Tumor Dormancy in Breast Cancer

Breast cancer has an unusual propensity of late recurrence even after decades of clinically undetectable disease. Regardless of wide scientific approaches, the biology behind this phenomenon of tumor dormancy, is still poorly understood. Currently, the selection of patients who would benefit from additional systemic therapies after surgical resection is based on their statistical risk of developing tumor recurrence. The risk estimation is based on the clinicopathological parameters and the immunohistochemical (IHC) evaluation of ER, PR, HER2 and KI67 expression of the primary tumor tissue, without knowledge of whether the patient actually harbour dormant minimal residual disease somewhere in organ parenchyma, or as disseminated/circulating tumor cells (DTCs/CTCs) in blood or lymph circulation. The aim of investigations of tumor dormancy is to identify new biomarkers, i.e. markers of tumor dormancy that would better foretell the reactivating of the dormant tumor cells to form life threatening metastazing cancer and help to develop new therapeutic strategies. This study is based on immunohistochemical (IHC) demonstration of the expression of different antigens in surgically removed breast cancer tissues. New cuts from the archival paraffin-embedded tissue blocks from the metastatic cancers and their corresponding primary tumors were stained. The expression levels of selected biomarkers were compared between early- and late-relapsing breast cancers in order to find out if their expression have association to the time of tumor recurrence. Altogether, among 73 breast cancer patients 19 had recurrence detected within 2 years of diagnosis, 33 had recurrence detected at 5 10 years, and 21 after 10 years of follow-up. In addition to the four established biomarkers used in clinical prognostication, ER, PR, HER2 and Ki67, nine other antibodies against proteins known to have a prognostic role in breast cancer were evaluated. These nine additional markers included the stanniocalcins (STC-1 and STC-2), maspin, bcl-2, p53, Bmi-1, c-myc, Snail and cytokeratin CK5. The most interesting proteins as target of further investigation of tumor dormancy, were those markers with significant association to late tumor recurrence, which were highly expressed in the metastases that manifested 10 years after the primary treatment, namely stanniocalcins, ER, bcl-2, and Bmi-1. Especially Bmi-1, which has supposed to have cancer stem cell properties, appears to be a promising marker to be investigated. Has the expression of Bmi-1 in blood association to progression of breast cancer?Rintasyöpäkasvaimen uinuminen Rintasyövän keskimääräinen ennuste on erinomainen, mutta taudin heterogeenisesta luonteesta johtuen sen uusiutumisaika voi vaihdella huomattavasti yksittäisillä potilailla. Rintasyöpä saattaa uusitua jopa vuosikymmenien oireettoman vaiheen jälkeen. Piilevää vaihetta, jossa potilaan elimistössä on pieniä syöpäjäänteitä, joita ei pystytä havaitsemaan, kutsutaan syövän uinumiseksi (tumor dormancy). Viimeisten kymmenen vuoden aikana on uinumista paljon tutkittu, mutta ilmiötä ei hyvin ymmärretä. Rintasyövän kirurgisen hoidon jälkeen suunnitellaan lääkehoito, joka perustuu kliinispatologisiin ennustetekijöihin ja kasvainkudoksesta määritettyjen proteiini- markkereiden esiintymiseen. Neljä vakiintunutta markkeria ovat estrogeenireseptori (ER), progesteronireseptori (PR), human epidermal growth factor reseptori HER2 ja proliferatiomarkkeri Ki67. Lääkehoitoa valittaessa ei ole tiedossa, onko potilaalla elimistössään hoidoille resistenttejä, elimistössä uinuvia tai veressä kiertäviä syöpäsoluja. Väitöstutkimuksessani määritettiin immunohistokemiallisesti vakiintuneiden markkereiden, ER, PR, HER2 ja Ki67 lisäksi yhdeksän muun proteiinin vasta-aineet, joilla tiedetään olevan ennusteellista merkitystä : stanniokalsiinit (STC-1 ja STC-2), maspin, bcl-2, p53, CK5 sekä potentiaaliset kantasolumarkkerit Bmi-1, c-myc ja Snail. Syöpämarkkereiden kirjavat lyhenteet voivat hämmentää. Kirjainyhdistelmät muodostuvat omista taustoistaan kuten kaikkien tuntema CEA. Aineisto valittiin siten, että etsittiin tiedostosta metastasoituneita kasvaimia, jotka olivat uusiutuneet nopeasti tai hitaasti. Näin voitiin proteiinimarkkereiden esiintymisvahvuuden eroista tehdä päätelmiä. Ensisijainen kiinnostus kohdistui siihen miten markkereiden esiintyminen korreloitui kasvaimen hitaaseen uusiutumiseen. Etsinnän tuloksena löytyi 73 potilasta, joista ensimmäisen syövän toteamisen jälkeen 19 syöpää uusiutui ennen kahta vuotta, 33 uusiutui viiden vuoden jälkeen, ja 21 syöpää kymmenen vuoden jälkeen. Tutkimuksen perusteella hitaaseen kasvaimen uusiutumiseen korreloivat stanniokalsiinit, ER, Bcl-2 ja Bmi-1. Nopeaan uusiutumiseen korreloivat HER2, Ki67, sytoplasminen maspin, p53 ja CK5. Kolme markkeria, PR, Snail ja c-myc eivät korreloineet kasvaimen uusiutumisnopeuteen. Nopean uusiutumisen merkittävimmät riskitekijät olivat kasvaimen koko ja HER2 positiivisuus. Uusiutumisriskiä pienensi merkittävämmin IHC luminal A (ER tai PR positiivinen HER2- negatiivinen) alatyyppi. Mielenkiintoista on tulevaisuudessa tutkia markkereita, joiden ekspressio oli voimakasta myöhään, yli 10 vuotta uusiutuneissa kasvaimissa. Erityisesti Bmi-1, jolla katsotaan olevan syöpäkantasolun ominaisuuksia, on mielenkiintoinen tutkimuskohde. Onko Bmi-1 markkerin esiintymisellä veressä yhteyttä rintasyövän etenemiseen

Core Needle Biopsy Enhances the Activity of the CCL2/CCR2 Pathway in the Microenvironment of Invasive Breast Cancer

The use of core needle biopsy (CNB) as a means to verify malignancy preoperatively is a paradigm in current breast cancer care, and the risk of enhancing tumor development by this procedure has been considered insignificant. Experimental work in mice has shown preoperative biopsies to increase tumor supportive elements in the microenvironment, whereas, in humans, the impact of CNB on the host’s immunologic response has not been investigated. In this pilot study, we compared the expression of CCL2/CCR2 pathway components at the protein level in samples from CNBs to those from the corresponding resected tumors from 52 patients with primary breast cancer. We found an increased expression of CD163, CD14 and CCR2 in monocytes/macrophages and a slight decrease of CCL2 in the malignant epithelium in the tumors after the biopsy. The increased infiltration of immunosuppressive monocytes/macrophages and the decreased tumor cell CCL2 expression, presumably due to the CCR2 availability-dependent CCL2 internalization, suggest that CNB enhances the activity of the CCL2/CCR2 pathway, and this finding warrants confirmatory examination. The switch in the context-dependent role of CCL2 on the polarization of macrophages may lead to increased tumor supportive function both locally and in the peripheral immune machinery. The future directions in breast cancer should include early interventions to support the tumor surveillance of the host

Quantities of CD3+, CD8+and CD56+lymphocytes decline in breast cancer recurrences while CD4+remain similar

Peer reviewe

High expression of CCL2 in tumor cells and abundant infiltration with CD14 positive macrophages predict early relapse in breast cancer

Macrophages are important for the function of the innate immune system, and in solid tumors, they represent a significant proportion of the tumor mass. Tumor-associated macrophages (TAM) have a M2 phenotype and show a multitude of pro-tumoral functions, promoting tumor cell survival, proliferation, and dissemination. CCL2, synthesized by tumor and stromal cells, initiates a chemokine cascade inducing these processes. We studied by immunohistochemistry (IHC) the frequency of TAMs and CCL2 expressing cells in three groups of primary tumor (PT)-recurrence (R) pairs, where relapse was recorded within 2years (group 1), between 5 and 10years (group 2), and after 10years (group 3). In our study all established breast cancers were heavily infiltrated by CD68 positive cells. Both in PTs and in R lesions the infiltration was more abundant in the peritumoral than in the intratumoral stroma. The mean frequency of M2 marker and CD14 positive cells in the intratumoral stroma and CCL2 expressing tumor cells was higher in the Rs as compared to the corresponding PTs. In PTs, a high frequency of CD14 positive cells and a high expression of CCL2 by tumor cells was associated with an early recurrence. The findings support the current understanding of immune cell orchestrated development, progression and metastatic spread of breast cancer. Our study showed that a high frequency of CCL2 positive tumor cells and CD14 positive TAMs are significant risk factors for rapid tumor recurrence. Potential targets for intervention are discussed.Peer reviewe

Clever-1 positive macrophages in breast cancer

Purpose Common Lymphatic Endothelial and Vascular Endothelial Receptor 1 (Clever-1) is expressed by a subset of immunosuppressive macrophages and targeting the receptor with therapeutic antibodies has been shown to activate T-cell-mediated anti-cancer immunity. The aim of this research was to study Clever-1 expression in breast cancer. Specifically, how Clever-1 + macrophages correlate with clinicopathologic factors, Tumor Infiltrating Lymphocytes (TILs) and prognosis. Methods Tissue microarray blocks were made from 373 primary breast cancer operation specimens. Hematoxylin and Eosin (H&E-staining) and immunohistochemical staining with Clever-1, CD3, CD4 and CD8 antibodies were performed. Differences in quantities of Clever-1 + macrophages and TILs were analyzed. Clever-1 + cell numbers were correlated with 25-year follow-up survival data and with breast cancer clinicopathologic parameters. Results Low numbers of intratumoral Clever-1 + cells were found to be an independent adverse prognostic sign. Increased numbers of Clever-1 + cells were found in high grade tumors and hormone receptor negative tumors. Tumors that had higher amounts of Clever-1 + cells also tended to have higher amounts of TILs. Conclusion The association of intratumoral Clever-1 + macrophages with better prognosis might stem from the function of Clever as a scavenger receptor that modulates tumor stroma. The association of Clever-1 + macrophages with high number of TILs and better prognosis indicates that immunosuppression by M2 macrophages is not necessarily dampening adaptive immune responses but instead keeping them in control to avoid excess inflammation.Peer reviewe

Clever-1 positive macrophages in breast cancer

Purpose: Common Lymphatic Endothelial and Vascular Endothelial Receptor 1 (Clever-1) is expressed by a subset of immunosuppressive macrophages and targeting the receptor with therapeutic antibodies has been shown to activate T-cell-mediated anti-cancer immunity. The aim of this research was to study Clever-1 expression in breast cancer. Specifically, how Clever-1 + macrophages correlate with clinicopathologic factors, Tumor Infiltrating Lymphocytes (TILs) and prognosis. Methods: Tissue microarray blocks were made from 373 primary breast cancer operation specimens. Hematoxylin and Eosin (H&E-staining) and immunohistochemical staining with Clever-1, CD3, CD4 and CD8 antibodies were performed. Differences in quantities of Clever-1 + macrophages and TILs were analyzed. Clever-1 + cell numbers were correlated with 25-year follow-up survival data and with breast cancer clinicopathologic parameters. Results: Low numbers of intratumoral Clever-1 + cells were found to be an independent adverse prognostic sign. Increased numbers of Clever-1 + cells were found in high grade tumors and hormone receptor negative tumors. Tumors that had higher amounts of Clever-1 + cells also tended to have higher amounts of TILs. Conclusion: The association of intratumoral Clever-1 + macrophages with better prognosis might stem from the function of Clever as a scavenger receptor that modulates tumor stroma. The association of Clever-1 + macrophages with high number of TILs and better prognosis indicates that immunosuppression by M2 macrophages is not necessarily dampening adaptive immune responses but instead keeping them in control to avoid excess inflammation.</p

Nurmi biokaasun raaka-aineena : RED II direktiivin mukainen kasvihuonekaasupäästöjen laskenta

Uusiutuvan energian direktiivi (RED II) vuosille 2021–2030 julkaistiin joulukuussa 2018. Direktiivi sisältää sitovat EU-tason kestävyyskriteerit biomassoille, joita käytetään energian tuotantoon. Kestävyyskriteereillä halutaan varmistaa, että bioenergian lisääntyvä käyttö EU:ssa tuottaa merkittäviä kasvihuonekaasupäästöjen vähennyksiä fossiilisiin polttoaineisiin verrattuna. Uudessa direktiivissä kestävyyskriteerit laajenevat koskemaan myös kiinteällä biomassalla sekä biokaasulla tuotettua sähkö-, lämpö-, ja jäähdytysenergiaa. Tämän työn tavoitteena oli tarkastella kestävyyskriteerejä ja raaka-ainekohtaisia kasvihuonepäästö-kertoimia Suomessa viljeltävälle nurmelle, kun nurmea käytetään biokaasun tuotannossa. Direktiivissä on biokaasun osalta oletusarvoja lietelannalle, maissille, lanta-maissi sekoituksille sekä biojätteelle. Tässä työssä päästökertoimet laskettiin viljellylle nurmelle (kivennäis- ja eloperäisellä maalla), suojavyöhykenurmelle sekä apilapitoiselle nurmelle. Lisäksi kertoimet laskettiin kolmannelle rehusadolle kun ensimmäinen ja toinen sato käytetään säilörehuksi sekä viljelykierrossa olevalle nurmelle (viherlannoitusnurmena). Lisäksi työssä laskettiin laitosesimerkkejä, joissa laskettiin laitoksen kokonaispäästö ja vertailtiin sitä direktiivissä annettuihin fossiilisiin vertailuarvoihin. Koska nurmen viljelykäytänteet vaihtelevat, on raportin tuloksista huomioitava, etteivät ne päde sellaisille tapauksille, missä viljelykäytänteet eroavat merkittävästi tässä esimerkissä käytetyistä lähtöoletuksista. Tässä työssä käytettyjen oletusten pohjalta voidaan kuitenkin päätellä, että jos nurmea viljellään pelkästään energiantuotantoa varten, on direktiivin mukaisiin päästövähennyksiin haastava päästä. Tämä pätee erityisesti sähkön- ja lämmöntuotannossa, missä päästövähennysvaatimukset ovat korkeimmat, kuin liikenteen polttoaineen tuotannossa. Päästövähennysvaatimus tulee myös kiristymään sähkön- ja lämmöntuotannon osalta vuoden 2026 jälkeen. Päästövähennyksiin on mahdollista kuitenkin päästä, jos prosessissa käytetään esimerkiksi viherlannoitukseen tarkoitettua nurmea, kolmannen sadon nurmea tai nurmea käytetään lisäsyötteenä esimerkiksi lannan kanssa. Ravinteiden kierrättämisen ja liikenteen uusiutuvien polttoaineiden tavoitteiden saavuttamiseksi lannan prosessointia olisikin tärkeä edistää ja nurmen käyttö lisä raaka-aineena edistäisi näitä tavoitteita, sillä nurmesta saatava lisäenergia parantaa biokaasulaitoksen taloudellista kannattavuutta.201

Evidence for the in vivo existence and mobilisation of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+CD45+CD31+CD34− MACs and CD14+CD45+NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1−1.7-fold in all patients 1 day after skin grafting (p < 0.01). In addition, histological analysis demonstrated effective vascularization of skin grafts, as well as presence of pericytes, and CD14 and CD45 expressing myeloid cells during wound healing. In conclusion, our data shows, for the first time, the presence and mobilisation of MACs and pericyte-like cells in human circulation.publishedVersionPeer reviewe

Evidence for the in vivo existence and mobilization of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+ CD45+ CD31+ CD34- MACs and CD14+ CD45+ NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1-1.7-fold in all patients 1 day after skin grafting (p </p

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells

Peer reviewe