Host Immune Response and Tumour Microenvironment in Pancreatic cancer : Impact on Patients’ Survival

Haiman duktaalinen adenokarsinooma on yleistyvä pahanlaatuinen sairaus. Kirurgia on edelleen ainoa hoitomuoto, johon liittyy pysyvän parantumisen mahdollisuus. Se on kuitenkin tarjolla vain pienelle osalle haimasyöpäpotilaista, koska tauti on yleensä diagnoosivaiheessa edennyt leikkaushoidon ulottumattomiin. Vain noin viidennes leikatuistakin potilaista elää yli viisi vuotta. Elimistön omalla immuunivasteella ja kasvaimen mikroympäristöllä on todettu olevan merkittävä vaikutus haimasyövän etenemiseen ja sitä kautta potilaiden selviytymiseen. Haimasyövän tiedetään kehittävän immuunivastetta vaimentavia mekanismeja. Haimasyöpäpotilaiden ennusteen arviointiin käytetyin menetelmä on kasvaimen kokoa sekä leviämisastetta kuvaava TNM -luokitus, johon kuitenkin liittyy merkittävää epätarkkuutta. Luokitus ei huomioi kasvaimen biologisia piirteitä eikä immuunivasteen voimakkuutta. Tämä väitöskirjatutkimus pyrki selvittämään kasvaimen mikroympäristön kuten kasvainkudoksen sisältämien immuunisolujen määrän ennusteellista vaikutusta haimasyöäpotilaan ennusteeseen. Lisäksi tutkittiin kasvaimen immuunisolumääriin perustuvan immune cell score (ICS) -luokituksen soveltuvuutta haimasyöpäpotilaiden selviytymisen ennustamiseen. Neljästä osatyöstä koostuva tutkimus perustui kahden eri sairaalan haimasyöpäleikkauksen läpikäyneiden potilaiden aineistoon. Ensimmäisessä osatyössä osoitettiin CD3+ ja CD8+ T -solujen määrään perustuvan ICS- luokituksen ennustavan haimasyöpäpotilaiden selviytymistä. Keski- Suomen Keskusssairaalassa leikatun 108:n haimasyöpäpotilaan aineistossa korkea ICS ennusti parempaa selviytymistä riippumatta TNM -luokasta. Toinen osatyö osoitti ICS:n toimivuuden toisessa, Oulun Yliopistollisessa sairaalassa leikatussa haimasyöpäpotilaiden aineistossa. Tässä työssä ICS -luokitus toteutettiin kahdella eri tekniikalla, käyttäen sekä kasvaimesta tehtyjä kokoleikkeitä että niin sanottua hot-spot -tekniikkaa, joista ensimmäinen osoittautui toimivammaksi. Kolmannessa osatyössä selvitti immuunivastetta vaimentavan CD73 -proteiinin ennustevaikutusta haimasyövässä. Tulokset osoittivat kasvainsolujen korkean CD73 -ekspression ennustavan huonoa selviytymistä. Lisäksi todettiin imusolmukemetastasoinnin olevan yleisempää niillä potilailla, joilla kasvainalueen immuunisolut ilmensivät runsaasti CD73 -proteiinia. Neljännessä osatyössä osoitettiin kasvaimen strooman hyaluronaanin suuren pitoisuuden liittyvän huonompaan ennusteeseen. Tämän todettiin myös olevan yhteydessä heikompiasteiseen immuunireaktioon. Johtopäätöksenä voidaan todeta haimasyövän etenemisen riippuvan vahvasti elimistön immuunivasteesta ja muista kasvaimen mikroympäristössä vaikuttavista tekijöistä kuten CD73 -proteiinin ja hyaluronaanin ylimäärästä. Immuunivasteen voimakkuutta kuvaava ICS vaikuttaa tämän tutkimuksen perusteella soveltuvan haimasyöpäpotilaiden selviytymisen ennustamiseen.Pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with an increasing incidence. Surgery is the only option to achieve cure, but it can be offered for a minority of patients since most patients present with unresectable disease. Five-year prognosis is only approximately 20 %, even for patients undergoing surgery with a curative aim. Host immune response and tumour microenvironment have a significant impact on the progression of the disease and on the survival of the patients with PDAC. PDAC is known to develop mechanisms for immune escape. Tumour-node- metastasis (TNM) classification has been the most used method for years when estimating the prognosis of the patients with PDAC. However, it describes only the stage of progression of the disease, not its’ biological features or the level of immune response leading to inadequate prognostic accuracy. This thesis aimed to study the prognostic role of immune cell infiltration and other microenvironmental factors in PDAC. The suitability of immune cell infiltration-based immune cell score (ICS) as a prognostic tool in PDAC was also assessed. The thesis comprises four studies based on two cohorts of PDAC patients who had undergone surgery with curative intent. The first part of the study demonstrates the prognostic value of CD3+ and CD8+ cell -based ICS in a cohort of 108 PDAC patients, operated on in Central Finland Central Hospital between 2000 and 2016. High ICS is shown to be an independent prognostic factor for prolonged survival regardless of TNM stage. In the second part of the study the prognostic value of ICS is shown in another cohort of 79 patients, operated on in Oulu University Hospital with curative intent between 1993 and 2015. ICS determination was performed using two different techniques in this part of the study. The study shows the superiority of whole tissue section technique over hot-spot technique. The third part focuses on the impact of immune suppressive protein CD73 on the survival of patients with PDAC. According to the results, high CD73 expression in tumour cells is an independent negative prognostic factor in PDAC. Moreover, high expression of CD73 in tumour infiltrating lymphocytes was associated with lymph node metastasis. The fourth part of the study shows the prognostic role of stromal hyaluronan accumulation in PDAC. The hyaluronan accumulation in stroma is shown to be associated with poor prognosis and low-level host immune response. As a conclusion, progression of PDAC is heavily dependent on host immune response and other microenvironmental factors such as overexpression of CD73 and hyaluronan. ICS as an indicator of immune response can be used as a predictor of the survival among patients with PDAC

Prognostic impact of CD73 expression and its relationship to PD-L1 in patients with radically treated pancreatic cancer

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Prognostic impact of CD73 expression and its relationship to PD-L1 in patients with radically treated pancreatic cancer

Immune suppressing molecule CD73 is overexpressed in various cancers and associated with poor survival. Little is so far known about the predictive value of CD73 in pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the prognostic significance of CD73 in PDAC. The study material consisted of 110 radically treated patients for PDAC. Tissue microarray blocks were constructed and stained immunohistochemically using CD73 antibody. Staining intensity and numbers of stained tumour cells, inflammatory cells, stroma, and blood vessels were assessed. High-level CD73 expression in tumour cells was positively associated with PD-L1 expression, perineural invasion, and histopathological grade. CD73 positivity in tumour-infiltrating lymphocytes was significantly associated with lymph node metastasis. Lymphocytic CD73 positivity was also associated with staining positivity in both stroma and vascular structures. In addition, CD73 positivity in vascular structures and stroma were associated with each other. There were no significant associations between CD73 positive tumour cells and CD73 positivity in any other cell types. PD-L1 expression was associated with CD73 staining positivity in stroma (p = 0.007) and also with histopathological grade (p = 0.033) and T class (p = 0.016) of the primary tumour. CD73 positivity in tumour cells was significantly associated with poor disease-specific (p = 0.021) and overall survival (p = 0.016). In multivariate analysis, CD73 positivity in tumour cells was an independent negative prognostic factor together with histopathological grade, TNM stage, and low immune cell score. In conclusion, high CD73 expression in tumour cells is associated with poor survival in PDAC independently of the number of tumour-infiltrating lymphocytes or TNM stage.peerReviewe

Immune cell score in pancreatic cancer-comparison of hotspot and whole-section techniques

Abstract An immune cell score (ICS) was introduced for predicting survival in pancreatic ductal adenocarcinoma (PDAC). Few studies have compared different methods of evaluating immune infiltrate. This study compared ICSs determined in whole sections or tissue microarray-like hotspots for predicting survival after PDAC surgery. We included in 79 consecutive patients from a single geographical area that underwent surgery for PDAC (R0/R1, stages I–III). We performed digital image analyses to evaluate CD3 and CD8 staining. ICSs were classified as low, moderate, or high, based on the numbers of immune cells in the tumour core and invasive margin. We compared ICS groups determined with the hotspot and whole-section techniques. Associations between ICS and survival were analysed with Cox regression models, adjusted for sex, age, tumour stage, differentiation grade, perineural invasion, and resection radicality. In hotspot ICS analysis, 5-year overall survival rates for low, moderate, and high groups were 12.1%, 26.3%, and 26.8%, respectively (p = 0.193). In whole-section analyses, overall survival rates were 5.3%, 26.4%, and 43.8%, respectively (p = 0.030). In the adjusted Cox model, whole-section ICS groups were inversely associated with the overall mortality hazard ratio (HR): low, moderate, and high ICS groups had HRs of 1.00, 0.42 (95% CI 0.20–0.88), and 0.27 (95% CI 0.11–0.67), respectively. The number of immune cells per square millimetre in the tumour core and the invasive margin were significantly higher and had a wider range in hotspots than in whole-tissue sections. Accordingly, ICS could predict survival in patients with PDAC after surgery. Whole tissue section ICSs exhibited better prognostic value than hotspot ICSs

Stromal hyaluronan accumulation is associated with low immune response and poor prognosis in pancreatic cancer

Abstract Hyaluronan (HA) accumulation has been associated with poor survival in various cancers, but the mechanisms for this phenomenon are still unclear. The aim of this study was to investigate the prognostic significance of stromal HA accumulation and its association with host immune response in pancreatic ductal adenocarcinoma (PDAC). The study material consisted of 101 radically treated patients for PDAC from a single geographical area. HA staining was evaluated using a HA-specific probe, and the patterns of CD3, CD8, CD73 and PD-L1 expression were evaluated using immunohistochemistry. HA staining intensity of tumour stromal areas was assessed digitally using QuPath. CD3- and CD8-based immune cell score (ICS) was determined. High-level stromal HA expression was significantly associated with poor disease-specific survival (p = 0.037) and overall survival (p = 0.013) In multivariate analysis, high-level stromal HA expression was an independent negative prognostic factor together with histopathological grade, TNM stage, CD73 positivity in tumour cells and low ICS. Moreover, high-level stromal HA expression was associated with low ICS (p = 0.017). In conclusion, stromal HA accumulation is associated with poor survival and low immune response in PDAC