Browsing by Subject "immunohistochemistry"

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  • Veija, Tuukka; Kero, Mia; Koljonen, Virve; Böhling, Tom (2019)
    Aims Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. Methods and results Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. Conclusions ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.
  • Kelppe, Jetta; Thoren, Hanna; Ristimäki, Ari; Haglund, Caj; Sorsa, Timo; Hagström, Jaana (2019)
    Objectives We aimed to investigate BRAF V600E percentage immunohistochemically in ameloblastomas of a single institute cohort. We were interested if age, location, histological properties, or tumor recurrence depend on the BRAF status. Subjects, materials and methods We had 36 formalin-fixed, paraffin-embedded ameloblastoma tissue samples of patients treated at the Helsinki University Hospital between the years 1983-2016. Tissue sections underwent immunohistochemistry by Ventana BenchMark XT immunostainer using Ms Anti-Braf V600E (VE1) MAB. We used R 3.4.2 and RStudio 1.1.383 to conduct statistical analysis for BRAF positivity and earlier onset as well as tumor location. We used chi-squared tests and 2-by-2 table functions to determine connections between BRAF positivity and recurrence, growth pattern, and type. Results BRAF-positive tumors occurred in younger patients compared to BRAF-negative tumors (p = 0.015) and they located mostly to the mandible (p <0.001). Growth patterns were limited to two in BRAF-negative tumors when BRAF-positive tumors presented with one to four growth patterns (p = 0.02). None of the maxillary tumors showed BRAF positivity and of these, 72.2% recurred. Conclusions An immunohistochemical BRAF marker could be a beneficial tool to predict the outcome of patients with this aggressive, easily recurring tumor.
  • Fagerstedt, K.W.; Salonen, T.; Zhao, F.; Kytölä, S.; Böhling, T.; Andersson, L.C. (2018)
    Myxoinflammatory fibroblastic sarcoma is a soft-tissue neoplasm most frequently found in the distal extremities of middle-aged adults. Most myxoinflammatory fibroblastic sarcoma are low-grade tumors with propensity for local recurrence after incomplete removal. We report a myxoinflammatory fibroblastic sarcoma which developed in the foot of a 41-year-old male and showed an exceptionally aggressive course with metastatic spread and fatal outcome within 16 months. We managed to establish a spontaneously transformed continuous cell line, called JU-PI, from a metastatic lesion. The JU-PI cells have a sub-tetraploid karyotype including the 1;10 chromosomal translocation and amplification of the proximal end of 3p; these features are considered genetic signatures of myxoinflammatory fibroblastic sarcoma. Both the primary tumor and the JU-PI cells showed nuclear expression of the TFE3 transcription factor but TFE3-activating chromosomal rearrangements were not found. To our knowledge, JU-PI is the first established myxoinflammatory fibroblastic sarcoma cell line. JU-PI cells offer a tool for investigating the molecular oncology of myxoinflammatory fibroblastic sarcoma. © 2018, © The Author(s) 2018.
  • Mohamed, Hesham; Haglund, Caj; Jouhi, Lauri; Atula, Timo; Hagström, Jaana; Mäkitie, Antti (2020)
    Oropharyngeal squamous cell carcinoma (OPSCC) is subclassified by the World Health Organization into two different entities: human papillomavirus (HPV)-positive and HPV-negative tumors. HPV infection promotes the epithelial-to-mesenchymal transition (EMT) and transformation of keratinocyte stem cells into cancer stem cells. EMT is a crucial process in the carcinogenesis of epithelial-derived malignancies, and we aimed to study the role of its markers in OPSCC. This study consists of 202 consecutive OPSCC patients diagnosed and treated with curative intent. We examined E-cadherin, beta-catenin, and vimentin expression using immunohistochemistry and compared these with tumor and patient characteristics and treatment outcome. We found that the cell-membranous expression of beta-catenin was stronger in HPV-positive than in HPV-negative tumors, and it was stronger in the presence of regional metastasis. The stromal vimentin expression was stronger among HPV-positive tumors. A high E-cadherin expression was associated with tumor grade. No relationship between these markers and survival emerged. In conclusion, beta-catenin and vimentin seem to play different roles in OPSCC: the former in the tumor tissue itself, and the latter in the tumor stroma. HPV infection may exploit the beta-catenin and vimentin pathways in carcinogenic process. More, beta-catenin may serve as a marker for the occurrence of regional metastasis:
  • Renkonen, Jutta; Toppila-Salmi, Sanna; Joenvaara, Sakari; Mattila, Pirkko; Parviainen, Ville; Hagstrom, Jaana; Haglund, Caj; Lehtonen, Mikko; Renkonen, Risto (2015)
    Toll-like receptors (TLRs) are important in barrier homeostasis, but their role in airborne allergies is not fully understood. The aim was to evaluate baseline and allergen-induced expression of TLR proteins in nasal epithelium during allergic rhinitis. Nineteen otherwise healthy non-smoking volunteers both allergic to birch pollen and non-allergic controls were enrolled. We took nasal biopsies before and after off-seasonal intranasal birch pollen or diluent challenge. The expression of epithelial TLR1-7, TLR9-10, and MyD88 proteins was immunohistochemically evaluated from the nasal biopsies. The TLR1-3 and TLR5-10 mRNAs were observed by RNA-microarray. Baseline epithelial expression of TLR proteins was wide and identical in controls and atopics. After off-seasonal intranasal birch pollen challenge, a negative change in the expression score of TLR1 and TLR6 proteins was detected in the atopic group. TLR mRNA expression was not affected by birch pollen challenge. Nasal epithelium seems to express all known TLRs. The mechanisms by which TLR1, and TLR6 proteins could affect pollen allergen transport need further studies.
  • Arffman, Maare (Helsingin yliopisto, 2021)
    Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.
  • Santti, Kirsi; Ihalainen, Hanna; Ronty, Mikko; Boehling, Tom; Karlsson, Christina; Haglund, Caj; Tarkkanen, Maija; Blomqvist, Carl (2018)
    Background and Objectives: Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry. Methods: The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed. Results: Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2). Conclusions: Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.
  • Peltonen, Reetta; Ahopelto, Kaisa; Hagström, Jaana; Böckelman, Camilla; Haglund, Caj; Isoniemi, Helena (2020)
    ABSTRACT Colorectal cancer (CRC) is the third most common cancer in the world. More than half of all affected patients develop liver metastases during the course of the disease, and over half experience recurrence despite radical primary surgery. Transketolase-like protein 1 (TKTL1) is a key enzyme in the glucose metabolism of cancer cells, and its expression in tumor tissue was previously shown to indicate a poor prognosis in colorectal cancer. In this study, we investigated the prognostic significance of TKTL1 in 111 patients with surgically resected colorectal liver metastases, with a minimum follow-up time of 10.3 years. TKTL1 expression was examined in tissue samples of both primary tumors and liver metastases, and compared to clinicopathological parameters, disease-free survival, and overall survival. We show that a high expression of TKTL1 in primary tumor tissue associated with poor disease-free survival in patients with synchronous liver metastases (P = .026, Kaplan-Meier log-rank test), but with better disease-free survival in patients with metachronous metastases, although not statistically significantly (P = .073). We found similar tendencies for TKTL1 expression in liver metastases. Thus, TKTL1 could serve as a candidate marker to identify patients who benefit from liver resection or who need more aggressive perioperative chemotherapy.
  • Tommola, Paivi; Unkila-Kallio, Leila; Paetau, Anders; Meri, Seppo; Kalso, Eija; Paavonen, Jorma (2016)
    BACKGROUND: Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia. OBJECTIVE: The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia. STUDY DESIGN: Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration. RESULTS: We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P = .006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P = .020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P = .004; R-2 = 0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P = .013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P = .053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers. CONCLUSION: Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia.
  • Remes, Satu M.; Leijon, Helena L.; Vesterinen, Tiina J.; Arola, Johanna T.; Haglund, Caj H. (2019)
    Neuroendocrine neoplasias (NENs) are known to express somatostatin receptors (SSTRs) 1-5, which are G-protein-coupled cell membrane receptors. Somatostatin receptor imaging and therapy utilizes the SSTR expression. Synthetic somatostatin analogs with radioligands are used to detect primary tumors, metastases, and recurrent disease. Receptor analogs are also used for treating NENs. Furthermore, commercially available SSTR antibodies can be used for the immunohistochemical (IHC) detection of SSTRs. We investigated different SSTR antibody clones applying diverse IHC protocol settings to identify reliable clones and feasible protocols for NENs. A tissue microarray including NENs from 12 different primary sites were stained. Only UMB clones were able to localize SSTR on the cell membranes of NENs. SSTR2 (UMB1) emerged as the most common subtype followed by SSTR5 (UMB4) and SSTR1 (UMB7). SSTR3 (UMB5) expression was mainly cytoplasmic. Yet, SSTR4 expression was weak and located primarily in the cytoplasm. Thus, appropriate IHC protocols, including proper positive and negative controls, represent requirements for high-quality NEN diagnostics and for planning personalized therapy.
  • Bishop, Hannah I.; Cobb, Melanie M.; Kirmiz, Michael; Parajuli, Laxmi K.; Mandikian, Danielle; Philp, Ashleigh M.; Melnik, Mikhail; Kuja-Panula, Juha; Rauvala, Heikki; Shigemoto, Ryuichi; Murray, Karl D.; Trimmer, James S. (2018)
    Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal alpha subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 alpha subunits. AMIGO-1 is coclustered with Kv2 alpha subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 alpha subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 alpha subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 alpha subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 alpha subunits, and that Kv2 alpha subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.
  • Abdel-Rahman, Wael Mohamed; Nieminen, Taina Tuulikki; Shoman, Soheir; Eissa, Saad; Peltomaki, Paivi (2014)
  • Markkanen, Anttoni (Helsingin yliopisto, 2021)
    Pleomorfinen adenooma (PA) on yleisin sylkirauhasten hyvänlaatuinen kasvain. Hoitamattomana niihin kehittyy maligniteetti noin 5-10% tapauksista. Malignisoituneista kasvaimista yleisin on carcinoma ex pleomorphic adenoma (CxPA). Tämän lisäksi niillä on taipumus uusiutua, recurrent pleomorphic adenoma (RPA) riittämättömän kirurgisen hoidon yhteydessä, mutta uusiutumia tavataan kuitenkin myös adekvaatin kirurgisen hoidon jälkeen. Uusiutuvat kasvaimet aiheuttavat hoidollisia haasteita. Tutkimuksemme tavoitteena oli selvittää ja tutkia mahdollisia histopatologisia muutoksia, sekä solusyklin säätelyyn liittyvän cyclin D1 proteiinin ja solujen proliferaatioon vaikuttavan Ki-67 proteiinin roolia liittyen pleomorfisen adenooman uusiutumiseen ja malignisoitumiseen. Cyclin D1 ja Ki-67 proteiinin ilmentymistä tutkittiin immunohistokemiallisin menetelmin. Ki-67 proteiinin ilmentymistä tutkittiin tutkimuksessamme patologiassa yleisesti käytetyn MIB-1 vasta-aineen avulla. Aineistona tutkimuksessamme käytettiin retrospektiivisesti kerättyä potilasaineistoa (n=65) ja potilasaineiston perusteella laadittiin tutkittavat kasvainryhmät. Tutkittavat kasvainryhmät koostuivat tavallisista hyvänlaatuisista PA kasvaimista (n=26), uusiutuneista kasvaimista (n=20) sekä maligneista CxPA kasvaimista (n=19). Uusiutuneiden kasvainten ryhmä jaettiin lisäksi alaryhmiksi, jotka koostuivat uusiutuneiden PA kasvainten primääreistä kasvamista (PA-prim) sekä primäärejä kasvaimia seuranneista uusiutuneista kasvaimista (PA-rec). Vertailimme kasvainryhmien välisiä eroavaisuuksia kasvainten kliinisten tietojen, histopatologisten ominaisuuksien sekä cyclin D1 ja MIB-1 ilmentymisen osalta. Tutkimuksemme perusteella MIB-1 ilmentyminen oli kohonnut CxPA ja PA-rec kasvaimissa, mutta ei PA-prim kasvaimissa. Tämä viittaa mahdollisesti siihen, että MIB-1 ilmentymisen eroavaisuudet näissä kasvaimissa voivat kehittyä vasta primäärin kasvaimen jälkeen. Cyclin D1 ilmentymisessä sekä histopatologiassa ei havaittu merkitseviä eroavaisuuksia ryhmien välillä.
  • Heikkinen, Aino (Helsingin yliopisto, 2018)
    Chronic psychosocial stress is a major risk factor for anxiety disorders, but the molecular background is still poorly known. Chronic social defeat stress (CSDS) is a mouse model simulating the psychosocial stress that humans face in their life. In CSDS, the examined mice are confronted by an aggressor mouse daily for 10 days, leading to defeat behavior and predisposing to anxiety-like symptoms. Some individuals develop these symptoms (susceptible) whereas others do not (resilient). Chronic stress has been shown to alter myelin-related gene transcription and myelin microstructure. Myelin is a membranous component around axons increasing the velocity of action potentials, and it is produced by oligodendrocytes (OLs). In this study, I investigated if CSDS affects the number of OLs or the size of the myelinated area (estimating the amount of myelin) in two inbred mouse strains that differ in their innate level of anxiety: the non-anxious C57BL/6NCrl (B6) and anxious DBA/2NCrl (D2). I studied three brain regions previously associated with anxiety: the medial prefrontal cortex (mPFC), bed nucleus of stria terminalis (BNST) and ventral hippocampus (vHP). The mice used in this study were previously exposed to CSDS and divided into resilient or susceptible phenotypes, and their brains were collected together with control mice. I performed two immunohistochemical staining experiments to calculate the number of OLs and to measure the myelinated area. I used anti-CNPase for OL cell counts and BlackGold II to stain myelin. I manually calculated the number of OLs using CNPase and cell morphology as markers. I built a macro to measure the BlackGold II stained myelinated area. I also measured the thickness of the corpus callosum (CC, major white matter tract) using the CNPase stained images to examine if the thickness is affected by CSDS. I observed a strain and region-specific effect of chronic stress in the BNST; B6 resilient mice had more OLs than susceptible mice whereas no differences were seen in the D2 strain, or other B6 brain regions. The size of the myelinated area did not differ between the phenotypes in either strain. Moreover, there was no significant correlation between the myelinated area and OL cell number. The CC thickness did not differ between the phenotypes. My findings indicate that myelin and OLs are affected by stress in a region specific manner and possibly contribute to the stress-resilient behavior. The response is genetic background-dependent, as I saw differences in B6 mice but not in D2 mice. Because CC thickness did not differ between the phenotypes, we suggest that CSDS does not induce extensive white matter atrophy in the mice brain. The mechanism underlying this dynamic myelin plasticity during stress requires more investigation, but this study provides evidence that alterations in OLs associate with chronic stress.
  • van der Vos, Wessel; Stein, Koen; Di-Poi, Nicolas; Bickelmann, Constanze (2018)
    Squamate reptiles constitute a major component of the world's terrestrial vertebrate diversity, encompassing many morphotypes related to ecological specialization. Specifically, Gekkota, the sister clade to most other squamates, have highly specialized autopodia, which have been linked to their ecological plasticity. In this study, a developmental staging table of the gecko Hemidactylus, housed at the Museum fur Naturkunde, is established. Twelve post-ovipositional stages are erected, monitoring morphological embryological transitions in eye, ear, nose, heart, limbs, pharyngeal arches, and skin structures. Ecomorphological specializations in the limbs include multiple paraphalanges, hypothesized to aid in supporting the strong muscles, that are situated adjacent to metacarpal and phalangeal heads. Furthermore, some phalanges are highly reduced in manual digits III and IV and pedal digits III, IV, and V. Development, composition, and growth of limb elements is characterized in detail via mu CT, histochemistry, and bone histological analysis. Using known life history data from two individuals, we found an average lamellar bone accretion rate in the humeral diaphysis comparable to that of varanids. Various adult individuals also showed moderate to extensive remodeling features in their long bone cortices, indicating that these animals experience a highly dynamic bone homeostasis during their growth, similar to some other medium-sized to large squamates. This study of in-ovo development of the gecko Hemidactylus and its ecomorphological specializations in the adult autopodia, enlarges our knowledge of morphological trait evolution and of limb diversity within the vertebrate phylum.
  • Rodrigues, Joana M.; Hassan, May; Freiburghaus, Catja; Eskelund, Christian W.; Geisler, Christian; Räty, Riikka; Kolstad, Arne; Sundstrom, Christer; Glimelius, Ingrid; Gronbaek, Kirsten; Kwiecinska, Anna; Porwit, Anna; Jerkeman, Mats; Ek, Sara (2020)
    Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients withTP53missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 andTP53status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression andTP53mutations on survival (hazard ratio of 3 center dot 1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.
  • 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.
  • Vesterinen, Tiina; Kuopio, Teijo; Ahtiainen, Maarit; Knuuttila, Aija; Mustonen, Harri; Salmenkivi, Kaisa; Arola, Johanna; Haglund, Caj (2019)
    Pulmonary carcinoid (PC) tumors are rare tumors that account for approximately 1% of all lung cancers. The primary treatment option is surgery, while there is no standard treatment for metastatic disease. As the number of PCs diagnosed yearly is increasing, there is a need to establish novel therapeutic options. This study aimed to investigate programmed death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) expression in PC tumors since blocking of the PD-1/PD-L1 pathway is a promising therapeutic option in various other malignancies. A total of 168 PC patients treated between 1990 and 2013 were collected from the Finnish biobanks. After re-evaluation of the tumors, 131 (78%) were classified as typical carcinoid (TC) and 37 (22%) as atypical carcinoid (AC) tumors. Primary tumor samples were immunohistochemically labeled for PD-1, PD-L1 and CD8. High PD-1 expression was detected in 16% of the tumors. PD-L1 expression was detected in 7% of TC tumors; all AC tumors were PD-L1 negative. PD-L1 expression was associated with mediastinal lymph-node metastasis at the time of diagnosis (P = 0.021) as well as overall metastatic potential of the tumor (P = 0.010). Neither PD-1 expression, PD-L1 expression nor CD8(+) T cell density was associated with survival. In conclusion, PD-1 and PD-L1 were expressed in a small proportion of PC tumors and PD-L1 expression was associated with metastatic disease. Targeting of the PD-1/PD-L1 pathway with immune checkpoint inhibitors may thus offer a treatment option for a subset of PC patients.
  • Howat, William J; Blows, Fiona M; Provenzano, Elena; Brook, Mark N; Morris, Lorna; Gazinska, Patrycja; Johnson, Nicola; McDuffus, Leigh-Anne; Miller, Jodi; Sawyer, Elinor J; Pinder, Sarah; van Deurzen, Carolien H M; Jones, Louise; Sironen, Reijo; Visscher, Daniel; Caldas, Carlos; Daley, Frances; Coulson, Penny; Broeks, Annegien; Sanders, Joyce; Wesseling, Jelle; Nevanlinna, Heli; Fagerholm, Rainer; Blomqvist, Carl; Heikkilä, Päivi; Ali, H Raza; Dawson, Sarah-Jane; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Cox, Angela; Brock, Ian W; Cross, Simon S; Reed, Malcolm W; Couch, Fergus J; Olson, Janet E; Devillee, Peter; Mesker, Wilma E; Seyaneve, Caroline M; Hollestelle, Antoinette; Benitez, Javier; Perez, Jose Ignacio Arias; Menéndez, Primitiva; Bolla, Manjeet K; Easton, Douglas F; Schmidt, Marjanka K; Pharoah, Paul D; Sherman, Mark E; García-Closas, Montserrat (2015)
    Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large-scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65–70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose-response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96–98%), but yielded many false positives (positive predictive value = 30–32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large-scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker-specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results.
  • Hannula, Mirva (Helsingfors universitet, 2010)
    Prolyl oligopeptidase (POP, prolyl endopeptidase, EC is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.