Onkolyyttiset adenovirukset gynegologisten syöpien hoidossa

Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly developing field is as well replication competent agents, which allow improved tumor penetration and local amplification of the anti-tumor effect. Adenoviral cancer gene therapy approaches lack cross-resistance with other treatment options and therefore synergistic effects are possible. This study focused on development of adenoviral vectors suitable for treatment of various gynecologic cancer types, describing the development of the field from non-replicating adenoviral vectors to multiple-modified conditional replicating viruses. Transcriptional targeting of gynecologic cancer cells by the use of the promoter of vascular endothelial growth factor receptor type 1 (flt-1) was evaluated. Flt-1 is not expressed in the liver and thus an ideal promoter for transcriptional targeting of adenoviruses. Our studies implied that the flt-1 promoter is active in teratocarcinomas.and therefore a good candidate for development of oncolytic adenoviruses for treatment of this often problematic disease with then poor outcome. A tropism modified conditionally replicating adenovirus (CRAd), Ad5-Δ24RGD, was studied in gynecologic cancers. Ad5-Δ24RGD is an adenovirus selectively replication competent in cells defective in the p16/Rb pathway, including many or most tumor cells. The fiber of Ad5-Δ24RGD contains an integrin binding arginine-glycine-aspartic acid motif (RGD-4C), allowing coxackie-adenovirus receptor independent infection of cancer cells. This approach is attractive because expression levels of CAR are highly variable and often low on primary gynecological cancer cells. Oncolysis could be shown for a wide variety of ovarian and cervical cancer cell lines as well as primary ovarian cancer cell spheroids, a novel system developed for in vitro analysis of CRAds on primary tumor substrates. Biodistribution was evaluated and preclinical safety data was obtained by demonstrating lack of replication in human peripheral blood mononuclear cells. The efficicacy of Ad5-Δ24RGD was shown in different orthotopic murine models including a highly aggressive intraperitoneal model of disseminated ovarian cancer cells, where Ad5-Δ24RGD resulted in complete eradication of intraperitoneal disease in half of the mice. To further improve the selectivity and specificity of CRAds, triple-targeted oncolytic adenoviruses were cloned, featuring the cyclo-oxygenase-2 (cox-2) promoter, E1A transcomplementation and serotype chimerism. Those viruses were evaluated on ovarian cancer cells for specificity and oncolytic potency with regard to two different cox2 versions and three different variants of E1A (wild type, delta24 and delta2delta24). Ad5/3cox2Ld24 emerged as the best combination due to enhanced selectivity without potency lost in vitro or in an aggressive intraperitoneal orthotopic ovarian tumor model. In summary, the preclinical therapeutic efficacy of the CRAds tested in this study, taken together with promising biodistribution and safety data, suggest that these CRAds are interesting candidates for translation into clinical trials for gynecologic cancer.Syöpä on toiseksi yleisin kuolinsyy Euroopassa aiheuttaen lähes neljänneksen kaikista kuolemantapauksista. Naisilla kohdunkaulansyöpä on yksi nuorten naisten yleisimmistä syövistä kun taas iäkkäämmillä naisilla sekä kohdunrunko- että munasarjasyöpä ovat yleistyneet. Vaikka hoitotulokset ovat parantuneet huomattavasti uusien hoitomuotojen myötä, pitkälle edenneeseen kohdunrunko- ja munasarjasyöpään ei usein ole parantavaa hoitoa. Tutkimuksemme tavoitteena oli uusien hoitomuotojen kehittäminen erityisesti pitkälle edenneisiin gynegologisiin syöpiin, joihin ei tällä hetkellä ole olemassa parantavaa hoitoa. Uusien hoitomuotojen kehittelyssä hyödynnettiin adenovirusta, joka on yksi yleisimmistä flunssaa aiheuttavista viruksista. Adenovirukset pystyvät monistumaan infektoimissaan kohdesoluissa, minkä seurauksena solu tuhoutuu vapauttaen ympäristöönsä uusia viruspartikkeleita, jotka voivat edelleen infektoida uusia kohdesoluja. Adenoviruksia voidaan myös muokata siten, että normaalien solujen sijaan virukset tunnistavat ja tuohoavat vain syöpäsoluja. Tällöin puhutaan ns. onkolyyttisistä adenoviruksista. Onkolyyttisiä adenoviruksia voidaan muokata syöpäsoluspesifiseksi pääasiassa kahdella eri tavalla. Yksi mahdollinen tapa on muokata virusten pintarakenteita siten, että virukset infektoivat vain syöpäsoluja. Toinen tapa perustuu adenovirusten genomin muokkaukseen, minkä seurauksena kyseiset virukset pystyvät monistumaan vain syöpäsoluissa. Nämä muokkaukset lisäävät sekä virusten tehokkuutta että vähentävät virusten mahdollisia sivuvaikutuksia. Tutkimuksessamme rakensimme ja testasimme onkolyyttisiä adenoviruksia, joita muokattiin syöpäsoluspesifisiksi erilaisia menetelmiä yhdistäen. Tutkimuksissamme havaitsimme, että kyseiset muokatut onkolyyttiset adenovirukset pystyivät monistumaan vain syöpäsoluissa, minkä seurauksena syöpäsolut tuhoutuivat tehokkaasti soluviljelymalleissa. Testasimme rakentamiamme onkolyyttisiä adenoviruksia myös kohdunakaula- ja munasarjasyöpien hiirimalleissa. Virushoidoilla pystyttiin estämään tehokkaasti sekä munasarja- että kohdunkaulasyöpäkasvainten kasvua ja joissain tapauksissa kasvaimet pystyttiin tuhoamaan täysin virushoitojen avulla. Lisäksi testasimme kyseisten virusten turvallisuutta sekä ihmisistä eristetyillä valkosoluilla että hiirimalleilla. Saamiemme tulosten perusteella onkolyyttisiä adenoviruksia muokkaamalla pystytään tuhoamaan syöpäsoluja hyvin tehokkaasti sekä soluviljely- että hiirimalleissa. Lisäksi erilaiset muokkausten avulla virusten tehokkuutta ja turvallisuutta pystytään parantamaan. Tutkimustulostemme perusteella muokatut onkolyyttiset adenovirukset ovat yksi lupaavista kehitteillä olevista gynegologisten syöpien hoitomuodoista

Adenoviruses with an alpha(v)beta integrin targeting moiety in the fiber shaft or the HI-loop increase tumor specificity without compromising antitumor efficacy in magnetic resonance imaging of colorectal cancer metastases

Peer reviewe

Risk for pelvic metastasis and role of pelvic lymphadenectomy in node-positive vulvar cancer - results from the AGO-VOP.2 QS vulva study

Simple Summary In node-positive vulvar squamous cell cancer, questions of when and how to perform pelvic lymphadenectomy (LAE) as well as the optimal extent of pelvic treatment in general have been surrounded by considerable controversy. In Germany, systematic pelvic LAE is currently recommended as a staging procedure in patients at risk for pelvic nodal involvement in order to prevent morbidity caused by pelvic radiotherapy (RT) in patients without histologically-confirmed pelvic involvement. However, the population at risk for pelvic metastases remains insufficiently described, resulting in the potential overtreatment of a considerable proportion of patients with groin-positive disease. This applies to the indication to perform surgical staging but also to adjuvant RT of the pelvis without previous pelvic staging. Our study aims to describe the risk for pelvic lymph node metastasis with regard to positive groin nodes and to clarify the indication criteria for pelvic treatment in node-positive vulvar cancer. Abstract The need for pelvic treatment in patients with node-positive vulvar cancer (VSCC) and the value of pelvic lymphadenectomy (LAE) as a staging procedure to plan adjuvant radiotherapy (RT) is controversial. In this retrospective, multicenter analysis, 306 patients with primary node-positive VSCC treated at 33 gynecologic oncology centers in Germany between 2017 and 2019 were analyzed. All patients received surgical staging of the groins; nodal status was as follows: 23.9% (73/306) pN1a, 23.5% (72/306) pN1b, 20.4% (62/306) pN2a/b, and 31.9% (97/306) pN2c/pN3. A total of 35.6% (109/306) received pelvic LAE; pelvic nodal involvement was observed in 18.5%. None of the patients with nodal status pN1a or pN1b and pelvic LAE showed pelvic nodal involvement. Taking only patients with nodal status ≥pN2a into account, the rate of pelvic involvement was 25%. In total, adjuvant RT was applied in 64.4% (197/306). Only half of the pelvic node-positive (N+) patients received adjuvant RT to the pelvis (50%, 10/20 patients); 41.9% (122/291 patients) experienced recurrent disease or died. In patients with histologically-confirmed pelvic metastases after LAE, distant recurrences were most frequently observed (7/20 recurrences). Conclusions: A relevant risk regarding pelvic nodal involvement was observed from nodal status pN2a and higher. Our data support the omission of pelvic treatment in patients with nodal status pN1a and pN1b

Systemic therapy for cervical cancer with potentially regulatable oncolytic adenoviruses

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Activation of G-Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces Progression of Vulvar Carcinoma Cells

Whether G protein-coupled estrogen receptor 1 (GPER1) is tumor-promoting or tumor-suppressive depends in part on tumor entity. Little is known about the function of GPER1 in vulvar carcinoma. In this work, we aim to clarify what role GPER1 plays in vulvar cancer, tumor-promoting or tumor-suppressive. Localization of GPER1 in A431 and CAL-39 vulvar carcinoma cells was examined by immunofluorescence. Using a tissue microarray of vulvar neoplasias, the correlation between GPER1 expression and grade of malignancy was investigated. A431 and CAL-39 cells were treated either with GPER1 agonist G1 or antagonist G36. Proliferation was quantified by BrdU assay and viability examined using Resazurin assay. Morphological changes were analyzed by microscopy and measured using ImageJ. Cell migration was analyzed by gap closure assay. Clonogenic potential was tested by colony and sphere formation. Expression of estrogen receptors was examined by Western blot. GPER1 was found consistently expressed in vulvar neoplasia tissues. The immune-reactive score was found to be significantly higher in tissue samples of lymph node metastases and neoplasias with grade 3. In A431 and CAL-39 vulvar carcinoma cells, GPER1 expression was mainly found in the cytoplasm and nuclei. Treatment of A431 and CAL-39 cells with GPER1 agonist G1 resulted in a decrease in proliferation and migration. In addition, colony formation and tumor sphere formation were reduced. Furthermore, morphological signs of necrosis and reduction in cell viability after G1 treatment were observed. The GPER1 antagonist G36 did not have significant effects on vulvar carcinoma cells. Neither agonist G1 nor antagonist G36 treatment resulted in altered expression of estrogen receptors. Activation of GPER1 with GPER1 agonist G1 reduces the tumorigenic potential of the vulvar carcinoma cells. It can be deduced from this that GPER1 appears to have a tumor-suppressive effect in vulvar carcinoma

Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells

Aggressive and mesenchymal-transformed breast cancer cells show high expression levels of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression significantly increased after the induction of mesenchymal transformation in breast cancer cells. Therefore, we investigated the influence of ARHGAP29 on the invasiveness of aggressive and mesenchymal-transformed breast cancer cells. After knock-down of ARHGAP29 using siRNA, invasion of HCC1806, MCF-7-EMT, and T-47D-EMT breast cancer cells was significantly reduced. This could be explained by reduced inhibition of RhoA and a consequent increase in stress fiber formation. Proliferation of the breast cancer cell line T-47D-EMT was slightly increased by reduced expression of ARHGAP29, whereas that of HCC1806 and MCF-7-EMT significantly increased. Using interaction analyses we found that AKT1 is a possible interaction partner of ARHGAP29. Therefore, the expression of AKT1 after siRNA knock-down of ARHGAP29 was tested. Reduced ARHGAP29 expression was accompanied by significantly reduced AKT1 expression. However, the ratio of active pAKT1 to total AKT1 remained unchanged or was significantly increased after ARHGAP29 knock-down. Our results show that ARHGAP29 could be an important factor in the invasion of aggressive and mesenchymal-transformed breast cancer cells. Further research is required to fully understand the underlying mechanisms