Human Glioblastomas: Histopathology, Tumor Growth, and MGMT Status

Glioblastomer er den vanligste og mest aggressive typen hjernesvulst som oppstår i hjernen hos voksne. Glioblastomene har en meget aggressiv og kompleks biologi, som medfører en dårlig prognose på omtrent 10 måneder etter diagnose. Svulstcellene infiltrerer også hjernevevet diffust, som gjør det umulig å fjerne hele svulsten under operasjon. Sammen med den aggressive biologien bidrar dette til at så godt som alle får tilbakefall etter behandling. Til tross for omfattende forskning har hverken overlevelsen eller standardbehandlingen endret seg i noen særlig grad de siste 15 årene. Likevel er det et håp om at bedre innsikt i tumorbiologien kan føre til bedre håndtering av pasientene gjennom identifisering av biologiske trekk som kan si noe om prognose, effekt av behandling, eller hva som kan være mulige angrepsmål for målrettet kreftbehandling. I denne doktorgradsavhandlingen har det overordnede målet vært å få bedre innsikt i den naturlige tumorbiologien i glioblastomer. Den naturlige biologien vil si at biologien er upåvirket av behandling. Dette har vi forsket på ved å undersøke biologiske mekanismer i vevspreparater og på MR-bilder som var tatt før pasientene fikk stråle- og/eller cellegiftbehandling. I alle de tre studiene i denne doktorgraden undersøkte vi en gruppe på 106 glioblastompasienter. Disse pasientene var valgt ut basert på om tumorveksthastigheten kunne beregnes utfra målinger av tumorvolum fra to MR bilder tatt før operasjon og tidsintervallet mellom dem. I Studie I og II var målet å se om det var sammenhenger mellom mikroskopiske vevsstrukturer sett i vevsprøver (histopatologiske trekk) og tumorveksthastighet beregnet fra MR bildene. Glioblastomene ble delt inn i to grupper basert på veksthastigheten: de som vokste raskere enn forventet og de som vokste saktere enn forventet. I Studie I undersøkte vi om tilstedeværelse av 27 ulike biologiske vevsstrukturer kunne si noe om veksthastighet, mens i Studie II så vi på om tetthet av kar i vevspreparatene hadde noen sammenheng med veksten. I begge studiene fant vi at tumorer med høy celletetthet og tumorer med blodpropper i tumorkar hadde en høyere sannsynlighet for å vokse raskere. Funnene våre tyder på at blodpropper i tumorkar kan utløse en mer aggressiv tumorbiologi ved at det blir et lavere oksygennivå i tumorvevet. I Studie II fant vi at kartetthet ikke var relatert til veksthastighet, som tyder på tumorvekst ikke nødvendigvis er avhengig av økt nydannelse av kar (angiogenese). I Studie III endret vi fokus til MGMT status, eller MGMT promoter metylerings status, som er en viktig genetisk markør som sier noe om hvor effektiv cellegiftbehandling vil være hos glioblastompasienter. MGMT (O6-metylguanin-DNA metyltransferase) er et enzym som opphever effekten av cellegift når det er til stede i vevet (umetylert), mens når genet for MGMT er metylert, hindres dannelsen av enzymet og cellegift har bedre effekt. Målet i Studie III var å se om den naturlige biologien var forskjellig mellom MGMT metylerte og MGMT umetylerte glioblastomer. De biologiske trekkene vi undersøkte var 24 ulike mikroskopiske vevsstrukturer, 4 strukturer på MR-bilder og tumorveksthastigheten. Vi fant ingen forskjeller i fordelingen av disse trekkene mellom de MGMT metylerte og de MGMT umetylerte glioblastomerne. Funnene våre tyder på at den økte overlevelsen hos MGMT metylerte pasienter ikke kan forklares av en mindre aggressiv tumorbiologi. Videre tilsier funnene at det ikke er ulikheter i biologien annet enn MGMT status som forklarer den ulike responsen på cellegift. Disse funnene tyder også på at man ikke kan bruke utseendet på MR-bilder tatt før operasjon til å forutsi MGMT status. Samlet sett har studiene i denne doktorgraden bidratt til økt innsikt i den naturlige tumorbiologien i glioblastomer hos pasienter. Likevel, så må funnene bekreftes i fremtidige studier før vi kan trekke endelige konklusjoner

The histological representativeness of glioblastoma tissue samples

Background Glioblastomas (GBMs) are known for having a vastly heterogenous histopathology. Several studies have shown that GBMs can be histologically undergraded due to sampling errors of small tissue samples. We sought to explore to what extent histological features in GBMs are dependent on the amount of viable tissue on routine slides from both biopsied and resected tumors. Methods In 106 newly diagnosed GBM patients, we investigated associations between the presence or degree of 24 histopathological and two immunohistochemical features and the tissue amount on hematoxylin-eosin (HE) slides. The amount of viable tissue was semiquantitatively categorized as “sparse,” “medium,” or “substantial” for each case. Tissue amount was also assessed for associations with MRI volumetrics and the type of surgical procedure. Results About half (46%) of the assessed histological and immunohistochemical features were significantly associated with tissue amount. The significant features were less present or of a lesser degree when the tissue amount was smaller. Among the significant features were most of the features relevant for diffuse astrocytic tumor grading, i.e., small necroses, palisades, microvascular proliferation, atypia, mitotic count, and Ki-67/MIB-1 proliferative index (PI). Conclusion A substantial proportion of the assessed histological features were at risk of being underrepresented when the amount of viable tissue on HE slides was limited. Most of the grading features were dependent on tissue amount, which underlines the importance of considering sampling errors in diffuse astrocytic tumor grading. Our findings also highlight the importance of adequate tissue collection to increase the quality of diagnostics and histological research

Microglia and macrophages in human glioblastomas: A morphological and immunohistochemical study

Glioblastomas (GBMs), a type of highly malignant brain tumour, contain various macrophages/microglia that are known as tumour‑associated macrophages (TAMs). These TAMs have various roles in tumour biology. Histopathological aspects of TAMs and associations with tumour growth assessed by magnetic resonance imaging (MRI) are poorly described. In the present study, 16 patients that had sufficient tumour tissue and histological hallmarks were examined. The tumours were classified as either slow‑ (n=7) or fast‑growing (n=9) based on the segmented tumour volumes from MRI scans taken at diagnosis and preoperatively. Using cluster of differentiation (CD)68 and ionized calcium-binding adaptor molecule 1 (Iba1) antibodies, the number, morphology, localization and distribution of TAMs in the GBM tissue were studied. TAMs were significantly more immunoreactive for anti‑Iba1 (TAMsIba1) compared with anti‑CD68 (TAMsCD68; P<0.001). In central tumour areas and around vessels in the infiltration zone there were more TAMsCD68 in slow‑growing tumours (P=0.003 and P=0.025, respectively). Central tumour areas contained more TAMs compared with the infiltration zone (P=0.001 for TAMsCD68 and P<0.001 for TAMsIba1). The majority of TAMs exhibited a ramified phenotype in the infiltration zone, whereas central TAMs were mostly amoeboid. TAMs were present in high numbers in most regions of the tumour, whereas there were few in necrotic areas. In conclusion, the present study demonstrated and confirmed that the high numbers of TAMs in GBMs assume a range of morphologies consistent with various activation states, and that slow‑growing GBMs seem to contain a TAM‑population different to their fast‑growing counterparts

Angiogenesis and radiological tumor growth in patients with glioblastoma

Abstract Background The preoperative growth of human glioblastomas (GBMs) has been shown to vary among patients. In animal studies, angiogenesis has been linked to hypoxia and faster growth of GBM, however, its relation to the growth of human GBMs is sparsely studied. We have therefore aimed to look for associations between radiological speed of growth and microvessel density (MVD) counts of the endothelial markers vWF (Factor VIII related antigen) and CD105 (endoglin). Methods Preoperative growth was estimated from segmented tumor volumes of two preoperative T1-weighted postcontrast magnetic resonance imaging scans taken ≥14 days apart in patients with newly diagnosed GBMs. A Gompertzian growth curve was computed from the volume data and separated the patients into two groups of either faster or slower tumor growth than expected. MVD counts of the immunohistochemical markers von Willebrand factor (vWF) (a pan-endothelial marker) and CD105 (a marker of proliferating endothelial cells) were assessed for associations with fast-growing tumors using Mann-Whitney U tests and a multivariable binary logistic regression analysis. Results We found that only CD105-MVD was significantly associated with faster growth in a univariable analysis (p = 0.049). However, CD105-MVD was no longer significant when corrected for the presence of thromboses and high cellular density in a multivariable model, where the latter features were significant independent predictors of faster growth with respective odds ratios 4.2 (95% confidence interval, 1.2, 14.3), p = 0.021 and 2.6 (95% confidence interval, 1.0, 6.5), p = 0.048. Conclusions MVDs of neither endothelial marker were independently associated with faster growth, suggesting angiogenesis-independent processes contribute to faster glioblastoma growth

MGMT Promoter Methylation Status Is Not Related to Histological or Radiological Features in IDH Wild-type Glioblastomas

O6-methylguanine DNA methyltransferase (MGMT) promoter methylation is an important favorable predictive marker in patients with glioblastoma (GBM). We hypothesized that MGMT status could be a surrogate marker of pretreatment tumor biology observed as histopathological and radiological features. Apart from some radiological studies aiming to noninvasively predict the MGMT status, few studies have investigated relationships between MGMT status and phenotypical tumor biology. We have therefore aimed to investigate such relationships in 85 isocitrate dehydrogenase (IDH) wild-type GBMs. MGMT status was determined by methylation-specific PCR and was assessed for associations with 22 histopathological features, immunohistochemical proliferative index and microvessel density measurements, conventional magnetic resonance imaging characteristics, preoperative speed of tumor growth, and overall survival. None of the investigated histological or radiological features were significantly associated with MGMT status. Methylated MGMT status was a significant independent predictor of improved overall survival. In conclusion, our results suggest that MGMT status is not related to the pretreatment phenotypical biology in IDH wild-type GBMs. Furthermore, our findings suggest the survival benefit of MGMT methylated GBMs is not due to an inherently less aggressive tumor biology, and that conventional magnetic resonance imaging features cannot be used to noninvasively predict the MGMT status