Matrix metalloproteinases and toll-like receptors in early-stage oral tongue squamous cell carcinoma

Predicting the clinical course of an early-stage oral tongue squamous cell carcinoma (OTSCC) is challenging, as even small tumors can behave aggressively. OTSCC often metastasizes to the cervical lymph nodes, and the presence of lymph node metastasis at the time of diagnosis is considered the most important tumor-related prognostic factor in OTSCC. The mechanisms of this disease progression are poorly understood. Despite slight improvement in the prognosis of OTSCC in recent decades, the outcome of these patients is still modest. Therefore, a deeper understanding of the phenomena behind tumor progression would enable medical professionals to evaluate the aggressiveness of the disease and to adjust its treatment more effectively. The extracellular matrix and basement membrane must be broken down before a tumor can invade surrounding tissues and further spread into blood and lymph vessels. This is a process that involves various proteolytic enzymes, the most important of which are matrix metalloproteinases (MMPs). Over 25 structurally related, but genetically distinct, human MMPs have been identified and characterized: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other MMPs. Toll-like receptors (TLRs) are pattern-recognition molecules involved in innate immunity that are also expressed in many types of cancer. TLRs apparently play a pivotal role in malignant disease: they are related to tumor progression and, conversely, to cancer inhibition. Their expression pattern and role in oral cancer, however, remains unclear. In this thesis we studied the expression of MMPs 2, 7, 8, 9, 13, and 25 and TLRs 2, 4, 5, 7, and 9 in early-stage OTSCC. The study comprised 73 consecutive clinically T1N0M0 and T2N0M0 OTSCC patients treated at the Helsinki University Hospital, Helsinki, Finland in 1992-2002. We prepared tissue array blocks from primary tumors and immunostained them. We also used whole section slides from patients with metastasized or recurrent disease. We compared immunoexpression of MMPs and TLRs to tumor and patient characteristics as well as to patient outcomes. We also used Western immunoblot to examine TLR-2 and -4 expression in the highly invasive and aggressive HSC-3 OTSCC cell line. In addition, we studied the effect of TLR-2 and -4 antagonist GIT27 (4,5-dihydro-5-isoxasoleacetic acid) on the invasion of the HSC-3 cell line in myoma organotypic invasion assay. OTSCC tumors expressed both MMPs and TLRs. Nuclear expression of MMP-13, but not cytoplasmic expression of MMP-2, -8, and -9, associated with deeper invasion and advanced tumor size. Furthermore, high nuclear MMP-13 expression predicted poor disease-specific survival. High MMP-7 protein expression associated with the presence of occult cervical metastases, increased invasion depth, and higher tumor grade, and also predicted poor outcome. Immunostaining of MMP-25 failed to correlate with any clinical parameters. High TLR-2, -4, and -9 expression correlated with deeper tumor invasion. Cytoplasmic expression of TLR-2 and -4 also correlated significantly with higher tumor grade, whereas high TLR-5 expression associated with lower tumor grade. High expression of TLR-9 correlated with advanced tumor size. Negative or mild TLR-5 expression predicted poor disease-specific survival. OTSCC primary tumors, neck metastases and recurrent tumors expressed TLR-2, -4, and -9. TLR-2 and -4 antagonist GIT27 did not affect the invasion of the HSC-3 cell line in myoma organotypic invasion assay. Thus, TLRs may operate under a different mechanism of action depending on whether they are activated by damage-associated molecular patterns in cancer or by pathogen-associated molecular patterns in infection. Our results suggest that MMP-7 and MMP-13 in particular may have prognostic value in OTSCC. Their use as prognostic biomarkers, however, calls for further study. TLR-2, -4, and -9 seemed to predict invasive tumor growth. Primary tumors and neck metastases as well as recurrent tumors of OTSCC express these TLRs, suggesting that TLRs seem to play a role in both the development and progression of tongue carcinoma.Kielisyövän käyttäytymisen ennustaminen on haastavaa, sillä jo pienet kasvaimet saattavat lähettää etäpesäkkeitä taudin varhaisessa vaiheessa. Etäpesäkkeiden esiintymistä kaulan imusolmukkeissa pidetään merkittävimpänä kasvaimeen liittyvänä tekijänä taudin ennustetta arvioitaessa. Huolimatta kielisyövän ennusteen hienoisesta paranemisesta viime vuosikymmenien aikana, taudin elossaoloennuste on edelleen varsin huono ja taudin etenemiseen vaikuttavat mekanismit tunnetaan huonosti. Näistä syistä johtuen olisi toivottavaa tuntea tarkemmin syövän taustalla piileviä biologisia mekanismeja, jotta taudin aggressiivisuutta voitaisiin arvioida paremmin ja mahdollisesti löytää myös kohdennettuja hoitomuotoja. Jotta kasvainsolut voivat edetä ympäröivään kudokseen sekä veri- ja imusuoniin, proteolyyttisten entsyymien pitää hajottaa soluväliainetta ja tyvikalvo. Matriksin metalloproteinaaseilla (engl. matrix metalloproteinases, MMP) on tässä keskeinen osuus. Nykyisin tunnetaan yli 25 rakenteellisesti toisiaan muistuttavaa, mutta geneettisesti erilaista, MMP:a, jotka jaetaan ryhmiin: kollagenaasit, gelatinaasit, stromelysiinit, matrilysiinit, solukalvoihin liittyvät ja muut MMP:t. Tollin kaltaiset reseptorit (engl. toll-like receptors, TLR) ovat proteiineja, jotka tunnistavat elimistölle vieraita ja myös elimistön omia rakenteita, ja jotka säätelevät luonnollista immuniteettia. Tiettyjen TLR:ien on todettu ilmentyvän myös eräissä syövissä. TLR:eilla on havaittu sekä syöpää edistäviä että hidastavia vaikutuksia, mutta niiden merkitys, ja osin myös ilmentyminen, suusyövässä on vielä epäselvä. Tässä väitöskirjatyössä tutkittiin MMP:ien 2, 7, 8, 9, 13 ja 25 sekä TLR:ien 2, 4, 5, 7 ja 9 ilmentymistä varhaisvaiheen kielisyövissä. Tutkimusmateriaalimme koostui 73 potilaasta, joilla oli todettu kliinisesti T1N0M0 tai T2N0M0 kielisyöpä. Potilaat oli hoidettu Helsingin yliopistollisessa keskussairaalassa vuosina 1992-2002. Emokasvaimista tehtiin tissue array -blokit, jotka värjättiin immunohistokemiallisin menetelmin. Kasvaimet, jotka olivat uusiutuneet tai lähettäneet etäpesäkkeitä, tutkittiin myös kokoleikkeitä käyttäen. Tutkittujen MMP:ien ja TLR:ien ilmentymistä syöpäkudoksessa verrattiin kasvaimen muihin ominaisuuksiin, potilaaseen liittyviin tekijöihin sekä taudin ennusteeseen. TLR-2:n ja -4:n ilmentymistä tutkittiin myös erittäin invasiivisessa ja aggressiivisessa HSC-3 kielisyöpäsolulinjassa. Lisäksi tutkimme TLR-2:n ja -4:n vastavaikuttaja GIT27:n (4,5-dihydro-5-isoxasoleacetic acid) vaikutusta kielisyöpäsolujen invaasioon myoomamallissa. MMP:t ja TLR:t ilmentyivät suuressa osassa kielisyöpäkasvaimia. MMP-13:n lisääntynyt ilmentyminen tumissa liittyi kasvaimen syvempään invaasioon ja suurempaan kokoon sekä ennusti potilaiden lyhyempää elossaoloaikaa. Samanlaista vaikutusta ei todettu MMP-2:n, -8:n ja -9:n ilmentymisellä syöpäsolujen solulimassa. MMP-7 ilmentyi voimakkaammin niillä potilailla, joilla todettiin kaulan etäpesäkkeet, kasvaimen syvempi invaasio tai huonompi erilaistumisaste. Lisääntynyt MMP-7-ekspressio ennusti myös potilaiden lyhyempää elossaoloaikaa. MMP-25:n ilmentyminen ei liittynyt mihinkään tutkittuun kliiniseen tekijään. Voimakas TLR-2, -4 ja -9 ilmentyminen liittyi kasvaimen syvempään invaasioon. Soluliman vahva TLR-2 ja -4 ilmentyminen liittyi lisäksi kasvaimen huonompaan erilaistumisasteeseen, kun taas TLR-5 proteiinia esiintyi enemmän korkeasti erilaistuneissa kasvaimissa. TLR-9:n ilmentyminen oli voimakkaampaa kookkaissa kasvaimissa. Puuttuva tai vähäinen TLR-5:n ilmentyminen puolestaan ennusti lyhyempää tautikohtaista elossaoloaikaa. TLR-2, -4 ja -9 ilmentyivät sekä kielisyövän emokasvaimissa että kaulan etäpesäkkeissä ja uusiutuneissa kasvaimissa. TLR-2:n ja -4:n vastavaikuttaja GIT27 ei vaikuttanut kielisyöpäsolujen invaasioon myoomamallissa. TLR:iden vaikutusmekanismi on mahdollisesti erilainen riippuen siitä, aktivoituvatko ne syövästä vai tulehduksesta johtuen. Tutkimuksemme perusteella etenkin MMP-7:lla ja -13:lla näyttäisi olevan ennusteellista merkitystä kielisyövässä. TLR-2, -4 ja -9 proteiinien ilmentyminen säätelee mahdollisesti kielisyövän invaasiota. Nämä TLR:t ilmentyivät sekä emokasvaimissa, kaulan etäpesäkkeissä että uusiutuneissa kasvaimissa, joten on todennäköistä, että TLR:eilla on jonkinlainen rooli niin kielisyövän synnyssä kuin sen etenemisessäkin

Kielisyöpä - varhainen toteaminen parantaa ennustetta

English summar

Epidemiology and aetiology of sport-related nasal fractures : Analysis of 599 Finnish patients

Peer reviewe

Changing trends in pediatric tonsil surgery

Objectives: We analyzed trends in tonsil surgery over a 10-year period in a single tertiary care hospital and evaluated the effects of these changes on use of hospital services and healthcare costs. Methods: This was a retrospective cohort study based on data from databases at the Department of Otorhinolaryngology, Helsinki University Hospital, Helsinki, Finland. Children under 16 years of age with tonsillectomy (TE) or tonsillotomy (TT) performed during 2007-2016 were included in the study. Results: In 10 years, 4979 tonsil surgeries were performed on 4951 children: TE in 3170 (64%) and TT in 1781 (36%) children. The total number of tonsil surgeries stayed nearly constant. TT operations commenced in the study hospital in 2009 and from 2012 onwards have been more common than TE procedures. Altogether 279 patients visited the emergency department because of complications; TE patients had 9.0 visits/100 surgeries and TT patients 1.8 visits/100 surgeries. The most common complication was postoperative hemorrhage: 200 cases (6.3%) in the TE group and 11 cases (0.6%) in the TT group. During the two-year follow-up after tonsil surgery the total costs of healthcare services were significantly lower in the TT group than in the TE group. Conclusion: Considerable changes have occurred in tonsil surgery in children during the 10-year study period; TT is today performed more often than TE. As a consequence, complications, readmissions to hospital, and number of patients treated in the operating room because of postoperative hemorrhage have decreased, lowering the costs of healthcare.Peer reviewe

The expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma

Publisher Copyright: © 2021 Scandinavian Societies for Medical Microbiology and Pathology.P-cadherin (CDH3) is a cell-to-cell adhesion molecule that regulates several cellular homeostatic processes in normal tissues. Lack of CDH3 expression is associated with aggressive behavior in oral squamous cell carcinoma (OSCC). Previous studies have shown that CDH3 is downregulated in high-grade OSCC and its reduced expression is predictive for poorer survival. The aim of this study was to evaluate the expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma (TSCC). A retrospective series of 211 TSCC and 50 lymph node samples were stained immunohistochemically with polyclonal antibody (anti-CDH3). CDH3 expression was assessed semi-quantitatively with light microscopy. Fisher’s exact test was used to compare patient and tumor characteristics, and the correlations were tested by Spearman correlation. Survival curves were drawn by the Kaplan–Meier method and analyzed by the log-rank test. Univariate and multivariate Cox regression was used to estimate the association between CDH3 expression and survival. CDH3 expression did not affect TSCC patient’s disease-specific survival or overall survival. Strong CDH3 expression in the primary tumor predicted poor disease-specific and overall survival in patients with recurrent disease. CDH3 expression in lymph nodes without metastasis was negative in all cases. CDH3 expression was positive in all lymph node metastases with extranodal extension. In contrast to previous report about the prognostic value of CDH3 in OSCC, we were not able to validate the result in TSCC.Peer reviewe

Small oral tongue cancers (ae 4 cm in diameter) with clinically negative neck : from the 7th to the 8th edition of the American Joint Committee on Cancer

One of the main changes in the 8th edition of the American Joint Committee on Cancer (AJCC) for staging of oral cancer is the inclusion of depth of invasion (DOI) in the T category. However, cancers in different oral subsites have variable behavior, with oral tongue squamous cell carcinoma (OTSCC) being the most aggressive one even at early stage. Thus, it is necessary to evaluate the performance of this new T category in homogenous cohort of early OTSCC. Therefore, we analyzed a large cohort of patients with a small (ae4 cm) OTSCC to demonstrate the differences in T stage between the AJCC 7th and 8th editions. A total of 311 early-stage cases (AJCC 7th) of OTSCC were analyzed. We used 5 mm and 10 mm DOI for upstaging from T1 to T2 and from T2 to T3 respectively, as in the AJCC 8th. We further reclassified the cases according to our own proposal suggesting 2 mm to upstage to T2 and 4 mm to upstage to T3. According to AJCC 7th, there were no significant differences in the survival analysis. When we applied the 8th edition, many cases were upstaged to T3 and thus associated with worse disease-specific survival (HR 2.37, 95% CI 1.12-4.99) and disease-free survival (HR 2.12, 95% CI 1.09-4.08). Based on our proposal, T3 cases were associated with even worse disease-specific survival (HR 4.19, 95% CI 2.27-7.74). The 8th edition provides better survival prediction for OTSCC than the 7th and can be further optimized by lowering the DOI cutoffs.Peer reviewe

The effect of fascin 1 inhibition on head and neck squamous cell carcinoma cells

Fascin 1 plays important pro-metastatic roles in head and neck carcinoma (HNSCC) migration, invasion, and metastasis. However, limited advancement in targeting metastasis remains a major obstacle in improving HNSCC patients' survival. Therefore, we assessed the therapeutic potential of fascin 1 targeted inhibition and its potential prognostic value in HNSCC patients. Using in vitro and in vivo approaches, we investigated the effect of compound G2, a novel fascin 1 inhibitor, on HNSCC cells migration, invasion, and metastasis. High-throughput screening (HTS) was used to assess cytotoxic activity of compound G2 alone or combined with irradiation. We also evaluated the prognostic potential of fascin 1 in HNSCC patients. Interestingly, compound G2 reduced carcinoma cells migration and invasion in vitro and inhibited metastasis in vivo. Moreover, HTS revealed a modest cytotoxic activity of the compound G2 on HNSCC cell lines. Irradiation did not synergistically enhance the compound G2-mediated cytotoxic activity. Survival analyses showed that high fascin 1 immunoexpression, at the tumor invasive front, was associated with cancer-specific mortality in the advanced stages of HNSCC. Collectively, our findings suggest that fascin 1 represents a promising anti-metastatic therapeutic target and a useful prognostic marker in patients with HNSCC. Novel anti-metastatic agents could provide a valuable addition to cancer therapy.</p

The Glanville fritillary genome retains ancient karyotype and reveals selective chromosomal fusions in Lepidoptera

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Effect of remdesivir post hospitalization for COVID-19 infection from the randomized SOLIDARITY Finland trial

We report the first long-term follow-up of a randomized trial (NCT04978259) addressing the effects of remdesivir on recovery (primary outcome) and other patient-important outcomes one year after hospitalization resulting from COVID-19. Of the 208 patients recruited from 11 Finnish hospitals, 198 survived, of whom 181 (92%) completed follow-up. At one year, self-reported recovery occurred in 85% in remdesivir and 86% in standard of care (SoC) (RR 0.94, 95% CI 0.47-1.90). We infer no convincing difference between remdesivir and SoC in quality of life or symptom outcomes (p > 0.05). Of the 21 potential long-COVID symptoms, patients reported moderate/major bother from fatigue (26%), joint pain (22%), and problems with memory (19%) and attention/concentration (18%). In conclusion, after a one-year follow-up of hospitalized patients, one in six reported they had not recovered well from COVID-19. Our results provide no convincing evidence of remdesivir benefit, but wide confidence intervals included possible benefit and harm.Peer reviewe