Vloga stresnega keratina 17 v odgovoru na zdravljenje z zaviralci imunskih kontrolnih točk pri raku glave in vratu

Ložar, Taja (avtor), Lambert, Paul F. (mentor), Grašič Kuhar, Cvetka (komentor)

Namen te doktorske naloge je bil ugotoviti povezanost med odgovorom na zdravljenje z zaviralci imunskih kontrolnih točk (ZIKT) in s stresnim keratinom 17 (K17) posredovanim izogibanjem imunskemu odzivu pri ploščatoceličnem raku glave in vratu (RGV). Vloga K17 kot napovednega označevalca odgovora na zdravljenje pri RGV, zdravljenem z ZIKT, še ni raziskana. V okviru predlagane naloge smo si zastavili sledeče hipoteze: 1. Izraženost K17 pri RGV je obratno povezana z izražanjem označevalcev aktiviranega imunskega odziva. 2. Nizka izraženost K17 je povezana z ugodnim odgovorom na zdravljenje z ZIKT pri RGV. V prvem delu naloge smo iskali razlike v izražanju z imunskim odzivom povezanih genov med tumorskimi regijami z visoko in nizko izraženostjo K17. Za ta namen smo izvedli prostorsko usmerjeno tarčno sekvenciranje transkriptoma z uporabo K17 kot morfološkega označevalca. Zaradi omejitev povezanih s to tehnologijo smo izvedli še visoko multipleksno imunofluorescenčno barvanje. V drugem delu naloge smo izvedli klinično raziskavo novega napovednega označevalca na dveh retrospektivnih kohortah: 1) pilotna kohorta, sestavljena iz 48 bolnikov z RGV, ki so bili zdravljeni z ZIKT na Univerzi v Wisconsin-Madisonu, ZDA, in 2) dodatna validacijska kohorta 22 bolnikov z RGV, ki so bili zdravljeni z ZIKT na Univerzi Yale. Izražanje K17 smo preiskovali z imunohistokemijskim barvanjem na parafinskih rezinah ter ga primerjali z odgovorom na zdravljenje in z izraženostjo drugih znanih napovednih dejavnikov (programmed death ligand 1 (PD-L1), T celična infiltracija). Rezultati našega dela kažejo na to, da imajo tumorji z nizko izraženostjo K17 visoko infiltracijo CD4+ in CD8+ T celic, B celic, makrofagov in T regulatornih celic. Med 48 bolniki iz pilotne kohorte (60 % moškega spola, srednja starost 61,5 let) je 21 bolnikov (44 %) imelo visoko izraženost K17 v tumorju, 27 (56 %) pa nizko. Sedemnajst bolnikov (35 %, od tega 77 % z nizko izraženostjo K17 ) je imelo od zdravljenja z ZIKT klinično dobrobit, 31 bolnikov (65 %, 45 % z nizk K17) pa od zdravljenja ni imelo dobrobiti. Visoka izraženost K17 v tumorju je bila statistično značilno povezana z odsotnostjo klinične dobrobiti (p=0,037), krajšim časom do odpovedi zdravljenja (p=0,025), časom do napredovanja bolezni (p = 0,004), ne pa tudi celokupnim preživetjem (p = 0,06). Visoka izraženost K17 v tumorju je bila statistično značilno povezana z odsotnostjo klinične dobrobiti v neodvisni potrditveni kohorti (p=0,011). Klinični izid in izraženost K17 v tumorju nista bili povezani s stanjem izraženosti PD-L1 ali HPV statusom. Naša raziskava predstavlja celostno oceno novega napovednega označevalca K17, vključno s standardizacijo platforme, oceno klinične uporabnosti označevalca ter usmerjenim eksperimentalnim delom, ki je vključeval prostorsko usmerjeno profiliranje transkriptoma s potrditvenim mIF. Naše ugotovitve kažejo na to, da je K17 neodvisni prediktivni označevalec slabšega odgovora na zdravljenje z ZIKT pri RGV. Prispevali smo k boljšemu razumevanju odnosa med K17 in imunskim mikrookoljem. Izsledki naše raziskave bodo prispevali k boljši izbiri bolnikov za zdravljenje z ZIKT.

• Jezik: Slovenski jezik
• Ključne besede: ploščatocelični rak glave in vratu, stresni keratin 17, citokeratin 17, prostorsko usmerjeno sekvenciranje transkriptoma, multipleksna imunofluorescenca
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2024
• PID: 20.500.12556/RUL-164289
• Datum objave v RUL: 19.10.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The role of stress keratin 17 in response to immune checkpoint inhibitors in head and neck carcinoma

Izvleček

The aim of this doctoral dissertation was to investigate the association between response to immune check-point blockade (ICB)-based therapy and stress keratin 17 (K17) mediated immune evasion axis in head and neck squamous cell carcinoma (HNSCC). The predictive role of K17 in ICB-treated HNSCC has not been investigated to date. As part of this work, we set out to investigate the following hypotheses: 1. K17 expression in HNSCC is inversely correlated with the expression of markers of immune activation in HNSCC. 2. Low K17 expression is associated with improved clinical response to ICB-based therapy in HNSCC. We investigated the differences in immune-related gene expression profile relative to K17 expression by employing targeted spatial transcriptomic profilling using K17 as a morphology marker. To bypass the limitations of spatial transcriptomics technology, we performed highly multiplex immunofluorescence staining. We next performed a clinical biomarker validation study using two independent retrospective cohorts: 1) a discovery cohort consisted of 48 patients with HNSCC that were treated with ICB-based therapy at the University of Wisconsin-Madison, followed by 2) a validation HNSCC cohort treated with ICB-based therapy at Yale University Hospital (n=22). The expression of K17 was determined by immunohistochemistry (IHC) on formalin fixed, parafin embedded (FFPE) samples, and subsequently correlated to clinical outcomes and the expression of other established predictive biomarkers (programmed death ligand 1 (PD-L1), T cell infiltration). We found K17 low expressing tumors had higher CD4, CD8, B-cell, macrophage and T-reg infiltration. Of the 48 patients in the discovery cohort (60% male, median age 61.5 years), 21 (44%) were K17 high, and 27 (56%) were K17 low. Seventeen patients (35%, 77% K17 low) had disease control, while 31 patients (65%, 45% K17 low) had progressive disease. High K17 expression was associated with lack of disease control (p=0.037), shorter time to treatment failure (p=0.025), progression-free (p = 0.004), but not overall survival (p = 0.06). High K17 expression was associated with lack of disease control in an independent validation cohort (p=0.011). PD-L1 expression or HPV status did not correlate with K17 expression or clinical outcome. This study represents a comprehensive evaluation of a novel predictive biomarker including a biomarker assay standardization and clinical utility assessment , and an analysis of the correlation between K17 and clinicopathologic characteristics, targetable genomic mutation profile, effect size analysis and a hypothesis-driven spatial transcriptomic experiment, followed by mIF validation. We demonstrate the independent predictive value of K17 in predicting non-response to pembrolizumab-based therapy in HNSCC. We further elucidate the relationship between K17 and the immune microenvironment. Our findings may inform clinicians treating HNSCC patients with ICB how to identify patients that are most likely to respond to this therapy.

• Ključne besede: head and neck squamous cell carcinoma, stress keratin 17, cytokeratin 17, biomarker, spatial transcriptomics, multiplex immunofluorescence