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Merkel cell polyomavirus infection and host defence in patients with Merkel cell carcinoma

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Title: Merkel cell polyomavirus infection and host defence in patients with Merkel cell carcinoma
Author: Sihto, Harri
Contributor: University of Helsinki, Faculty of Medicine, Institute of Clinical Medicine, Syöpätautien osastoMolecular Cancer Biology Program
Thesis level: Doctoral dissertation (article-based)
Abstract: Background and purpose: Merkel cell carcinoma (MCC) is a rare and often aggressive skin cancer that usually arises in elderly individuals. MCC is frequently associated with ultraviolet radiation exposure and immunosuppressive conditions. The majority of MCCs harbor Merkel cell polyomavirus (MCPyV), a newly discovered human cancer virus. Several studies have indicated its importance in MCC tumorigenesis.

The aim of the thesis was to evaluate the frequency of MCPyV infection in MCC and to investigate its associations with patient and tumor characteristics, and with survival. In addition, we aimed to investigate the associations between MCPyV infection with cell cycle regulatory protein expression, platelet-derived growth factor receptor (PDGFR) family protein expression, TP53, KIT and PDGFRA mutations, and their associations with clinicopathological factors. We also determined the frequency and type of leukocytes that infiltrate MCC, and their associations with the presence of MCPyV DNA in MCC and clinicopathological factors including disease outcome.

Experimental design: The study was based on a population-wide MCC patient series from Finland, with the MCCs diagnosed in 1979 to 2004, and the corresponding archival formalin-fixed paraffin-embedded tumor tissue samples. The patients were identified from the files of the Finnish Cancer Registry. MCPyV DNA was detected using polymerase chain reaction (PCR) and quantitative PCR, tumor infiltrating immune cells were identified and the expression of MCPyV large T (LT) antigen and other proteins was assessed by immunohistochemistry. Gene mutations were investigated using PCR and DNA sequencing. Protein expression was usually assessed from tissue microarray (TMA) sections, while the numbers of tumor infiltrating leukocytes were counted from full tumor tissue sections. The associations between the molecular and host response factors studied and the clinicopathological factors including survival were investigated using conventional statistical tests, such as Kaplan-Meier survival analyses and Cox s proportional hazards models.

Results: We found that most (approximately 80%) MCCs harbor MCPyV DNA and that the MCPyV LT antigen was expressed in 67% of the tumors. The presence of MCPyV DNA in tumor was associated with better disease-specific (5-year survival: 75.9% vs. 41.1%, p < 0.001) and overall survival (5-year survival: 45.0% vs. 13.0%, p < 0.001) as compared to MCPyV-negative MCCs. The MCPyV DNA-positive MCCs were located more often in a limb than in a trunk or head and neck region, they had less often metastasized to regional lymph nodes at the time of the diagnosis, and less often expressed p53 and KIT than MCPyV-negative tumors (p-values < 0.05). LT antigen expression in tumor cells was associated with the female gender, location of the tumor in a limb, low cell proliferation rate, and absence of p53 expression in tumor (p-values < 0.05). Retinoblastoma protein (RB) expression was almost invariably associated with presence of MCPyV DNA and LT expression in MCC (p-values < 0.0001), whereas TP53 mutations were found exclusively in MCPyV-negative tumors (p=0.001). The presence of MCPyV DNA and LT antigen expression in tumor were independent prognostic factors for favorable overall survival in a Cox multivariable analysis, when gender and the nodal status, or the post-surgical stage were included as covariables in the analyses.

No KIT or PDGFRA mutations were found in MCC. Tumors with p53 expression were associated with worse MCC-specific and overall survival as compared to p53-negative tumors, whereas tumor RB expression was associated with favorable survival. MCPyV DNA-positive MCCs contained significantly higher numbers of tumor infiltrating CD3+, CD8+, CD16+, FoxP3+, and CD68+ cells in comparison to MCPyV DNA-negative MCCs. A higher than the median number of CD3+, CD8+ and FoxP3+ T lymphocytes, and high CD8+/CD4+ and FoxP3+/CD4+ cell ratios in tumor were associated with favorable overall survival. Both a higher than the median number of intratumoral CD3+ cells and the presence of MCPyV DNA in tumor were independently associated with favorable overall survival in a Cox multivariable analysis that included also the nodal status and gender as covariables.

Conclusions: MCPyV-positive and -negative MCCs differ in molecular features. They show important differences also in their clinical outcomes and associations with several clinicopathological factors, as well as in host immune response. A high number of tumor infiltrating T lymphocytes and presence of MCPyV DNA in tumor were identified as novel independent prognostic factors in MCC.Merkelinsolukarsinooma on harvinainen, ennusteeltaan melko huono ihosyöpätyyppi, jota tavataan yleisimmin vanhuksilla tai immuunipuutostiloista kärsivillä potilailla. Se syntyy useimmiten ultraviolettivalolle altistuville ihoalueille. Merkelinsolupolyoomavirus (Merkel cell polyomavirus, MCPyV) on uusi ihmisen syöpää aiheuttava virus, jolla uskotaan olevan tärkeä merkitys merkelinsolukarsinoomien synnyssä.

Väitöstutkimuksessa määritettiin MCPyV-infektion yleisyys merkelinsolukarsinoomissa, sekä tutkittiin virusinfektion vaikutusta potilaan taudinkulkuun. Tutkimuksessa selvitettiin lisäksi virusinfektion yhteyttä useisiin syöpäsolujen kasvua ja jakaantumista ohjaavien valkuaisaineiden ilmentymiseen sekä eräiden syöpään liittyvän geenien (TP53, KIT ja PDGFRA) mutaatioiden esiintymiseen. Tutkimme myös merkelinsolukasvaimeen tunkeutuvien valkosolujen määrää ja tyyppejä, sekä näiden yhteyttä MCPyV-infektioon ja syövän ennusteeseen.

Virusinfektion tunnistamiseksi kehitettiin virukselle spesifinen tunnistusmenetelmä (kvantitatiivinen polymeraasiketjureaktio, qPCR). Menetelmän avulla voitiin määrittää MCPyV DNA:n määrä syöpäkudoksissa. Toisessa infektion tunnistusmentelmässä tunnistettiin syöpäsoluissa ilmentyvä viruksen tuottama valkuaisaine (LT-antigeeni) immunohistokemiallisen analyysin avulla. Väitöstutkimuksen tarpeisiin kerättiin sairaaloiden patologian laitosten arkistoista koko Suomen kattava kudosaineisto, joka sisälsi maassamme vuosina 1979-2004 todetut merkelinsolukarsinoomat.

Tutkimuksessa todettiin, että noin 80% kaikista merkelinsolukarsinoomista sisältää MCPyV:n DNA:ta. Virusnegatiiviset kasvaimet sisälsivät usein tunnetun kasvunrajoitegeeniin TP53:n mutaatioita toisin kuin viruspositiiviset kasvaimet, eivätkä virusnegatiiviset kasvaimet ilmentäneet solujen jakaantumista säätelevää retinoblastoomaproteiinia (RB).

Useiden valkosolutyyppien määrä oli merkitsevästi suurempi MCPyV-positiivisissa kuin MCPyV-negatiivisissa syövissä. Sekä kasvaimen viruspositiivisuus että korkea T-lymfosyyttien määrä olivat itsenäisesti yhteydessä merkelinsolusyöpään sairastuneiden potilaiden keskimääräistä parempaan eloonjäämisennusteeseen.

Tutkimuksen tuloksista voidaan päätellä, että MCPyV-positiiviset ja MCPyV-negatiiviset merkelinsolukarsinoomat eroavat toisistaan sekä molekyylitasolla että taudin ennusteen osalta. Merkelinsolusyövän synnyn ja kehittymisen kannalta keskeisten mekanismien tunnistaminen saattaa edistää uusien hoitomuotojen kehittämistä merkelinsolusyöpään sairastuneille potilaille.
URI: URN:ISBN:978-952-10-8365-5
http://hdl.handle.net/10138/37307
Date: 2012-11-12
Copyright information: This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
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