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  • Lopes, Nair; Bergsland, Christian Holst; Bjornslett, Merete; Pellinen, Teijo; Svindland, Aud; Nesbakken, Arild; Almeida, Raquel; Lothe, Ragnhild A.; David, Leonor; Bruun, Jarle (2020)
    Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and beta-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.
  • Mpindi, John Patrick; Sara, Henri; Haapa-Paananen, Saija; Kilpinen, Sami; Pisto, Tommi; Bucher, Elmar; Ojala, Kalle; Iljin, Kristiina; Vainio, Paula; Bjorkman, Mari; Gupta, Santosh; Kohonen, Pekka; Nees, Matthias; Kallioniemi, Olli (2011)
    Background Meta-analysis of gene expression microarray datasets presents significant challenges for statistical analysis. We developed and validated a new bioinformatic method for the identification of genes upregulated in subsets of samples of a given tumour type (‘outlier genes’), a hallmark of potential oncogenes. Methodology A new statistical method (the gene tissue index, GTI) was developed by modifying and adapting algorithms originally developed for statistical problems in economics. We compared the potential of the GTI to detect outlier genes in meta-datasets with four previously defined statistical methods, COPA, the OS statistic, the t-test and ORT, using simulated data. We demonstrated that the GTI performed equally well to existing methods in a single study simulation. Next, we evaluated the performance of the GTI in the analysis of combined Affymetrix gene expression data from several published studies covering 392 normal samples of tissue from the central nervous system, 74 astrocytomas, and 353 glioblastomas. According to the results, the GTI was better able than most of the previous methods to identify known oncogenic outlier genes. In addition, the GTI identified 29 novel outlier genes in glioblastomas, including TYMS and CDKN2A. The over-expression of these genes was validated in vivo by immunohistochemical staining data from clinical glioblastoma samples. Immunohistochemical data were available for 65% (19 of 29) of these genes, and 17 of these 19 genes (90%) showed a typical outlier staining pattern. Furthermore, raltitrexed, a specific inhibitor of TYMS used in the therapy of tumour types other than glioblastoma, also effectively blocked cell proliferation in glioblastoma cell lines, thus highlighting this outlier gene candidate as a potential therapeutic target. Conclusions/Significance Taken together, these results support the GTI as a novel approach to identify potential oncogene outliers and drug targets. The algorithm is implemented in an R package (Text S1).
  • Ollila, Hely; Paajanen, Juuso; Wolff, Henrik; Ilonen, Ilkka; Sutinen, Eva; Välimäki, Katja; Östman, Arne; Anttila, Sisko; Kettunen, Eeva; Räsänen, Jari; Kallioniemi, Olli; Myllärniemi, Marjukka; Mäyränpää, Mikko I.; Pellinen, Teijo (2021)
    Malignant pleural mesothelioma (MPM) has a rich stromal component containing mesenchymal fibroblasts. However, the properties and interplay of MPM tumor cells and their surrounding stromal fibroblasts are poorly characterized. Our objective was to spatially profile known mesenchymal markers in both tumor cells and associated fibroblasts and correlate their expression with patient survival. The primary study cohort consisted of 74 MPM patients, including 16 patients who survived at least 60 months. We analyzed location-specific tissue expression of seven fibroblast markers in clinical samples using multiplexed fluorescence immunohistochemistry (mfIHC) and digital image analysis. Effect on survival was assessed using Cox regression analyses. The outcome measurement was all-cause mortality. Univariate analysis revealed that high expression of secreted protein acidic and cysteine rich (SPARC) and fibroblast activation protein in stromal cells was associated with shorter survival. Importantly, high expression of platelet-derived growth factor receptor beta (PDGFRB) in tumor cells, but not in stromal cells, was associated with shorter survival (hazard ratio [HR] = 1.02, p <0.001). A multivariable survival analysis adjusted for clinical parameters and stromal mfIHC markers revealed that tumor cell PDGFRB and stromal SPARC remained independently associated with survival (HR = 1.01, 95% confidence interval [CI] = 1.00-1.03 and HR = 1.05, 95% CI = 1.00-1.11, respectively). The prognostic effect of PDGFRB was validated with an artificial intelligence-based analysis method and further externally validated in another cohort of 117 MPM patients. In external validation, high tumor cell PDGFRB expression associated with shorter survival, especially in the epithelioid subtype. Our findings suggest PDGFRB and SPARC as potential markers for risk stratification and as targets for therapy.
  • Thunnissen, Erik; Weynand, Birgit; Udovicic-Gagula, Dalma; Brcic, Luka; Szolkowska, Malgorzata; Hofman, Paul; Smojver-Jezek, Silvana; Anttila, Sisko; Calabrese, Fiorella; Kern, Izidor; Skov, Birgit; Perner, Sven; Dale, Vibeke G.; Eri, Zivka; Haragan, Alex; Leonte, Diana; Carvallo, Lina; Prince, Spasenja Savic; Nicholson, Siobhan; Sansano, Irene; Ryska, Ales (2020)
    A questionnaire on biomarker testing previously used in central European countries was extended and distributed in Western and Central European countries to the pathologists participating at the Pulmonary Pathology Society meeting 26-28 June 2019 in Dubrovnik, Croatia. Each country was represented by one responder. For recent biomarkers the availability and reimbursement of diagnoses of molecular alterations in non-small cell lung carcinoma varies widely between different, also western European, countries. Reimbursement of such assessments varies widely between unavailability and payments by the health care system or even pharmaceutical companies. The support for testing from alternative sources, such as the pharmaceutical industry, is no doubt partly compensating for the lack of public health system support, but it is not a viable or long-term solution. Ideally, a structured access to testing and reimbursement should be the aim in order to provide patients with appropriate therapeutic options. As biomarker enabled therapies deliver a 50% better probability of outcome success, improved and unbiased reimbursement remains a major challenge for the future.
  • Remes, Satu Maria; Leijon, Helena; Vesterinen, Tiina; Louhimo, Johanna; Pulkkinen, Ville; Ezer, Sini; Kere, Juha; Haglund, Caj; Arola, Johanna (2020)
    Neuroendocrine tumors (NETs) are often diagnosed from the metastases of an unknown primary tumor. Specific immunohistochemical (IHC) markers indicating the location of a primary tumor are needed. The proprotein convertase subtilisin/kexin type 2 (PCSK2) is found in normal neural and neuroendocrine cells, and known to express in NETs. We investigated the tissue microarray (TMA) of 86 primary tumors from 13 different organs and 9 metastatic NETs, including primary tumor-metastasis pairs, for PCSK2 expression with polymer-based IHC. PCSK2 was strongly positive in all small intestine and appendiceal NETs, the so-called midgut NETs, in most pheochromocytomas and paragangliomas, and in some of the typical and atypical pulmonary carcinoid tumors. NETs showing strong positivity were re-evaluated in larger tumor cohorts confirming the primary observation. In the metastases, the expression of PCSK2 mirrored that of the corresponding primary tumors. We found negative or weak staining in NETs from the thymus, gastric mucosa, pancreas, rectum, thyroid, and parathyroid. PCSK2 expression did not correlate with Ki-67 in well-differentiated NETs. Our data suggest that PCSK2 positivity can indicate the location of the primary tumor. Thus, PCSK2 could function in the IHC panel determined from screening metastatic NET biopsies of unknown primary origins.
  • Pasanen, Annukka; Ahvenainen, Terhi; Pellinen, Teijo; Vahteristo, Pia; Loukovaara, Mikko; Bützow, Ralf (2020)
    Programmed death-ligand 1 (PD-L1) is a biomarker that may predict the response to anti-programmed death 1/PD-L1 immunotherapy. We evaluated the expression of PD-L1 in carcinoma cells (Ca) and immune cells (ICs) across histopathologic and The Cancer Genome Atlas (TCGA) molecular subgroups of endometrial carcinoma (EC). Our study included 842 patients with EC. Direct sequencing of polymerase epsilon (POLE) exonuclease domain hot spots and conventional immunohistochemistry (MLH1, PMS2, MSH2, MSH6, p53) were conducted to identify TCGA classification-based molecular subgroups of EC: POLE-mutated, mismatch repair deficient, no specific molecular profile, and p53 aberrant. Multiplex immunohistochemistry was performed to evaluate PD-L1 expression in Ca and tumor-infiltrating ICs. PD-L1 expression in Ca and in ICs was detected in 8.6% and 27.7% of the cases, respectively. A combined positive score (CPS) was >= 1% in 19.4% of the samples. PD-L1 positivity in Ca and ICs, and CPS correlated with tumor T-cell density (P= 1% (P=0.037) positivity compared with early disease. In conclusion, PD-L1 expression profiles differ between molecular subclasses, histologic subtypes, and disease stage of EC. Prospective studies are needed to explore the predictive value of various PD-L1 scoring systems within the subgroups of EC. CPS presents methodological advantages over cell type-specific scoring systems.
  • Keinänen, Arvi; Marinescu-Gava, Magdalena; Uittamo, Johanna; Hagström, Jaana; Marttila, Emilia; Snäll, Johanna (2020)
    Objectives We assessed the periodontal situation radiologically according to tumour p16 status. Materials and methods Patients with a diagnosis of tonsillar cancer and availability of a digital panoramic radiograph (DPR) during a 5-year period were included in this retrospective study. The predictor variables were periodontal stability, marginal bone loss, marginal bone loss without periodontal stability and total number of teeth. Periodontal status was compared with p16 status, age, gender, smoking and alcohol use. Results Among 115 patients included in the analyses (p16-negative,n = 24; p16-positive,n = 91), smoking (p <.0001), heavy alcohol use (p <.0001) and total number of teeth (p = .0001) were significantly associated with p16 status. Current smoking (OR = 7.3) and heavy alcohol use (OR = 10.1) increased the risk of p16-negative cancer. Conclusions Patients with p16-negative tonsillar carcinoma had less teeth than patients with p16-positive tumours. Other periodontal findings were common in both groups without statistical significance. Heavy alcohol use and smoking were the most important risk factors for p16-negative tonsillar carcinoma.
  • Storvall, Sara; Leijon, Helena; Ryhänen, Eeva; Louhimo, Johanna; Haglund, Caj; Schalin-Jäntti, Camilla; Arola, Johanna (2019)
    Introduction: Parathyroid carcinoma represents a rare cause of primary hyperparathyroidism. Distinguishing carcinoma from the benign tumors underlying primary hyperparathyroidism remains challenging. The diagnostic criteria for parathyroid carcinoma are local and/or metastatic spreading. Atypical parathyroid adenomas share other histological features with carcinomas but lack invasive growth. Somatostatin receptors are commonly expressed in different neuro endocrine tumors, but whether this also holds for parathyroid tumors remains unknown. Aim: Our aim is to examine the immunohistochemical expression of somatostatin receptor 1-5 in parathyroid typical adenomas, atypical adenomas and carcinomas. Methods: We used a tissue microarray construct from a nationwide cohort of parathyroid carcinomas (n = 32), age- and gender-matched typical parathyroid adenomas (n = 72) and atypical parathyroid adenomas (n = 27) for immunohistochemistry of somatostatin receptor subtypes 1-5. We separately assessed cytoplasmic, membrane and nuclear expression and also investigated the associations with histological, biochemical and clinical characteristics. Results: All parathyroid tumor subgroups expressed somatostatin receptors, although membrane expression appeared negligible. Except for somatostatin receptor 1, expression patterns differed between the three tumor types. Adenomas exhibited the weakest and carcinomas the strongest expression of somatostatin receptor 2, 3, 4 and 5. We observed the largest difference for cytoplasmic somatostatin receptor 5 expression. Conclusions: Parathyroid adenomas, atypical adenomas and carcinomas all express somatostatin receptor subtypes 1-5. Somatostatin receptor 5 may serve as a potential tumor marker for malignancy. Studies exploring the role of somatostatin receptor imaging and receptor-specific therapies in patients with parathyroid car cinomas are needed.
  • Rosin, Gustaf; Hannelius, Ulf; Lindstrom, Linda; Hall, Per; Bergh, Jonas; Hartman, Johan; Kere, Juha (2012)
  • Johansson, Benjamin; Sahi, Helka; Koljonen, Virve; Böhling, Tom (2019)
    Background: Merkel cell carcinoma (MCC) tumor samples frequently express B-lymphoid lineage markers. However, the reasons for expression of specific B-lymphoid lineage markers are still unclear. We studied the expression of TdT and PAX5 (two B-cell lymphoid lineage markers) in a large pool of MCC tissue microarray samples. Methods: Immunoexpression and staining intensities of TdT and Pax-5 were statistically correlated with patient, tumor, Merkel cell polyomavirus (MCV), and disease-specific parameters. Results: In a cohort of 117 MCC patients and their corresponding tumor samples, TdT was expressed in 37 (31.6%) samples and PAX5 in 26 (22.2%). Simultaneous immunostaining for TdT and PAX5 was observed in 13 (11.1%) samples. A statistically significant relationship was observed between MCV virus copy number and positive TdT expression (P = 0.0056). Similarly, a significant relationship was also observed between positive TdT and tumor MCV virus positivity (P = 0.000495). Conclusion: We observed frequent TdT and PAX5 immunoexpression in MCC tumor samples. However, simultaneous immunoexpression of these markers was scarce. TdT expression was statistically significantly associated with MCV positivity. The absence of a statistically significant association between tumor parameters and disease progression markers undermines the systemic use of these markers in clinical practice.