Biomarkers in Pulmonary Carcinoid Tumors

Pulmonary carcinoid (PC) tumors are rare neuroendocrine lung neoplasms that have in general an indolent clinical course. Based on morphology, PC tumors are categorized into typical carcinoid (TC) tumors and atypical carcinoid (AC) tumors. The primary treatment is surgery, while there is no consensus on the treatment of metastatic disease. Thus, effective and more targeted therapies are needed for the PC patients who are inoperable or not curable with surgery alone. While histologic subtype and stage are important prognostic factors, other predictive and prognostic factors or biomarkers are urgently needed for individualized treatment and patient-tailored surveillance protocols. The main aim of this thesis was to find tissue-based biomarkers in PC tumors by utilizing the Finnish hospital-integrated biobanks as a source of the study material. Moreover, the performance of these biobanks in distributing material for a research of a rare cancer was evaluated. The nationwide study cohort consisted of 224 PC tumor patients operated on between 1990 and 2013 in Finland. Eighty-one per cent (n=182) of the tumors were classified as TCs and 19% (n=42) as ACs. Hilar/mediastinal lymph node involvement at the time of primary surgery as well as metastatic disease in general was more common in AC patients than in TC patients. The follow-up time of the patients varied between <1 and 28.0 years. Of 224 patients, 14 died with evidence of disease and 33 died from unrelated causes. The 10-year disease-specific survival rate was 98% for TC patients and 81% for AC patients. Five clinicopathological factors were recognized to be associated with the risk of disease-specific mortality. These were age over 56 at diagnosis, tumor size over 2.5 cm, atypical subtype, hilar/mediastinal lymph node involvement at diagnosis, and presence of metastatic disease. Similarly, lack of somatostatin receptor (SSTR) 1 expression and presence of SSTR3 expression as well as a Ki-67 proliferation index over 2.5% were identified as risk factors for shorter DSS. In addition, lack of SSTR2 expression and presence of SSTR4 expression was associated with a risk of shorter DSS in AC patients. Metastasized PC tumors expressed significantly more SSTR4 and programmed death ligand 1 (PD-L1) than non-metastasized tumors. In addition, lack of SSTR1 or SSTR2 expression was associated with metastatic disease. Based on biobank registry data, Finnish hospital-integrated biobanks were able to identify 88% of the histologically verified PC tumor patients registered by the FCR. However, they were only able to deliver 63% of the tumor samples because samples were missing in the archives or they were too scarce. The most challenging part of the biobanks’ workflow was collecting the clinical data.Keuhkon karsinoidikasvaimet ovat harvinaisia diffuusin neuroendokriinisen järjestelmän kasvaimia. Suomessa niitä todetaan vuosittain 20–30 potilaalla. Morfologiansa perusteella keuhkokarsinoidit jaetaan tyypillisiin (TC) ja atyyppisiin karsinoideihin (AC). Molempien kasvainten ensisijainen hoitomuoto on leikkaus. Keuhkokarsinoidipotilaiden ennuste on tavallisesti hyvä. Kuitenkin osa kasvaimista lähettää etäpesäkkeitä muualle elimistöön tai uusiutuu paikallisesti siten, että ne eivät ole enää leikattavissa. Levinnyttä keuhkokarsinoidia sairastaville potilaille ei ole tällä hetkellä vakiintunutta hoitopolkua. Keuhkokarsinoidien ennustetekijöistä tärkein on kasvaimen alatyyppi, sillä atyyppiset karsinoidit ovat kliiniseltä kuvaltaan aggressiivisempia kuin tyypilliset karsinoidit. Kasvainkudoksesta määritettäviä hoitojen tehoa tai potilaan taudin kulkua ja elinaikaa ennustavia tekijöitä tunnetaan vain niukalti. Tämän väitöskirjatyön tavoite olikin löytää tällaisia kudosmerkkiaineita. Keuhkokarsinoidikasvainten harvinaisuuden vuoksi kasvainnäytemateriaali kerättiin suomalaisen sairaalabiopankkiverkoston kautta. Samalla arvioitiin tämän verkoston toimivuutta harvinaista syöpää tutkittaessa. Tutkimusaineisto koostui vuosina 1990–2013 Suomessa leikattujen keuhkokarsinoidipotilaiden kasvainnäytteistä ja kliinisistä tiedoista. Tyypillisiä karsinoideja oli 182 ja atyyppisia 42. Potilaiden seuranta-aika vaihteli alle vuodesta 28 vuoteen. Seurannan aikana 14 potilasta kuoli keuhkokarsinoidin vuoksi ja 33 muiden syiden takia. Tautispesifinen kymmenen vuoden elossaoloennuste oli TC-potilaille 98 % ja AC-potilaille 81 %. Viisi kliinis-patologista tekijää liittyi potilaan huonoon ennusteeseen. Ne olivat yli 56 vuoden ikä primaarileikkaushetkellä, kasvaimen koko yli 2,5 cm, kasvaimen atyyppinen alatyyppi, paikalliset imusolmuke-etäpesäkkeet leikkaushetkellä sekä levinnyt tauti. Kasvaimessa ilmentyvistä merkkiaineista somatostatiinireseptoriprofiilin sekä Ki-67-proliferaatioindeksin todettiin liittyvän potilaan ennusteeseen. Lisäksi somatostatiinireseptoriprofiili ja PD-L1-proteiinin (programmed death ligand 1) ilmentyminen liittyivät levinneeseen tautiin. Biopankit tunnistivat rekistereistään 88 % Suomen Syöpärekisteriin talletetuista keuhkokarsinoidipotilaista. Tähän tutkimukseen saatiin 63 % näiden potilaiden kasvainnäytteistä. Keskeisimmät syyt noin kolmanneksen poisjäämiseen olivat näytemateriaalin niukkuus ja näyteblokkien puuttuminen biopankkien arkistoista. Biopankkien näkökulmasta haastavinta oli kerätä näytteisiin liittyvät tiedot potilastietojärjestelmistä

Keuhkon karsinoidikasvaimet ja suomalaisen aineiston löydökset

Vertaisarvioitu.Keuhkokarsinoidit ovat harvinaisia keuhkojen neuroendokriinisiä kasvaimia, jotka kuuluvat WHO-luokituksen mukaan pahanlaatuisten keuhkokasvainten ryhmään. Niiden esiintyvyys on viime vuosikymmenten aikana lisääntynyt. Tämä johtuu ainakin osittain tarkentuneesta diagnostiikasta, kun kuvantamismenetelmät ovat kehittyneet ja niiden käyttö lisääntynyt. Keuhkokarsinoidit jaetaan morfologiansa perusteella tyypillisiin ja atyyppisiin karsinoideihin. Potilaat ovat diagnoosihetkellä verrattain nuoria, keskimäärin 50-vuotiaita, eikä kasvaimen kehittymisellä ole havaittu olevan yhteyttä tupakointiin. Keuhkokarsinoideista suurin osa on oireettomia, ja kasvain todetaankin sattumalöydöksenä muun syyn takia tehdyssä kuvantamistutkimuksessa. Ensisijainen hoito on leikkaushoito. Keuhkokarsinoidipotilaan ennuste on yleensä hyvä.Peer reviewe

Automated assessment of Ki-67 proliferation index in neuroendocrine tumors by deep learning

The Ki-67 proliferation index (PI) is a prognostic factor in neuroendocrine tumors (NETs) and defines tumor grade. Analysis of Ki-67 PI requires calculation of Ki-67-positive and Ki-67-negative tumor cells, which is highly subjective. To overcome this, we developed a deep learning-based Ki-67 PI algorithm (KAI) that objectively calculates Ki-67 PI. Our study material consisted of NETs divided into training (n = 39), testing (n = 124), and validation (n = 60) series. All slides were digitized and processed in the Aiforia(R) Create (Aiforia Technologies, Helsinki, Finland) platform. The ICC between the pathologists and the KAI was 0.89. In 46% of the tumors, the Ki-67 PIs calculated by the pathologists and the KAI were the same. In 12% of the tumors, the Ki-67 PI calculated by the KAI was 1% lower and in 42% of the tumors on average 3% higher. The DL-based Ki-67 PI algorithm yields results similar to human observers. While the algorithm cannot replace the pathologist, it can assist in the laborious Ki-67 PI assessment of NETs. In the future, this approach could be useful in, for example, multi-center clinical trials where objective estimation of Ki-67 PI is crucial.Peer reviewe

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p < 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST

Tensin2 Is a Novel Diagnostic Marker in GIST, Associated with Gastric Location and Non-Metastatic Tumors

GIST is a rare soft tissue sarcoma, for which KIT and DOG1 are used as highly sensitive diagnostic markers. Other diagnostic markers include CD34, protein kinase C θ, deficiency of succinate dehydrogenase complex subunit B, carbonic anhydrase II, and type I insulin-like growth factor receptor. We investigated the role of TNS2 as a diagnostic biomarker by using immunohistochemistry in 176 GISTs and 521 other sarcomas. All GISTs expressed TNS2, with intermediate or high expression in 71.4% of samples. The majority (89.8%) of other sarcomas were negative for TNS2, and intermediate to strong staining was only seen in 2.9% of samples. Strong TNS2 staining was associated with gastric location (gastric 52.8% vs. non-gastric 7.2%; p p &lt; 0.001), absence of metastases (non-metastatic tumors 44.3% vs. metastatic tumors 5.9%; p = 0.004), female sex (female 45.9% vs. male 33.8%; p = 0.029), and tumors of lower risk categories (very low or low 46.9% vs. intermediate 51.7% vs. high 29.0%; p = 0.020). TNS2 expression did not correlate with overall survival or metastasis-free survival. No associations between TNS2 expression and KIT/PDGFRA mutation status, tumor size, mitotic count, or age of the patient were detected. The results provide conclusive evidence for the value of TNS2 as a sensitive and specific diagnostic biomarker for GIST.Peer reviewe

Immunohistochemical Expression of Somatostatin Receptor Subtypes in a Panel of Neuroendocrine Neoplasias

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.Peer reviewe

PD-1 and PD-L1 expression in pulmonary carcinoid tumors and their association to tumor spread

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.Peer reviewe

Lateralization in C-11-Metomidate PET and outcome of adrenalectomy in primary aldosteronism

Introduction Subtype classification method is essential when considering adrenalectomy as a possible treatment for primary aldosteronism. We aimed to retrospectively evaluate surgical outcomes of primary aldosteronism in patients who had undergone C-11-metomidate positron emission tomography (C-11-MTO-PET) for subtype classification. Methods Postoperative clinical and biochemical cure and histopathological diagnosis from biobank samples were retrospectively evaluated in 44 patients who had all undergone preoperative C-11-MTO-PET with or without adrenal venous sampling (AVS). We compared those operated based on C-11-MTO-PET alone and those with concordant or discordant lateralization in C-11-MTO-PET and AVS studies according to postoperative immunohistochemical findings and biochemical and clinical cure. Results Adrenalectomy side was based on C-11-MTO-PET alone in 14 cases and on AVS in 30 cases of whom 42 achieved complete and two partial biochemical cures. Among those who underwent AVS and were operated according to it, the two lateralization methods were concordant in 22 cases and discordant in 8 cases. Similar immunohistochemical profiles and cure rates were seen after C-11-MTO-PET alone or AVS-based operations. Respectively, those with concordant or discordant C-11-MTO-PET and AVS lateralization did not differ in surgical outcome. Together, we found errors of lateralization diagnostics with C-11-MTO-PET in 18% and with AVS in 3% among those eligible for adrenal surgery. Conclusions Outcomes of adrenalectomy based on clinically significant lateralization in C-11-MTO-PET alone correspond to those based on C-11-MTO-PET with concordant AVS lateralization. However, our results suggest that diagnosis of unilateral PA should be performed with caution with C-11-MTO-PET in case of discordant lateralization studies.Peer reviewe