Hypoxia-associated biomarkers in rectal cancer treated by preoperative radiotherapy or chemoradiotherapy

Hypoksiaan liittyvät biologiset merkkiaineet leikkausta edeltävällä sädehoidolla tai kemosädehoidolla hoidetussa peräsuolisyövässä Peräsuolensyöpä on yleinen pahanlaatuinen kasvain. Leikkausta edeltävä sädehoito annetaan yleensä T3-T4-kasvaimille. Tutkimuksella pyrittiin selvittämään, voidaanko kasvaimen hapenpuutteeseen liittyvillä biologisilla merkkiaineilla arvioida peräsuolisyövän ennustetta leikkausta edeltävän sädehoidon tai kemosädehoidon jälkeen. Tällaisia merkkiaineita ovat hapenpuutteen vaikutuksesta aktivoituva HIF-1alfa hiilihappoanhydraasi IX (CA IX), sokerin kuljetukseen solussa osallistuva GLUT-1 sekä solun tukirankaproteiini ezrin. Tutkimukseen otettiin 178 potilasta, jotka olivat saaneet ennen leikkausta lyhyen (n=77) tai pitkän sädehoidon (n=10), pitkän sädehoidon ja solunsalpaajahoidon (n=37) tai ei mitään hoitoa (n=54). Lisäksi osalta leikkausta edeltävää sädehoitoa saaneelta potilaalta tutkittiin hoitoja edeltävät, diagnostiset näytteet (n=80). Tutkimuksessa käytettiin immunehistokemiallisia värjäysmenetelmiä. Kasvaimen regressiota (TRG) arvioitiin pitkän sädehoidon jälkeisistä näytteistä. Leikkausnäytteissä negatiivinen/heikko CA IX intensiteetti liittyi sekä pidempään tautispesifiseen (p=0.034) että tautivapaaseen elinaikaan (p=0.003) ja pitkän sädehoidon jälkeen HIF-1alfa-negatiivisuus pidempään tautispesifiseen (p=0.001) sekä negatiivinen/heikko GLUT-1 pidempään tautivapaaseen elinaikaan (p=0.066). Voimakas ezrin-ilmentymä diagnostisissa näytteissä liittyi lyhyempään tautivapaaseen ja tautispesifiseen (p=0.027 ja p=0.002) ennusteeseen. Monimuuttuja-analyysissä vahva CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä selviytymistä. Erinomainen TRG liittyi negatiiviseen/heikkoon CA IX- (p=0.057), ezrin- (p=0.012) ja GLUT-1 -ilmentymään (p=0.013) leikkausnäytteissä. Kun kaikki neljä merkkiainetta analysoitiin yhdessä monimuuttuja-analyysissä, CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti tautispesifistä elinaikaa. Voimakas CA IX-ilmentymä leikkausnäytteissä ja positiivinen HIF-1alfa- ja vahva GLUT-1-ilmentymä pitkän sädehoidon jälkeisissä leikkausnäytteissä sekä vahva ezrin-ilmentymä diagnostisissa näytteissä liittyivät epäsuotuisaan ennusteeseen. Monimuuttujaanalyysissä kohtalainen/voimakas CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä elinaikaa. CA IX on vahva biologinen merkkiaine peräsuolisyövässä.

Molecular markers for progression of squamous cell carcinoma of the skin

Incidence of nonmelanoma skin cancer (NMSC) is increasing. Ultraviolet (UV) –light is a major risk factor for the development of cutaneous SCC. Cutaneous SCCs that develop to chronic ulcers are known to progress and metastasize more easily than UV-induced SCCs. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are suggested to have a role in cancer growth and invasion. The molecular background for progression of cutaneous SCC was examined by immunohistochemistry (IHC) using tissue samples of recessive dystrophic epidermolysis bullosa (RDEB) –associated SCC, sporadic UV-induced SCC, and SCC precursors. IHC studies using tissue microarray (TMA) technique revealed overexpression of MMP-7 and MMP-13 in SCC tumor cells. MMP-7 expression was enhanced especially in the SCC tumor cells of the RDEB –associated SCCs. Studies with SCC cell lines showed that tumor cell derived MMP-7 activated heparin binding epidermal growth factor –like growth factor (HB-EGF) which enhanced the growth of SCC tumor cells. Further, it was shown that type VII collagen (COL7) is expressed in sporadic SCC tumor cells. Interestingly, it was shown that SCC –associated MMP-13 is capable of cleaving COL7 in vitro. COL7 cleavage may have a role in the progression of cutaneous SCC. Studies on serine proteinase inhibitor gene family using SCC tumor cell gene array, quantitative real-time PCR, SCC cell lines, normal human epidermal keratinocytes and IHC of TMA samples showed that serine proteinase inhibitor clade A, member 1 (serpinA1, alpha-1-antitrypsin) is expressed and produced by human SCC tumor cells but not by normal keratinocytes. Moreover, serpinA1 expression was shown to correlate with the progression of cutaneous SCC using transformed HaCaT-cell lines and mouse chemically induced skin SCC model. SerpinA1 may serve as a novel biomarker for the progression of cutaneous SCC. This study elucidated putative mechanisms of the progression of cutaneous SCC and revealed novel biomarker candidates for the progression of SCC of the skin.

Libyan breast cancer: Health services and biology. Diagnosis delay and prognostic value of DNA pploidy, S-phase fraction, and Ki-67 and Bcl-2 immunohistochemistry

The aim of this study was to investigate the diagnosis delay and its impact on the stage of disease. The study also evaluated a nuclear DNA content, immunohistochemical expression of Ki-67 and bcl-2, and the correlation of these biological features with the clinicopathological features and patient outcome. 200 Libyan women, diagnosed during 2008–2009 were interviewed about the period from the first symptoms to the final histological diagnosis of breast cancer. Also retrospective preclinical and clinical data were collected from medical records on a form (questionnaire) in association with the interview. Tumor material of the patients was collected and nuclear DNA content analysed using DNA image cytometry. The expression of Ki-67 and bcl-2 were assessed using immunohistochemistry (IHC). The studies described in this thesis show that the median of diagnosis time for women with breast cancer was 7.5 months and 56% of patients were diagnosed within a period longer than 6 months. Inappropriate reassurance that the lump was benign was an important reason for prolongation of the diagnosis time. Diagnosis delay was also associated with initial breast symptom(s) that did not include a lump, old age, illiteracy, and history of benign fibrocystic disease. The patients who showed diagnosis delay had bigger tumour size (p<0.0001), positive lymph nodes (p<0.0001), and high incidence of late clinical stages (p<0.0001). Biologically, 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF of tumors was 11% while the median positivity of Ki-67 was 27.5%. High Ki-67 expression was found in 76% of patients, and high SPF values in 56% of patients. Positive bcl-2 expression was found in 62.4% of tumors. 72.2% of the bcl-2 positive samples were ER-positive. Patients who had tumor with DNA aneuploidy, high proliferative activity and negative bcl-2 expression were associated with a high grade of malignancy and short survival. The SPF value is useful cell proliferation marker in assessing prognosis, and the decision cut point of 11% for SPF in the Libyan material was clearly significant (p<0.0001). Bcl-2 is a powerful prognosticator and an independent predictor of breast cancer outcome in the Libyan material (p<0.0001). Libyan breast cancer was investigated in these studies from two different aspects: health services and biology. The results show that diagnosis delay is a very serious problem in Libya and is associated with complex interactions between many factors leading to advanced stages, and potentially to high mortality. Cytometric DNA variables, proliferative markers (Ki-67 and SPF), and oncoprotein bcl-2 negativity reflect the aggressive behavior of Libyan breast cancer and could be used with traditional factors to predict the outcome of individual patients, and to select appropriate therapy.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.