types in bulk cultures or on isolated CD4 T cells that can be labeled and reintroduced into their tissue environment. Importantly, CD4 T cells maintain their tissue-specific properties such as viability, activation state, or immunocompetence following reassembly into lymphoid aggregates. This highly efficient and versatile gene editing workflow for tonsillar CD4 T cells enables the dissection of molecular mechanisms in ex vivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types.

Anterior Access to the Cervicothoracic Junction via Partial Sternotomy: A Clinical Series Reporting on Technical Feasibility, Postoperative Morbidity, and Early Surgical Outcome.

Surgical access to the cervicothoracic junction (CTJ) is challenging. The aim of this study was to assess technical feasibility, early morbidity, and outcome in patients undergoing anterior access to the CTJ via partial sternotomy. Consecutive cases with CTJ pathology treated via anterior access and partial sternotomy at a single academic center from 2017 to 2022 were retrospectively reviewed. Clinical data, perioperative imaging, and outcome were assessed with regards to the aims of the study. A total of eight cases were analyzed: four (50%) bone metastases, one (12.5%) traumatic instable fracture (B3-AO-Fracture), one (12.5%) thoracic disc herniation with spinal cord compression, and two (25%) infectious pathologic fractures from tuberculosis and spondylodiscitis. The median age was 49.9 years (range: 22-74 y), with a 75% male preponderance. The median Spinal Instability Neoplastic Score (SINS) was 14.5 (IQR: 5; range: 9-16), indicating a high degree of instability in treated cases. Four cases (50%) underwent additional posterior instrumentation. All surgical procedures were performed uneventfully, with no intraoperative complications. The median length of hospital stay was 11.5 days (IQR: 9; range: 6-20), including a median of 1 day in an intensive care unit (ICU). Two cases developed postoperative dysphagia related to stretching and temporary dysfunction of the recurrent laryngeal nerve. Both cases completely recovered at 3 months follow-up. No in-hospital mortality was observed. The radiological outcome was unremarkable in all cases, with no case of implant failure. One case died due to the underlying disease during follow-up. The median follow-up was 2.6 months (IQR: 23.8; range: 1-45.7 months). Our series indicates that the anterior approach to the cervicothoracic junction and upper thoracic spine via partial sternotomy can be considered an effective option for treatment of anterior spinal pathologies, exhibiting a reasonable safety profile. Careful case selection is essential to adequately balance clinical benefits and surgical invasiveness for these procedures.

Intensity modulated proton therapy for early-stage glottic cancer: high-precision approach to laryngeal function preservation with exceptional treatment tolerability.

BACKGROUND: Due to the increasing expertise in transoral laser surgery and image-guided radiation therapy, treatment outcomes have recently improved in patients with early-stage glottic cancer. The objective of the current study was to evaluate intensity-modulated proton therapy (IMPT) as novel treatment option. METHODS: A total of 15 patients with T1-2N0 glottic squamous cell carcinoma, treated between 2017 and 2020, were evaluated. Toxicity was recorded according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.03. RESULTS: The majority were T1a/b tumors (66.7%) and no patient had lymph node or distant metastases. The median total dose was 70 Gy relative biological effectiveness (RBE) (range 66-70 Gy RBE). The one- and two-year OS and metastases-free survival were 100%. One patient developed local failure and received salvage laryngectomy. No higher-grade acute or late toxicity was reported. The mean number of CTCAE grade I and II overall toxicity events per patient was 4.1 (95%-[confidence interval] CI 3.1-5.3) and 1.0 (95%-CI 0.5-1.5). CONCLUSION: High-precision proton therapy of T1-2N0 glottic cancer resulted in exceptional treatment tolerability with high rates of laryngeal function preservation and promising oncological outcome. IMPT has the potential to become a standard treatment option for patients with early-stage laryngeal cancer.

Human Leucocyte Antigens as Prognostic Markers in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The induction and regulation of immune responses depend on human leucocyte antigen (HLA) molecules that present peptides derived from mutated neoantigens or tumor-associated antigens to cytotoxic T cells. The natural variation of HLA molecules might differ between tumor patients and the normal population. Thus, there might be associations between the frequencies of HLA alleles and the survival of tumor patients. METHODS: This issue was studied in a cohort of 84 patients with head and neck squamous cell carcinomas (HNSCCs) of different localizations. The cohort was followed up for more than 10 years. HLA-A/B/C CTS-PCR-SSP typing at 1 field level from blood samples was performed, and the results were correlated with survival. RESULTS: HLA-A*02 was the most prevalent allele in our cohort and was present in 51.1% of patients. The HLA-A*25 and HLA-C*06 alleles exhibited a significantly higher frequency in cancer patients than in the normal population of 174 blood and kidney donors (p = 0.02 and p = 0.01, respectively, Fisher's exact test). For HLA-C*04, a negative impact on overall survival in univariate analysis (p = 0.045) and a negative, but statistically insignificant effect on survival toward poorer survival in multivariate analysis (HR: 1.82; 95% CI: 0.99-3.34, p = 0.053) were observed. In addition, HLA-A*02 was also beneficial for overall survival and progression-free survival in multivariate analysis (HR 0.54; 95% CI: 0.31-0.92; p = 0.023). CONCLUSION: HLA-A*02 allele expression might not only predict better survival but might also indicate superior tumor antigen presentation and, thus, help to select patients who could benefit from T-cell-dependent immunotherapies.

Conjoint analysis of OPRPN and SMR3A protein expression as potential predictive biomarkers for head and neck squamous cell carcinoma after radiotherapy.

Increased submaxillary gland androgen­regulated protein 3A (SMR3A) expression was previously shown to serve as an independent risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and as a surrogate biomarker for active estrogen receptor 2 signaling in radioresistant tumor cells. In the present study, it was aimed to unravel the expression and clinical significance of another member of the opiorphin family, opiorphin prepropeptide (OPRPN), in the radiotherapy for head and neck squamous cell carcinoma (HNSCC). Expression of SMR3A and OPRPN were analyzed for the prior and post fractionated irradiation (4x2 Gy) by double immunofluorescence staining in established HNSCC cell lines as well as by immunohistochemical (IHC) staining in ex vivo tumor tissues. Next, in a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n=96), who received definitive surgery and adjuvant radiotherapy were reviewed, and expression levels of OPRPN protein were detected by IHC. Immunoreactivity scores (IRS) were associated with pathological and clinical risk factors by Chi­square analysis. Survival analysis was performed by using the Kaplan­Meier plot, log­rank test and Cox regression analysis. The expression levels of OPRPN and SMR3A protein were both induced by fractionated irradiation in vitro and ex vivo. In primary tumor samples, IRS of OPRPN was significantly higher than scores of SMR3A expression and positively correlated with expression patterns of SMR3A. SMR3A was confirmed to serve as an unfavorable factor, while OPRPN protein had no significant association with the clinical outcome of patients with OPSCC. A combinational analysis revealed that the subgroup with SMR3AhighOPRPNlow staining pattern had the worst clinical outcome among the various subgroups. Multivariate Cox regression analyses indicated that high expression of SMR3A serves as an independent unfavorable biomarker, while increased expression of OPRPN appears to exert protective function. In summary, the present study indicated that SMR3A and OPRPN serve as potential prognostic markers for HNSCC after radiotherapy.

[Larynx preservation: recommendations for decision-making in T3 laryngeal cancer patients]. / Organerhalt: Entscheidungskriterien für Patienten mit T3-Larynxkarzinom.

BACKGROUND: By today's standard, the optimal treatment of every individual tumor patient is discussed and determined in an interdisciplinary tumor board. According to the new S3 guidelines, larger volume T3 laryngeal cancers which are no longer safely resectable with larynx-sparing surgery are ideal candidates for a larynx preservation approach using primary chemoradiation (pCRT). So far, no clear criteria have been defined under what circumstances primary radiotherapy alone (pRT) might be acceptable in case chemotherapy (CT) is prohibited or in what cases, even in T3, upfront total laryngectomy with risk-adapted adjuvant treatment (TL±a[C]RT) should be recommended. METHOD: The literature was searched for parameters chosen as criteria for an inclusion in the surgical rather than the conservative arm in non-randomized LP studies or which proved to be significant prognostic markers after conservative treatment. Development of a counselling tool for therapeutic decision making. RESULTS: Significant prognostic markers were tumor volume (< 3.5 ccm/< 6 ccm vs. 6-12 ccm vs. > 12 ccm), presence and kind of vocal cord fixation (none vs. Succo I/II vs. Succo III/IV), extent of cartilage infiltration (none vs. minimal vs. multiple/gross), nodal status (N0­1 vs. N2-3), and laryngeal dysfunction (pretreatment necessity of feeding tube or tracheostomy). CONCLUSION: For T3 laryngeal cancers, pRT could be acceptable when the tumor volume is < 3.5 ccm for glottic and < 6 ccm for supraglottic tumors and there are no further risk factors. pCRT can be regarded as the standard for LP for tumors between 6 ccm and 12 ccm, vocal cord fixation Succo pattern I/II, only minimal cartilage infiltration and a high nodal burden. For tumor > 12 ccm, vocal cord fixation Succo pattern III/IV, gross or multiple cartilage infiltration or clinically relevant laryngeal dysfunction, upfront TL±a[C]RT should be considered.

[Larynx preservation up to T4 laryngeal cancer?] / Larynxorganerhalt bis zum T4­Larynxkarzinom?

Could primary chemoradiotherapy (pCRT) possibly be viewed as an alternative standard therapy to upfront total laryngectomy (TL)? According to the new German S3 guideline, despite higher rates of local recurrence, there would be no survival disadvantage and salvage surgery would be a curative option. In several large database studies and case series, statistically significant survival disadvantages of more than 30% between pCRT and TL have been reported for T4 laryngeal cancer. According to the literature, the success rate of salvage TL for T4 laryngeal cancer is only about 25-50%. Larynx preservation (LP) studies which could qualify the recommendation of pCRT as an alternative standard therapy to TL in T4 carcinomas should 1) evaluate T4a cancers within the T4 category; 2) perform subgroup analysis of laryngeal and hypopharyngeal cancers; 3) be sufficiently highly powered; 4) provide long-term outcomes of at least 5 years; 5) with oncological and 6) functional outcomes (duration of the need for tracheostomy and/or feeding tube dependency; necessity and success of salvage laryngectomies). 7) Specification of the criteria of the respective T4 classification (invasion through the outer cortex of the cartilage, or infiltration of which extralaryngeal structures) and 8) evaluation of pretreatment laryngeal function (at least: tracheostomy, feeding tube dependency). Collection of all the aforementioned data of T4 patients treated with pCRT in a large prospective observational cohort study in German-speaking countries is suggested. In case of rejection of TL by T4 laryngeal cancer patients, differentiation between primary spontaneous reluctance and a definitive, carefully considered decision is important. This distinction should be achieved by sensitive discussions. Not only oncological but also functional outcome probabilities should be included in the overall decision-making process.

Chemoradiotherapy but Not Radiotherapy Alone for Larynx Preservation in T3. Considerations from a German Observational Cohort Study.

For advanced laryngeal cancers, after randomized prospective larynx preservation studies, nonsurgical therapy has been applied on a large scale as an alternative to laryngectomy. For T4 laryngeal cancer, poorer survival has been reported after nonsurgical treatment. Is there a need to fear worse survival also in T3 tumors? The outcomes of 121 T3 cancers treated with pCRT, pRT alone, or surgery were evaluated in an observational cohort study in Germany. In a multivariate Cox regression of the T3 subgroup, no survival difference was noted between pCRT and total laryngectomy with risk-adopted adjuvant (chemo)radiotherapy (TL ± a(C)RT) (HR 1.20; 95%-CI: 0.57-2.53; p = 0.63). However, survival was significantly worse after pRT alone than after TL ± a(C)RT (HR 4.40; 95%-CI: 1.72-11.28, p = 0.002). A literature search shows that in cases of unfavorable prognostic markers (bulky tumors of 6-12 ccm, vocal cord fixation, minimal cartilage infiltration, or N2-3), pCRT instead of pRT is indicated. In cases of pretreatment dysphagia or aspiration requiring a feeding tube or tracheostomy, gross or multiple cartilage infiltration, or tumor volume > 12 ccm, outcomes after pCRT were significantly worse than those after TL. In these cases, and in cases where pCRT is indicated but the patient is not suitable for the addition of chemotherapy, upfront total laryngectomy with stage-appropriate aRT is recommended even in T3 laryngeal cancers.

Contact-dependent inhibition of HIV-1 replication in ex vivo human tonsil cultures by polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMNs), the most abundant white blood cells, are recruited rapidly to sites of infection to exert potent anti-microbial activity. Information regarding their role in infection with human immunodeficiency virus (HIV) is limited. Here we report that addition of PMNs to HIV-infected cultures of human tonsil tissue or peripheral blood mononuclear cells causes immediate and long-lasting suppression of HIV-1 spread and virus-induced depletion of CD4 T cells. This inhibition of HIV-1 spread strictly requires PMN contact with infected cells and is not mediated by soluble factors. 2-Photon (2PM) imaging visualized contacts of PMNs with HIV-1-infected CD4 T cells in tonsil tissue that do not result in lysis or uptake of infected cells. The anti-HIV activity of PMNs also does not involve degranulation, formation of neutrophil extracellular traps, or integrin-dependent cell communication. These results reveal that PMNs efficiently blunt HIV-1 replication in primary target cells and tissue by an unconventional mechanism.

Could Primary Chemoradiotherapy in T2 Glottic Cancers Yield Results Comparable to Primary Radiotherapy in T1? Considerations from 531 German Early Stage Patients.

T1 glottic cancer is a highly treatable disease with local control (LC) rates over 90% by either primary radiotherapy (pRT) or transoral laser microsurgery (TLM). LC of T2 glottic cancers is 15 percent points poorer on average. However, salvage after pRT entails more than 50% total laryngectomy. Therefore, there is a need for enhanced LC. Altered fractionation regimens improved LC in T1 but not in T2. For this reason, for T2, alternative strategies must be considered. In a large observational cohort study including 531 early-stage laryngeal cancers, a small number of patients were treated with primary chemoradiotherapy (pCRT). In multivariable analysis, factors associated with significantly poorer outcomes included age, comorbidities, supraglottic localization, and T category. While there was a significant difference between pRT and surgery (HR 1.79; 95%-CI: 1.15-2.79), there was none between pCRT and surgery (HR 0.70; 95%-CI: 0.33-1.51). There is evidence from the literature that pCRT in early glottic cancers could yield results that surpass the limits so far experienced in radiotherapy alone with acceptable toxicity. Thus, prospective randomized studies with larger numbers of patients are warranted.

An Observational Cohort Study on 194 Supraglottic Cancer Patients: Implications for Laser Surgery and Adjuvant Treatment.

Supraglottic laryngeal cancer is characterized by poor prognosis. In contrast, excellent outcomes have been published in early-stage supraglottic cancers after laser surgery in single-institutional series in centers of excellence. Are these results reproducible in the normal clinical practice of less specialized facilities? As part of an observational cohort study, the outcomes of 194 supraglottic cancer patients were assessed after treatment by larynx-preserving surgery (transoral laser microsurgery [TLM] or open partial laryngectomy [OPL]) or total laryngectomy (TL), with each having risk-adopted adjuvant treatment, or primary (chemo-)radiotherapy (pCRT or pRT). In early-stage supraglottic cancers, TLM achieved a 5-year overall survival (5-year OS) of 62.0%. No significant survival difference could be discerned between patients with and without adjuvant treatment (HR 1.47; 95% CI: 0.80 2.69). The comparison between pCRT and pRT patients suggests that CRT is more effective in supraglottic cancer. The 5-year OS rate achieved in our multiinstitutional setting is comparable to that reached in laser surgery centers of excellence (59.4-76.0%). According to our data and supported by the literature, adjuvant RT (aRT) is not sufficiently effective in supraglottic cancers. In case adjuvant therapy is indicated, adjuvant chemoradiation (aCRT) could be recommended.

Functional role of miR-148a in oropharyngeal cancer: influence on pregnane X receptor and P-glycoprotein expression.

MicroRNAs are short noncoding RNAs of about 19-25 nucleotides that usually target the 3' untranslated regions of mRNAs thus mediating post-transcriptional regulation of gene expression. Previous data indicate a role for miR-148a in the regulation of the pregnane X receptor (PXR/NR1I2), a nuclear receptor that regulates the expression of drug transporters like P-glycoprotein (P-gp/ABCB1). Our study investigated the effect of miR-148a on the post-transcriptional regulation of PXR and its target gene ABCB1 in oropharyngeal cancer cell lines (OPSCC). miR-148a was over-expressed and knocked-down in three OPSCC cell lines (HNO41, HNO206, and HNO413) by transfection with miR-148a mimic and miR-148a antagomir, respectively. Expression of miR-148a, NR1I2, and ABCB1 mRNA was quantified via real-time qPCR, protein expression of PXR was assessed by immunoblotting. Transfection of miR-148a mimic led to increased miR-148a levels in all cell lines and transfection of miR-148a antagomir reduced miR-148a expression in HNO206 and HNO413. Whereas these changes had no significant effect on PXR mRNA expression, protein expression was reduced in HNO41 by transfection with miR-148a and increased in HNO413 by transfection with miR-148a antagomir. Transfection of miR-148a downregulated ABCB1 mRNA in all cell lines, whereas antagonizing miR-148a had no significant effect. Our data demonstrate a modulation of PXR/NR1I2 and ABCB1 expression in OPSCC by miR-148a, however the effect was not uniform in all cell lines and depended on the range of expression of miR-148 and the genotype of rs1054190 SNP in NR1I2 3'UTR. Thus, our findings argue against an unequivocal association between miR-148a and PXR levels in OPSCC.

Antibody Responses to Cancer Antigens Identify Patients with a Poor Prognosis among HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Patients.

PURPOSE: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. EXPERIMENTAL DESIGN: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. RESULTS: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). CONCLUSIONS: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.

Absence of disruptive TP53 mutations in high-risk human papillomavirus-driven neck squamous cell carcinoma of unknown primary.

BACKGROUND: To enforce the evidence for causality between high-risk human papillomavirus (hrHPV) infections and neck squamous cell carcinoma from unknown primary (NSCCUP) and provide biological basis for treatment de-intensification, we searched for TP53 mutations in association with HPV status. METHODS: TP53 mutations were searched for by amplification of exons 4 to 10. RESULTS: Of the 70 NSCCUP, 27 (39%) harbored HPV infection. TP53 sequencing resulted in the identification of 19 patients harboring single mutations including 16 disruptive alterations (84%). The association of TP53 mutations and HPV could be evaluated in 48 NSCCUP including those with disruptive mutation in any exon (n = 16) and those without mutations but with complete sequence of exons 4 to 9 (n = 32): no disruptive mutations were found in the 17 HPV-driven NSCCUP but in 16 of the 31 non-HPV-driven NSCCUP (P = .0002). CONCLUSION: In a fraction of cases, NSCCUP is an HPV-driven entity harboring wild-type TP53 gene or nondisruptive TP53 mutations. HPV-driven NSCCUP might benefit from treatment de-intensification.

Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status.

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC.

DNA methylation at an enhancer of the three prime repair exonuclease 2 gene (TREX2) is linked to gene expression and survival in laryngeal cancer.

BACKGROUND: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). RESULTS: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. CONCLUSIONS: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies.

Differential Activation of ERK Signaling in HPV-Related Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) forms a distinct tumor entity with better survival clinical outcome. Numerous underlying molecular mechanisms have been postulated for differences in treatment response, but the impact of MEK/ERK signaling, a main driver of carcinogenesis in various cancers including OPSCC and key player mediating therapy resistance remains elusive. In a retrospective experimental cohort study, primary tumor samples from OPSCC patients (n = 124) were available on tissue microarrays (TMAs) and expression levels of phosphorylated ERK1/2 (pERK1/2) were detected by immunohistochemical staining. Correlations of pERK1/2 expression patterns with clinicopathological features and clinical outcome were evaluated by statistical analysis. A low pERK1/2 expression was strongly associated with HPV-related OPSCC, while primary tumors with high pERK1/2 staining showed a distinctly worse survival outcome and were associated with higher cellular differentiation. Co-activation of both ERK1/2 and AKT was a common event and was associated with unfavorable prognosis in our cohort. However, the combinatorial analysis of pAKT (Ser473) and pERK1/2 did not strengthen the predictive power of pERK1/2, suggesting that pERK1/2 plays a more significant function in OPSCC. In summary, our data provide a compelling experimental and statistical evidence that low levels of tumor cell intrinsic ERK1/2 activation contribute at least in part to the favorable outcome of HPV-related OPSCC. On the other hand, presented findings indicate that non-HPV-related OPSCC with elevated ERK phosphorylation are at high risk for treatment failure and might benefit from targeted therapy of MEK/ERK signaling.

Cortactin expression: Association with disease progression and survival in oral squamous cell carcinoma.

BACKGROUND: Cortactin (CTTN) is located on chromosome 11q13 and is associated with invasiveness in various cancer entities. CTTN protein expression could be a prognosticator of oral squamous cell carcinoma (OSCC) in terms of recurrence and survival. METHODS: CTTN-dependent invasion was performed using migration assay in human papillomavirus-negative head and neck squamous cell carcinoma (HNSCC) cells. Cortactin protein analysis in tissue microarrays was used for correlation with clinical parameters, as well as for survival analysis. Gene expression profiling in HNSCC cells was performed to unreveal CTTN signaling. RESULTS: Knockdown of CTTN in HNSCC cells showed less invasion in vitro. Gene expression profiling showed various deregulated genes known to be involved in progression. We confirmed the link between CTTN overexpression and progression in a large clinical cohort. High expression was associated with worse overall and progression-free survival. CONCLUSIONS: We propose CTTN for managing OSCC in terms of adjuvant therapy and aftercare. Furthermore, our study reveals new potential targets in CTTN signaling for individualized OSCC therapy.

Human papilloma virus (HPV) 18 proteins E6 and E7 up-regulate ABC transporters in oropharyngeal carcinoma. Involvement of the nonsense-mediated decay (NMD) pathway.

Oropharyngeal cancer incidence increased dramatically in the last decades, being infection with human papillomaviruses (HPV) a determinant of this trend. Concerning etiology, treatment response and prognosis, HPV+ and HPV- oropharyngeal cancers constitute different disease entities. The underlying molecular background is not completely understood. ATP-binding cassette (ABC) transporters mediate the efflux of anticancer drugs and are regulated by changes in the intracellular milieu. Furthermore, a role in cancer pathogenesis besides drug transport was reported. We evaluated the effect of transfection with E6 and E7 oncogenes from HPV16 and HPV18 on ABC transporters in oropharyngeal cancer cells. HPV18E6/E7 up-regulated P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and MRP2 expression in HNO206 cells and breast cancer resistance protein (BCRP) in HNO206 and HNO413 cells. While P-gp was regulated translationally, MRP1, MRP2 and BCRP up-regulation resulted from mRNA stabilization. For MRP1 and MRP2, the nonsense-mediated decay pathway was involved. In general, resistance to substrates of up-regulated transporters was increased. Transfection with oncogenes individually indicated a major role of HPV18E7. Our findings suggest ABC transporters as molecular players leading to differences in the pathogenesis of HPV+ and HPV- oropharyngeal cancer.