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Head and neck squamous cell carcinoma (HNSCC) include malignancies that arise in the oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx. HNSCC is the eighth most common cancer worldwide. The 5-year overall survival rate of HNSCC patients is approximately 50%, but varies depending on the anatomical site of the tumor. Treatment approaches for HNSCC include surgery in combination with radiotherapy, chemotherapy, targeted therapy, immunotherapy, or combinations thereof. Although radiotherapy is an effective nonsurgical single treatment, resistance to radiotherapy often occurs. More effective therapies and personalized treatment approaches for HNSCC patients are urgently needed. This doctoral thesis aimed to I) identify a histological/immunohistological marker to predict which patients are more likely to respond to radio- or chemoradiotherapy; II) investigate how culturing conditions affect the gene expression profile of freshly isolated cancer cells using RNA sequencing transcriptome profile and the effect of these changes on the cancer cell responses to anti-cancer treatments, including radiotherapy; III) conduct a systematic review to investigate the effectiveness of combining angiogenesis inhibitors with radiotherapy for HNSCC treatment; IV) study the possible anti-metastatic effect of fascin 1 inhibitor, compound G2, and to determine if radiotherapy can synergistically enhance this effect; V) screen and identify a possible new effective targeted therapy compound that could have a synergistic effect with radiotherapy. In study I, we obtained formalin-fixed paraffin-embedded (FFPE) samples from 71 oral tongue squamous cell carcinoma (OTSCC) patients treated with radio- or chemoradiotherapy and used immunohistochemistry (IHC) to study different prognostic markers. In study II, we isolated single cells from fresh tissue samples from 7 HNSCC patients, obtained a transcriptome profile via RNA sequencing, and performed a cell viability assay to determine how culturing matrices affect the gene expression profile and drug response of cancer cells. In study III, we performed a systematic review and found 301 records, of which 38 were clinical trials that studied the effectiveness of angiogenesis inhibitors in the treatment of HNSCC patients. In study IV, the anti-metastatic effect of compound G2 with radiotherapy was investigated using high-throughput drug screening (HTS), migration and invasion assays, and a zebrafish larvae assay of HNSCC cells. We also studied the prognostic value of fascin 1 in OTSCC tissue samples. In study V, we used 13 primary and metastatic HNSCC cell lines and performed a HTS drug screen on 396 Food and Drug Administration (FDA)-approved compounds and tested them with and without irradiation. In study I, we did not identify any significant associations between the evaluated markers and disease-free survival (DFS) of OTSCC patients treated with surgery and adjuvant radio- or chemoradiotherapy. Nevertheless, a trend of association between depth of invasion (DOI) and budding and DFS was observed, where low DOI or non-budding resulted in improved DFS. In study II, we showed that after isolating and culturing freshly obtained carcinoma cells, the gene expression profile significantly changed regardless of culturing conditions. Furthermore, results of anti-cancer treatment testing varied between the different culturing conditions. In study III, although most studies did not show benefit of angiogenesis inhibitors in treating HNSCC, endostatin and lenvatinib were well tolerated and their anticancer effects appeared promising. Study IV revealed that targeted inhibition of fascin 1 with compound G2 diminished HNSCC cell migration and invasion both in vivo and in vitro. Although we did not detect any cytotoxic effects of compound G2 in the HTS or in zebrafish assay, the anti-metastatic effect was clear. No synergy between compound G2 and irradiation was observed. In study V, HTS screening identified several radiosensitive and radioresistant compounds. The most promising synergistic effect was seen with navitoclax. Although we have previously shown that DOI and budding are reliable prognostic markers for OTSCC, we saw only a trend of association between these markers and DFS, presumably due to the small number of patients in study I. Thus, larger cohorts are needed to explore the prognostic value of DOI and budding for OTSCC patients treated with postoperative radio- or chemoradiotherapy. Study II revealed the challenges of in vitro testing of cancer treatments using enzymatic cell digestion. The artificial upregulation of some pathways in cultured cells compared with fresh carcinoma tissue may partially explain the common clinical failures of targeted therapies that appear promising in in vitro testing. In study III, the clinical trials of HNSCC patients in the systematic review examined various treatment combinations. However, as only 12 out of 38 studies included radiotherapy, we were not able to determine the effectiveness of combining angiogenesis inhibitors with irradiation. Nevertheless, considering the promising effects and tolerability of endostatin and lenvatinib, further investigations of these agents are warranted, particularly as a combination therapy for HNSCC patients. The findings of compound G2 in study IV indicated that targeting fascin 1 mainly affects the metastatic properties of the HNSCC but not cancer cell proliferation. We also confirmed the prognostic power of fascin 1 for HNSCC patients with advanced disease. In study V, we identified navitoclax as a promising agent and recommend that it is tested in clinical trials of HNSCC patients in combination with irradiation. Pään ja kaulan alueen levyepiteelikarsinooma (head and neck squamous cell carcinoma, HNSCC) on maailmanlaajuisesti kahdeksanneksi yleisin syöpätyyppi ja se voi esiintyä suuontelossa, nenäontelossa, nielussa tai kurkunpäässä. Pään ja kaulan alueen levyepiteelikarsinoomapotilaiden viiden vuoden eloonjäämisennuste on vain noin 50 %, mutta se vaihtelee anatomisen sijainnin mukaan. Hoitomuotoja ovat leikkaus yhdistettynä sädehoitoon, kemoterapiaan, täsmälääke- ja/tai immunoterapiahoitoon. Sädehoito on yksi tehokkaimmista konservatiivisista hoitomuodoista, mutta vaste sädehoitoon vaihtelee potilaiden välillä ja joidenkin kohdalla vaste jää alhaiseksi. Tämän takia on suuri tarve kehittää tehokkaampia lääkkeitä sekä henkilökohtaisempia hoitomuotoja pään ja kaulan alueen levyepiteelikarsinoomapotilaille. Tämän väitöskirjan tavoitteena oli: I) löytää histologinen/immunohistologinen markkeri, jonka avulla voidaan ennustaa mitkä potilaat todennäköisimmin hyötyvät säde- tai kemosädehoidosta; II) tutkia kuinka kasvatusolosuhteet vaikuttavat tuumorista eristettyjen karsinoomasolujen geeniekspressioon, sekä kuinka nämä muutokset vaikuttavat syöpäsolujen vasteeseen yleisesti käytettyihin syöpälääkkeisiin; III) tehdä systemaattinen kirjallisuuskatsaus, jossa selvitetään angiogeneesi-inhibiittoreiden tehokkuus pään ja kaulan alueen levyepiteelikarsinoomapotilailla sekä olisiko sädehoito yhdistettynä näihin lääkkeisiin tehokas hoitomuoto; IV) tutkia minkälainen anti-mestastaattinen vaikutus fascin 1 inhibiittori G2:della on, sekä voisiko sädehoito toimia synergisesti tämän lääkkeen kanssa; V) etsiä ja identifioida mahdollinen uusi tehokas täsmälääke, joka voisi toimia synergistisesti yhdessä sädehoidon kanssa. Tutkimuksessa I käytimme 71 säde- tai kemosähdehoitoa saaneen kielisyöpäpotilaan kudosnäytteitä ja tutkimme erilaisia prognostisia markkereita sekä histologian että immunohistokemian avulla. Tutkimuksessa II eristimme syöpäsoluja seitsemän suusyöpäpotilaan kasvaimesta otetusta näytteestä ja tutkimme, kuinka kasvatusolosuhteet vaikuttavat solujen geeniekspressiioon sekä lääkevasteeseen RNA sekvensoinnin sekä viabiliteettikokeiden avulla. Tutkimuksessa III teimme systemaattisen kirjallisuuskatsauksen, jossa löysimme 301 artikkelia joista 38 olivat kliinisiä tutkimuksia, joissa tutkittiin angiogeneesi-inhibiittoreiden tehokkuutta HNSCC potilaiden hoidossa. Tutkimuksessa IV, tutkimme fascin inhibiittori G2:den tehokkuutta HNSCC soluihin suuritehoisen seulonnan, migraatio ja invaasio kokeiden, sekä seeprakalamallin avulla. Tutkimme myös fascin 1 prognostista arvoa kielisyöpäpotilaiden kudosnäytteiden avulla. Tutkimuksessa V käytimme 13 HNSCC primaari- ja metastaasisolulinjaa ja toteutimme suuritehoisen seulonnan käyttäen 396 lääkettä, jotka sisälsivät sekä Yhdysvaltain elintarvike- ja lääkeviraston (FDA) hyväksymiä että tutkittavia lääkevalmisteita. Tutkimuksessa I emme pystyneet osoittamaan merkitsevää yhteyttä minkään eri markkerin ja taudittoman hoidon jälkeisen jakson välillä säde- tai kemosädehoitoa saaneilla kielisyöpäpotilailla. Tuloksissa oli kuitenkin havaittavissa trendi taudittoman hoitojakson sekä invaasiosyvyys ja silmuilu analyysitulosten välillä, jossa alhainen invaasiosyvyys sekä silmuilu johtivat pidempään taudittomaan jaksoon. Tutkimuksessa II osoitimme, että karsinoomasolujen geeniekspressio muuttui merkittävästi eristyksen ja kasvatuksen jälkeen, riippumatta kasvatusolosuhteista. Lisäksi totesimme tulosten perusteella, että eri kasvuympäristöt eivät merkittävästi eroa lääkevasteen kannalta. Tutkimuksessa III, suurin osa kliinisistä tutkimuksista eivät osoittaneet merkittävää hyötyä angiogeneesi-inhibiittoreista HNSCC potilailla, mutta endostatiini sekä lenvatiniibi olivat hyvin siedettyjä, ja niiden teho oli lupaava. Tutkimuksessa IV fascinin 1 kohdennettu inhibiitio G2:den avulla vähensi selkeästi HNSCC solujen migraatiota ja invaasiota in vitro ja in vivo. Vaikka emme kyenneet osoittamaan G2:della olevan sytotoksisia vaikutuksia suuritehoisessa seulonnassa tai seeprakalamallissa, lääkeaineen antimetastaattinen vaikutus oli selkeä. Emme pystyneet osoittamaan synergistä vaikutusta G2:den ja sädehoidon välillä. Tutkimuksessa V löysimme suuritehoisen seulonnan avulla useita radiosensitiivisiä sekä sädehoitoon reagoimattomia lääkkeitä. Lupaavin synergistinen vaikutus sädehoidon kanssa oli navitoklaks-lääkkeellä. Vaikka olemme aikaisemmissa tutkimuksissa osoittaneet, että sekä invaasiosyvyys että silmuilu ovat luotettavia prognostisia markkereita kielisyöpäpotilailla, pystyimme tässä kyseisessä tutkimuksessa osoittamaan vain trendin näiden markkereiden ja taudittoman hoitojakson välillä. Tämä johtuu luultavasti tutkimuksen pienestä potilasmäärästä. Näiden tulosten perusteella lisätutkimukset isommilla kohorteilla ovat perusteltuja, jotta voidaan tutkia invaasiosyvyys- ja silmuilumarkkereiden prognostista arvoa säde- ja kemosädehoitoa saaneilla kielisyöpäpotilailla. Tulokset tutkimuksessa II osoittavat syöpälääkkeiden in vitro testauksessa yleisesti käytettyjen enstymaattisten digestiomenetelmien haasteet. Joidenkin geenireittien keinotekoinen aktivoituminen kasvatetuissa soluissa verrattuna tuoreeseen kudosnäytteeseen, voi osittain selittää joidenkin täsmälääkkeiden huono vaste kliinisissä kokeissa, vaikka edeltävät in vitro kokeet ovat olleet lupaavia. Tutkimuksen III kirjallisuuskatsaus sisälsi kliinisiä tutkimuksia HNSCC potilailla erilaisilla hoitomuotokombinaatioilla. Kuitenkin, vain 12 kaikista 38:sasta tutkimuksesta sisälsi sädehoitoa, joten emme voineet vetää johtopäätöksiä angiogeensi inhibiittoreiden ja sädehoidon yhdistelmän tehokkuudesta. Tästä huolimatta, endostatiinin ja lenvatiniibin osoittivat lupaavia tuloksia tehokkuuden ja siedettävyyden kannalta, minkä takia lisätutkimukset angiogeneesi-inhibiittoreista HNCSS potilailla ovat yhä perusteltuja. Tutkimuksen IV tulosten perusteella voidaan todeta, että fascin 1 inhibiittori G2 vaikuttaa pääasiassa solujen metastaattisiin ominaisuuksiin, eikä niinkään vähennä solujen proliferaatiota. Tutkimuksessa todettiin myös, että fascin 1 on lupaava prognostinen markkeri HNSCC potilailla pitkälle edenneessä taudissa. Tutkimuksessa V löysimme lupaavan navitoklas-lääkkeen, ja suosittelemme kliinisiä tutkimuksia navitoklaksin ja sädehoidon yhdistelmästä HNSCC potilailla.

Background Head and neck squamous cell carcinoma (HNSCC) carries poor survival outcomes despite recent progress in cancer treatment in general. Angiogenesis is crucial for tumour survival and progression. Therefore, several agents targeting the pathways that mediate angiogenesis have been developed. We conducted a systematic review to summarise the current clinical trial data examining angiogenesis inhibitors in HNSCC. Methods We carried out a literature search on three angiogenesis inhibitor categories-bevacizumab, tyrosine kinase inhibitors and endostatin-from Ovid MEDLINE, Cochrane Library, Scopus and ClinicalTrials.gov database. Results Here, we analysed 38 clinical trials, total of 1670 patients, investigating 12 angiogenesis inhibitors. All trials were in phase I or II, except one study in phase III on bevacizumab. Angiogenesis inhibitors were used as mono- and combination therapies together with radio-, chemo-, targeted- or immunotherapy. Among 12 angiogenesis inhibitors, bevacizumab was the most studied drug, included in 13 trials. Although bevacizumab appeared effective in various combinations, it associated with high toxicity levels. Endostatin and lenvatinib were well-tolerated and their anticancer effects appeared promising. Conclusions Most studies did not show benefit of angiogenesis inhibitors in HNSCC treatment. Additionally, angiogenesis inhibitors were associated with considerable toxicity. However, some results appear encouraging, suggesting that further investigations of angiogenesis inhibitors, particularly in combination therapies, for HNSCC patients are warranted. Systematic Review Registration PROSPERO (https://www.crd.york.ac.uk/prospero/), identifier CRD42020157144.

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting.

Additional file 1. Supplementray tables.

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI), Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University., Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance., Funder: The Canadian Cancer Society, Funder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194, Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

Funder: Breast Cancer Research Foundation (BCRF); doi: https://doi.org/10.13039/100001006, Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting.