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Background Malignant pleural mesothelioma (MPM) is a rare but fatal cancer, which is largely caused by exposure to asbestos. Reliable information about the incidence of MPM prior the influence of asbestos is lacking. The nationwide regional incidence trends for MPM remain poorly characterized. We use nationwide MPM data for Denmark (DK), Finland (FI), Norway (NO) and Sweden (SE) to assess incidence, mortality and survival trends for MPM in these countries. Methods We use the NORDCAN database for the analyses: incidence data were available from 1943 in DK, 1953 in FI and NO and 1958 in SE, through 2016. Survival data were available from 1967 through 2016. World standard population was used in age standardization. Results The lowest incidence that we recorded for MPM was 0.02/100,000 for NO women and 0.05/100,000 for FI men in 1953–57, marking the incidence before the influence of asbestos. The highest rate of 1.9/100,000 was recorded for DK in 1997. Female incidence was much lower than male incidence. In each country, the male incidence trend for MPM culminated, first in SE around 1990. The regional incidence trends matched with earlier asbestos-related industrial activity, shipbuilding in FI and SE, cement manufacturing and shipbuilding in DK and seafaring in NO. Relative 1-year survival increased from about 20 to 50% but 5-year survival remained at or below 10%. Conclusion In the Nordic countries, the male incidence trends for MPM climaxed and started to decrease, indicating that the prevention of exposure was beneficial. Survival in MPM has improved for both sexes but long-term survival remains dismal.

Cigarette smoking epidemic, which started before the World War II, completely changed the cancer landscape. Reliable incidence data spanning the stepwise spreading epidemic are rare, but the Nordic cancer registries are unique sources in being able to catch the pre-epidemic situation in the female population where smoking became more prevalent after the War. For Swedish men, smoking prevalence has decease early and cancer rates may herald postsmoking rates. We used data from the NORDCAN database, constructed by the cancer registries of Denmark, Finland, Norway and Sweden, for the analysis of incidence changes in lung and bladder cancers from year 1943 (Denmark), from 1953 (Finland and Norway) and from 1960 (Sweden) until year 2016. The analyses revealed four novel observation relevant to the smoking epidemic. (1) The incidence of lung cancer in Norwegian women in the 1950s, when the smoking prevalence was very low, was 1.8/100 000 (world standard rate), which is at the level of lowest global female rates known to-date; (2) the earliest lung-to-bladder incidence ratio among Norwegian women was 0.64, probably benchmarking the incidence rates prior to the smoking epidemic; (3) bladder cancer incidence for Finnish women diagnosed in the 1950s was 1.2/100 000 which is at the level of the lowest rates currently known and (4) Swedish men with the lowest smoking prevalence in Europe, showed an epochal crossing of lung and bladder cancer incidence rates before year 2015. The data suggest that the approaching of the incidence rates for lung and bladder cancer can be expected in the course of the abating smoking epidemic.

Background Many cancers are increased in immunosuppressed patients and evidence is accumulating that immune dysfunction may be a contributing risk factor for second primary cancers (SPCs). The aim of this study was to explore the potential influence of immune mechanisms in SPC. Methods We used the Swedish Cancer Registry (1990‐2015) to select 13 male and 14 female first primary cancers (FPCs) that are known to be related to immune suppression. We assessed relative risks (RRs) for any of these as concordant (same first and second cancer) and discordant FPC‐SPC pairs. Hierarchical clustering of significant RRs was performed for cancers as FPC and SPC. Results Concordant risks for SPCs were excessive in men and women for nasal (RRs 59.3 for men and 150.6 for women), tongue/mouth (51.7 and 100.8), and lip (32.4 and 61.2) cancers. Heatmaps showed that some cancers, such as skin cancer, tongue/mouth cancers, and non‐Hodgkin lymphoma had multiple bidirectional associations as FPC and SPC. Nasal cancer and chronic lymphocytic leukemia had associations mainly as FPC while liver and kidney cancers showed most associations as SPC. Conclusions Immune dysfunction may be a plausible contributing factor for most of the associations, which calls for experimental verification., Relative risks for a single second primary cancer after any first primary cancer (top), and RRs for any second primary cancer after a single first primary cancer (bottom). Male data are blue and female data in yellow symbols. The vertical bars show 95%CIs. NHL, non‐Hodgkin lymphoma, CLL, chronic lymphocytic leukemia.

Background: Most literature on second primary cancers (SPCs) focuses on possible factors, which may increase the risk of these cancers, and little attention has been paid for the overall incidence differences between first primary cancers (FPCs) and same SPCs. We wanted to compare the incidence rates for all common cancers when these were diagnosed as FPCs and SPCs after invasive and in situ squamous cell carcinoma (SCC) of the skin, which are usually treated by surgery only. Methods: Cancers were identified from the Swedish Cancer Registry from the years 1990 through to 2015, and they included, in addition to skin cancers, 20 male cancers totaling 484,850 patients and 22 female cancers totaling 452,909 patients. Standardized incidence rates and relative risks (RRs) were calculated for sex-specific common cancers as FPC and as SPC after skin SCC. Spearman rank correlations were used in the analysis of incidence ranking of FPC and SPC. Results: Of total, 29,061 men and 23,533 women developed invasive SCC and 27,842 men and 36,383 women in situ SCC. The total number of 20 other male cancers was 484,850 and of 22 female cancers it was 452,909. Rank correlations ranged from 0.90 to 0.96 (P~5×10−6), indicating that overall skin SCC did not interfere with SPC formation. The exceptions were increased SPC risks for melanoma, sharing risk factors with skin SCC, and non-Hodgkin and Hodgkin lymphoma, and cancers of the upper aerodigestive tract, connective tissue, and male and female genitals suggesting contribution by skin cancer initiated immune dysfunction. Conclusion: The incidence ranking of SPCs after skin cancers largely follows the incidence ranking of FPCs indicating that overall skin SCC does not greatly interfere with the intrinsic carcinogenic process. The main deviations in incidence between FPC and SPC appeared to be due to shared risk factors or immunological processes promoting immune responsive cancer types. (Less)

Background: Survival in malignant cutaneous melanoma has improved but increasing survival will result in an increased likelihood of the occurrence of second primary cancers (SPCs). SPCs may adversely interfere with survival. We quantified survival in patients with different types of SPCs, in comparison to known poor prognostic indicators of metastatic disease. Methods: Data for melanoma and any SPCs were obtained from the Swedish Cancer Registry for years 2003 through 2015, including clinical TNM classification. SPCs were grouped into three ‘prognostic groups’ based on 5-year relative survival of these cancers as first primary cancer. Kaplan-Meier survival curves were generated and hazard ratios were estimated using Cox regression, adjusted for a number of variables and treating diagnosis of SPC as a time-dependent variable. Results: The total number of first melanoma patients was 28,716 followed by 3,202 (11.1%) SPCs, 1/3 of which had a second melanoma while 2/3 had other SPCs. Among men diagnosed at age over 70 years, who survived at least 10 years, 31.4% had SPC. HRs (95% CI) for survival increased systematically from the reference rate of 1.00 (no SPC) to 1.59 (1.35–1.87) with SPC of good prognosis (78.6% of SPCs) to 3.49 (2.58–4.72) of moderate prognosis (12.0%) and to 7.93 (5.50–11.44) of poor prognosis (9.4%). In patients without SPC, the HRs increased to 2.62 (2.02–3.39) with any nodal metastases and to 5.88 (4.57–7.57) with any distant metastases compared to patients without local or distant metastases. Conclusion: The data showed that SPCs are an increasingly common negative prognostic factor for melanoma. Future attempts to improve melanoma survival need to target SPCs. (Less)

The notion of developing variants of the classic interleukin 2 (IL-2) cytokine has emerged from the limitations observed with the systemic use of human IL-2 in the clinic: severe adverse events accompanied by low therapeutic response rate in treated patients. Modifications made in the IL-2 receptor-binding structure leads to preferential binding of IL-2 variant cytokine to receptors on effector anti-tumor lymphocytes over T regulatory (TReg) cells. Because of their inherent immunogenicity, oncolytic adenoviruses are useful for expression of immunomodulatory molecules in tumors, for induction of a pro-inflammatory state in the tumor microenvironment. In the present study, we constructed an adenovirus coding for an IL-2 variant (vIL-2) protein, Ad5/3-E2F-d24-vIL2. Functionality of the new virus was tested in vitro, and anti-tumor efficacy and mechanism of action studies were performed in immunocompetent hamsters bearing pancreatic tumors. Ad5/3-E2F-d24-vIL2 treatment elicited efficient anti-tumor response, with 62.5% monotherapy complete response. Moreover, it promoted substantial repression of genes associated with myeloid cells mediated immunosuppression (CD11b, ARG1, CD206). This was seen in conjunction with upregulation of genes associated with tumor-infiltrating lymphocyte (TIL) cytotoxicity (CD3G, SAP, PRF1, GZMM and GZMK). In summary, Ad5/3-E2F-d24-vIL2 demonstrates therapeutic potential by counteracting immunosuppression and in efficiently coordinating lymphocytes mediated anti-tumor response in immunosuppressive tumors. Thus, Ad5/3-E2F-d24-vIL2 is a promising candidate for translation into clinical trials in human immunosuppressive solid tumors.

After the discovery and characterization of the adenovirus in the 1950s, this prevalent cause of the common cold and other usually mild diseases has been modified and utilized in biomedicine in several ways. To date, adenoviruses are the most frequently used vectors and therapeutic (e.g., oncolytic) agents with a number of beneficial features. They infect both dividing and nondividing cells, enable high-level, transient protein expression, and are easy to amplify to high concentrations. As an important and versatile research tool, it is of essence to understand the limits and advantages that genetic modification of adenovirus vectors may entail. Therefore, a retrospective analysis was performed of adenoviral gene therapy constructs produced in the same laboratory with similar methods. The aim was to assess the impact of various modifications on the physical and functional titer of the virus. It was found that genome size (designed within "the 105% golden rule") did not significantly affect the physical titer of the adenovirus preparations, regardless of the type of transgene (e.g., immunostimulatory vs. other), number of engineered changes, and size of the mutated virus genome. One statistically significant exception was noted, however. Chimeric adenoviruses (5/3) had a slightly lower physical titer compared to Ad5-based viruses, although a trend for the opposite was true for functional titers. Thus, 5/3 chimeric viruses may in fact be appealing from a safety versus efficacy viewpoint. Armed viruses had lower functional and physical titers than unarmed viruses, while five genomic modifications started to decrease functional titer. Importantly, even highly modified armed viruses generally had good titers compatible with clinical testing. In summary, this paper shows the plasticity of adenovirus for various vector, oncolytic, and armed oncolytic uses. These results inform future generations of adenovirus-based drugs for human use. This information is directly transferable to academic laboratories and the biomedical industry involved in vector design and production optimization.

Second primary cancers (SPCs) account for an increasing proportion of all cancer diagnoses and family history of cancer may be a risk factor for SPCs. Using the Swedish Family-Cancer Database on non-Hodgkin lymphoma (NHL), we assessed the influence of family history on risk of SPCs and of SPCs on survival. NHL patients were identified from the years 1958 to 2015 and generalized Poisson models were used to calculate relative risks (RRs) for SPCs and familial SPCs. Among 14,393 NHL patients, a total of 1,866 (13.0%) were diagnosed with SPC. Familial risk of nine particular cancers were associated with risks of these cancers as SPCs, with 2 to 5-fold increases in RRs. At the end of a 25-year follow-up period, the survival probability for persons with SPC was only 20% of that for patients without SPC; the hazard ratio for SPC was 1.59 (95% CI: 1.46 ? 1.72). Survival could be predicted by the prognostic groups based on first cancers and HRs increase systematically with worse prognosis yielding a trend of P = 4.6x10-5. SPCs had deleterious consequences for survival in NHL patients. Family history was associated with increasing numbers of SPCs. Prevention of SPCs and their early detection is an important target in the overall strategy to improve survival in NHL patients. Counseling for avoidance of risk factors and targeted screening based on family history are feasible steps in risk reduction. This article is protected by copyright. All rights reserved.

Introduction Oncolytic adenoviruses are among the most studied oncolytic viruses because of their tumor selectivity, safety, and transgene-delivery capability. With a growing number of different immunotherapies against cancer, the extraordinary immunogenicity of the adenovirus has emerged as a differentiating strength. Enabling T-cell related therapies with oncolytic adenoviruses appears a promising approach due to its inherent ability to elicit responses from the adaptive immune compartment. Areas covered These viruses have successfully enhanced both adoptive T-cell therapies and immune-checkpoint therapies. Oncolytic viruses induce several effects at the tumor and on the systemic level that help to circumvent current limitations of T-cells and related therapies, such as T-cell trafficking, tumor immune suppressivity and antigen spreading Expert opinion Taking into account the multitude of possibilities of treating cancer with immunotherapies, learning to optimize the combinations and administration strategies of these drugs, could lead to durable responses in patients with currently incurable cancers.

Background With continuous increases in survival rates following breast cancer (BC) diagnosis, the challenge of multiple primary cancers has become an issue. The data on familial risk of SPCs after BC diagnosis and the related mortality in BC patients are scarce. Methods A total of 87 752 female BC patients were followed for SPC diagnoses and records of death. Relative risks (RRs) of SPC in BC patients who had first‐degree relatives (parents or siblings) affected by the same cancer were compared to the patients without family history. Causes of death were compared between patients with and without SPC. Results After a median follow‐up of 5 years, 14 952 BC patients developed SPCs, among which 10 280 (68.8%) had first‐degree relatives diagnosed with cancer. Familial risks were significant for 14 site‐specific SPCs, and the highest risk was for second ovarian cancer (RR = 6.28, 95%CI: 4.50‐8.75), compared to those without family history (1.49, 1.34‐1.65). In patients with SPC, SPC was the main cause of death, including diverse cancers and BC in approximately equal proportions. Conclusions Family history contributed to the excess number of patients with SPCs, and SPC was the leading cause of death in patients with SPC. Taking family history at diagnosis of BC may provide warning signs with regard to possible subsequent SPCs and may offer possibilities for counseling, intervention and management., Family history contributed to the excess number of BC patients with SPC. SPC was found the leading cause of death in BC patients with SPC.

Guoqiao Zheng,1– 3 Kristina Sundquist,3– 5 Jan Sundquist,3– 6 Tianhui Chen,7 Asta Försti,1,3,8,9 Akseli Hemminki,10,11 Kari Hemminki1– 3,12 1Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany; 3Center for Primary Health Care Research, Lund University, Malmö, Sweden; 4Department of Family Medicine and Community Health; 5Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY, USA; 6Center for Community-Based Healthcare Research and Education (CoHRE), Department of Functional Pathology, School of Medicine, Shimane University, Matsue, Shimane, Japan; 7Department of Cancer Prevention, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer, Chinese Academy of Sciences, Hangzhou, 310022, People’s Republic of China; 8Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany; 9Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany; 10Cancer Gene Therapy Group, Translational Immunology Research Program, University of Helsinki, Helsinki, Finland; 11Comprehensive Cancer Center, Helsinki University Hospital, Helsinki, Finland; 12Biomedical Center, Faculty of Medicine and Biomedical Center in Pilsen, Charles University in Prague, Pilsen, 30605, Czech RepublicCorrespondence: Kari HemminkiBiomedical Center, Charles University Medical Faculty in Pilsen, Pilsen, 30605, Czech RepublicEmail K.Hemminki@dkfz.deBackground: Second primary cancers (SPCs) are increasing, which may negatively influence patient survival. Gastric cancer (GC) has poor survival and when it is diagnosed as SPC it is often the cause of death. We wanted to analyze the risk of SPCs after GC and the risk of GC as SPC after any cancer. Such bidirectional analysis is important in relation to fatal cancers because SPCs may be under-reported in the short-term survival period.Methods: Cancers were obtained from the Swedish Cancer Registry from years 1990 through 2015. Standardized incidence ratios (SIRs) were used to estimate bidirectional relative.Results: We identified 23,137 GC patients who developed 1042 SPCs (4.5%); 2158 patients had GC as SPC. While the risk for three SPCs was increased after GC, seven first primary cancers were followed by an increased risk of GC as SPC, including esophageal, colorectal, bladder, squamous cell skin and breast cancers and non-Hodgkin lymphoma. Breast cancer, which was followed by a diagnosis of second GC, showed an excess of lobular histology.Conclusion: Multiple primary cancers in the same individuals may signal genetic predisposition. Accordingly, the association of GC with breast cancer may be related to mutations in the CDH1 gene, and clustering of colorectal, small intestinal and bladder cancers could be related to Lynch syndrome. The third line of findings supports a contribution of immune dysfunction on the increased risk of GC as SPC after skin cancer and non-Hodgkin lymphoma. Early detection of GC in the risk groups could save lives.Keywords: cancer incidence, relative risk, second primary cancer, cancer etiology, stomach cancer

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20–3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.

In this review, we discuss the use of oncolytic viruses in cancer immunotherapy treatments in general, with a particular focus on adenoviruses. These serve as a model to elucidate how versatile viruses are, and how they can be used to complement other cancer therapies to gain optimal patient benefits. Historical reports from over a hundred years suggest treatment efficacy and safety with adenovirus and other oncolytic viruses. This is confirmed in more contemporary patient series and multiple clinical trials. Yet, while the first viruses have already been granted approval from several regulatory authorities, room for improvement remains.As good safety and tolerability have been seen, the oncolytic virus field has now moved on to increase efficacy in a wide array of approaches. Adding different immunomodulatory transgenes to the viruses is one strategy gaining momentum. Immunostimulatory molecules can thus be produced at the tumor with reduced systemic side effects. On the other hand, preclinical work suggests additive or synergistic effects with conventional treatments such as radiotherapy and chemotherapy. In addition, the newly introduced checkpoint inhibitors and other immunomodulatory drugs could make perfect companions to oncolytic viruses. Especially tumors that seem not to be recognized by the immune system can be made immunogenic by oncolytic viruses. Logically, the combination with checkpoint inhibitors is being evaluated in ongoing trials. Another promising avenue is modulating the tumor microenvironment with oncolytic viruses to allow T cell therapies to work in solid tumors.Oncolytic viruses could be the next remarkable wave in cancer immunotherapy.

Checkpoint inhibitors have revolutionized cancer therapy and validated immunotherapy as an approach. Unfortunately, responses are seen in a minority of patients. Our objective is to use engineered adeno- viruses designed to increase lymphocyte trafficking and cytokine production at the tumor, to assess if they increase the response rate to checkpoint inhibition, as these features have been regarded as predictive for the responses. When Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (an oncolytic adenovirus coding for TNFa and IL-2, also known as TILT-123) and checkpoint inhibitors were used together in fresh urological tumor histo- cultures, a significant shift toward immune activity (not only tumor necrosis alpha and interleukin-2 but also interferon gamma and granzyme B) and increased T-cell trafficking signals (CXCL10) was observed. In vivo, our viruses enabled an anti-PD-L1 (a checkpoint inhibitor) delivering complete responses in all the treated animals (hazard ratios versus anti-PD-L1 alone 0.057 [0.007; 0.451] or virotherapy alone 0.067 [0.011; 0.415]). To conclude, when an engineered oncolytic adenovirus was utilized to modify the tumor microenvironment towards what meta-analyses have pointed as predictive markers for checkpoint inhibitory therapy, the response to them increased synergistically. Of note, key findings were confirmed in fresh patient-derived tumor explants.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation. ispartof: JOURNAL FOR IMMUNOTHERAPY OF CANCER vol:8 issue:1 ispartof: location:England status: published

Despite some promising results, the majority of patients do not benefit from T cell therapies, as tumors prevent T cells from entering the tumor, shut down their activity, or downregulate key antigens. Due to their nature and mechanism of action, oncolytic viruses have features that can help overcome many of the barriers currently facing T cell therapies of solid tumors. This study aims to understand how four different oncolytic viruses (adenovirus, vaccinia virus, herpes simplex virus, and reovirus) perform in that task. For that purpose, an immunocompetent in vivo tumor model featuring adoptive tumor-infiltrating lymphocyte (TIL) therapy was used. Tumor growth control (p < 0.001) and survival analyses suggest that adenovirus was most effective in enabling T cell therapy. The complete response rate was 62% for TILs + adenovirus versus 17.5% for TILs + PBS. Of note, TIL biodistribution did not explain efficacy differences between viruses. Instead, immunostimulatory shifts in the tumor microenvironment mirrored efficacy results. Overall, the use of oncolytic viruses can improve the utility of T cell therapies, and additional virus engineering by arming with transgenes can provide further antitumor effects. This phenomenon was seen when an unarmed oncolytic adenovirus was compared to Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123). A clinical trial is ongoing, where patients receiving TIL treatment also receive TILT-123 (ClinicalTrials.gov: NCT04217473)., Graphical Abstract

Second primary cancers (SPCs) are becoming a common cancer entity, which may interfere with survival in relatively benign first primary cancers. We examined the hypothesis that immune dysfunction may contribute to SPCs by assessing SPCs associated with known immune responsive skin cancers, invasive and in situ squamous cell carcinoma, Kaposi sarcoma, and Merkel cell carcinoma. Cancers were identified from the Swedish Cancer Registry from the year 1958 to 2015. Standardized relative risks were calculated bidirectionally for any SPC after skin cancer and for skin cancer as SPC. Over 80,000 first primary cancers were identified for each invasive and in situ squamous cell carcinoma of the skin. Bidirectional increased risks were observed for 26 cancers associated with invasive skin cancer; the Spearman rank correlation was 0.72 (P = 4.6 x 10(-5)). The highest bidirectional relative risks were for invasive and in situ skin cancer as SPCs (14.59 and 16.71, respectively). Remarkably high risks for second in situ squamous cell carcinoma of the skin were found after Kaposi sarcoma (685.68) and Merkel cell carcinoma (117.23). The high systematic bidirectional risks between immune responsive skin cancers and most other cancers suggest that immune suppression is a key mechanism contributing to an increased risk of SPCs.

BackgroundOvarian cancers often contain significant numbers of tumor-infiltrating lymphocytes (TILs) that can be readily harnessed for adoptive T-cell therapy (ACT). However, the immunosuppressive ovarian tumor microenvironment and lack of tumor reactivity in TILs can limit the effectiveness of the therapy. We hypothesized that by using an oncolytic adenovirus (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) to deliver tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2), we could counteract immunosuppression, and enhance antitumor TIL responses in ovarian cancer (OVCA).MethodsWe established ex vivo tumor cultures freshly derived from patients with advanced OVCA and evaluated the effects of Ad5/3-E2F-D24-hTNFa-IRES-hIL2 or Ad5/3-E2F-D24 (the control virus without TNFa and IL-2) on TILs, cytokine response and tumor viability. Tumor reactivity was assessed by determining interferon gamma (IFNg) response of clinically relevant TILs towards autologous T-cell-depleted ex vivo tumor cultures pretreated with or without the aforementioned oncolytic adenoviruses.ResultsTreatment of ex vivo tumor cultures with Ad5/3-E2F-D24-hTNFa-IRES-hIL2 caused a substantial rise in proinflammatory signals: increased secretion of IFNg, CXCL10, TNFa and IL-2, and concomitant activation of CD4+ and CD8+ TILs. Potent tumor reactivity was seen, as clinically relevant TIL secreted high levels of IFNg in response to autologous T-cell-depleted ovarian ex vivo tumor cultures treated with Ad5/3-E2F-D24-hTNFa-IRES-hIL2. This phenomenon was independent of PD-L1 expression in tumor cells, a factor that determined the variability of IFNg responses seen in different patient samples.ConclusionsOverall, oncolytic adenovirus Ad5/3-E2F-D24-hTNFa-IRES-hIL2 was able to rewire the ovarian tumor microenvironment to accommodate heightened antitumor TIL reactivity. Such effects may improve the clinical effectiveness of ACT with TILs in patients with advanced OVCA.

Despite exciting proof-of-concept data mediated by adoptive T-cell transfer and checkpoint blockade, major challenges imposed by the tumor microenvironment restrict clinical benefits to a minority of patients with advanced or metastatic solid malignancies. While employment of toxic pre- and postconditioning regimens to circumvent the inefficacy of T-cell transfer presents a fundamental problem for heavily pretreated cancer patients, for checkpoint blockade, the main issue relates to low single-agent response rates. To overcome these hurdles, combination therapy with oncolytic adenovirus is becoming an attractive solution given multiple intrinsic modulatory effects on the intratumoral immune compartment, engineering capabilities and safety profile. Here, we provide a short overview on the tumor microenvironmental challenges in solid tumors, and how oncolytic adenoviruses can counteract these barriers. Finally, the immunotherapeutic potential of oncolytic adenoviruses will be discussed in the context of clinical experience with adoptive T-cell therapy and immune checkpoint inhibitors.

Summary Background Patients with squamous cell skin cancer (SCC) have an excellent prognosis but second primary cancers (SPCs) weaken survival prospects. Family history is a known risk factor for cancer but whether it is a risk factor for SPC in patients with SCC is not known. Objectives To quantify the risk of family history on SPCs in patients with SCC and estimate survival probabilities of patients with SPCs depending on family history. Methods With 13 945 histologically verified SCCs, relative risks (RRs) were estimated for family history using a generalized regression model. For survival analysis, hazard ratios (HRs) were assessed using a multivariable Cox proportional-hazards model. Results Family history of invasive SCC increased risk of second invasive SCC [RR = 42·92, 95% confidence interval (CI) 33·69?50·32] compared with risk without family history (RR 19·12, 95% CI 17·88?21·08). Family history of any nonskin cancer in invasive SCC increased risk of the same cancers to be diagnosed as SPC (RRFH = 1·48, 95% CI 1·35?1·61 vs. RRno FH = 1·40, 95% CI 1·32?1·48); significant increases were observed for seven different nonskin cancers. Most results were replicated for in situ SCC. SPC was deleterious for survival irrespective of family history; HR for patients with SPC was 4·28 (95% CI 3·83?4·72) vs. those without SPC (1·04). Conclusions Family history of nonskin cancer was associated with approximately a doubling of risk for SPCs in patients with SCC. SPC increases the death rate in patients with SCC 3?4 times, irrespective of family history. Taking family history into account at SCC diagnosis may help prevention or early detection of SPCs. What's already known about this topic? Second primary cancers (SPCs) are frequently diagnosed in patients with invasive and in situ squamous cell carcinoma (SCC); some epidemiological studies suggest a link to immune dysfunction. Family history of cancer is a risk factor for practically all first primary cancers but whether it also influences risk of SPCs in patients with SCC is not known. The possible influence of family history on survival in patients with SCC remains to be established.

The first US Food and Drug Administration (FDA)- and EMA-approved oncolytic virus has been available since 2015. However, there are no markers available that would predict benefit for the individual patient. During 2007–2012, we treated 290 patients with advanced chemotherapy-refractory cancers, using 10 different oncolytic adenoviruses. Treatments were given in a Finnish Medicines Agency (FIMEA)-regulated individualized patient treatment program (the Advanced Therapy Access Program [ATAP]), which required long-term follow-up of patients, which is presented here. Focusing on the longest surviving patients, some key clinical and biological features are presented as “oncograms.” Some key attributes that could be captured in the oncogram are suggested to predict treatment response and survival after oncolytic adenovirus treatment. The oncogram includes immunological laboratory parameters assessed in peripheral blood (leukocytes, neutrophil-to-lymphocyte ratio, interleukin-8 [IL-8], HMGB1, anti-viral neutralizing antibody status), features of the patient (gender, performance status), tumor features (histological tumor type, tumor load, region of metastases), and oncolytic virus-specific features (arming of the virus). The retrospective approach used here facilitates verification in a prospective controlled trial setting. To our knowledge, the oncogram is the first holistic attempt to identify the patients most likely to benefit from adenoviral oncolytic virotherapy. Keywords: oncolytic adenovirus, oncolytic virotherapy, immunotherapy, cancer, immunostimulation, anti-cancer, adenovirus, oncoimmunology, immunogram, oncogram

Male breast cancer is associated with female breast cancer in families but whether male breast cancer clusters with other discordant cancers has not been studied. As concordant male breast cancers are utterly rare, discordant associations of male breast cancer with other cancers may reveal genetic and possible environmental risk factors contributing to male breast cancer susceptibility. We calculated relative risks (RRs) for male breast cancer in families with discordant cancers, and conversely, for discordant cancers in families of male breast cancer patients, based on 15.7 million individuals in the Swedish Family-Cancer Database. Among 1428 male breast cancer patients, 16.2% had a female relative diagnosed with breast cancer. Ovarian and female anal cancers showed the strongest associations with male breast cancer (p value

Relatives of cancer patients are at an increased risk of the same (concordant) cancer but whether they are at a risk for different (discordant) cancers is largely unknown - beyond well characterized hereditary cancer syndromes - but would be of major scientific and clinical interest. We therefore decided to resolve the issue by analyzing familial risks when family members were diagnosed with any discordant cancers. We compared the population impact of concordant to discordant familial cancer. The Swedish Family-Cancer Database (FCD) was used to calculate familial relative risks (RRs) for family members of cancer patients, for the 27 most common cancers. Population attributable fractions (PAFs) were estimated for concordant and discordant family histories. Discordant cancers in the family were detected as significant risk factors for the majority of cancers, although the corresponding RRs were modest compared to RRs for concordant cancers. Risks increased with the number of affected family members with the highest RRs for pancreatic (2.31), lung (1.69), kidney (1.98), nervous system (1.79) and thyroid cancers (3.28), when 5 or more family members were diagnosed with discordant cancers. For most cancers, the PAF for discordant family history exceeded that for concordant family history. Our findings suggest that there is an unspecific genetic predisposition to cancer with clinical consequences. We consider it unlikely that shared environmental risk factors could essentially contribute to the risks for diverse discordant cancers, which are likely driven by genetic predisposition. The identification of genes that moderately increase the risk for many cancers will be a challenge.

Aim: We combined anti-androgen therapy with radiotherapy in a first-line setting for metastatic prostate cancer aiming to cause maximal cancer-cell death to delay the emergence of castration-resistant disease. Materials and Methods: In this non-randomized retrospective series of 46 patients, the initial median prostate-specific antigen (PSA) was 98.5 μg/l (range=6.7-15,500), median Gleason score 9 and most men had at least T3N1M1 disease. All patients received luteinizing hormone releasing hormone analog or degarelix with bicalutamide. If PSA remained above 1 μg/l, docetaxel was initiated. At PSA nadir, all patients received radical radiotherapy of the prostate. Results: The median follow-up time was 4.38 years (range=0.36-11.24). Most radiotherapy-related adverse events were grade 1 and transient. There were no grade 4 events. Overall survival (OS) at 5 years was 81.3%. Conclusion: The feasibility and safety of aggressive multimodality treatment were good resulting in an excellent median OS of 8.35 years.

Background The ovarian tumour microenvironment is an abundant source of tumour-infiltrating lymphocytes (TILs) which can be readily harnessed for infusion in the context of adoptive TIL therapy. This strategy has been proven feasible, but it appears clinically ineffective in ovarian cancer patients due to the absence of tumour-reactive TILs and the presence of immunosuppression. Hence, we hypothesized that an oncolytic adenovirus expressing Tumour Necrosis Factor (TNF)-alpha(a) and Interleukin (IL)-2 (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) could overcome this by generating a proinflammatory microenvironment and reinvigorating TIL anti-tumour activity. Methods Fresh explants from metastatic or primary tumour sites were obtained from stage III-IV ovarian cancer patients and short-term cultures were established in the absence or presence of oncolytic adenovirus. The remainder of the tumour explant was used to generate clinically relevant TILs which were co-cultured with autologous T cell-depleted single cell suspensions pre-treated with or without oncolytic adenoviruses. Cytokine content changes and TIL reactivity was assessed during culture by flow cytometry or interferon (IFN) gamma(g) enzyme-linked immune sorbent assay. Results Treatment of short-term cultures with oncolytic adenovirus coding for TNFa and IL-2 introduced profound changes within the microenvironment, which were characterized by an increase in proinflammatory cytokines and decrease in suppressive cytokines. Further benefits were seen in T-cell depleted ovarian cancer single-cell suspensions infected with cytokine-coding oncolytic adenovirus, which enabled significant production of IFNg by autologous TILs. Such levels were not seen in co-cultures where no virus or the backbone oncolytic adenovirus (no cytokine transgenes) was added. Conclusion These data illustrate the potential of oncolytic adenovirus coding for TNFa and IL-2 to rewire the ovarian tumour microenvironment for effective TIL anti-tumour reactivity. This approach may improve the efficacy of adoptive TIL therapy in ovarian cancer patients, thus warranting further clinical investigation. Legal entity responsible for the study TILT Biotherapeutics Ltd. Funding TILT Biotherapeutics Ltd. Disclosure J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. M. Siurala: Full / Part-time employment: TILT Biotherapeutics Ltd. T. de Gruijl: Advisory / Consultancy: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.

BACKGROUND: Many studies have indicated that colon and rectal cancers differ in etiology and histology. OBJECTIVE: The aim of this study was to investigate whether the associations of colon and rectal cancers with any other (discordant) cancer were site specific. DESIGN: A novel approach was implemented in which cancer risks were analyzed in families with increasing numbers of family members diagnosed with defined cancers. The novel assumption was that, for a true familial association, the risk should increase by the number of affected family members. In separate analyses, familial risks were calculated after the exclusion of putative families with hereditary nonpolyposis colorectal cancer. SETTINGS: The study was conducted using the Swedish Family-Cancer Database. MAIN OUTCOME MEASURES: The outcome measure was relative risk. RESULTS: Relative risks of colorectal cancer and colon cancer were higher when family members were diagnosed with colon cancer than when family members were diagnosed with rectal cancer (incidence rate ratio for colorectal: 1.82 (95% CI, 1.74-1.90) vs 1.61 (95% CI, 1.51-1.71); incidence rate ratio for colon: 1.92 (95% CI, 1.83-2.02) vs 1.56 (95% CI, 1.45-1.69)). Relative risks for 10 discordant cancers were increased in colon or rectal cancer families, whereas none of the relative risks differed significantly between colon and rectal cancers. After deleting hereditary nonpolyposis colorectal cancer families, the relative risks of endometrial and ovarian cancers were no longer significant. LIMITATIONS: Genetic data are unavailable in the database. CONCLUSIONS: Our results suggested that familial risks for colon cancer were higher than risks for rectal cancer in families of patients with colorectal cancer and colon cancer. The relationships of lung cancer and nervous system cancer with colorectal cancer were site specific. The associations of colon and rectal cancers with lung cancer, myeloma, and cancer of unknown primary appeared not to point out known syndromes and may suggest involvement of a novel predisposition. See Video Abstract at http://links.lww.com/DCR/A791. (Less)

Improvement of survival in lymphocytic leukaemia has been accompanied by the occurrence of second primary cancer (SPCs). Based on Swedish Family Cancer Database, we applied bi-directional analyses in which relative risks (RRs) were calculated for any SPCs in patients with chronic lymphocytic leukaemia (CLL), acute lymphoblastic leukaemia (ALL) and hairy cell leukaemia (HCL) and the risks of these leukaemias as SPCs. After CLL, RRs were significant for 20 SPCs, and high for skin squamous cell cancer (2458 for insitu and 763 for invasive), Merkel cell carcinoma (1436), Hodgkin lymphoma (716) and Kaposi sarcoma (676). Conversely, 15 CLL cancer pairs were reciprocally increased. The increased risks were reciprocal for ALL and four cancers. RR for ALL was 1535 after myeloid neoplasia. HCL showed reciprocally increased RRs with non-Hodgkin lymphoma and melanoma. The concordance between RRs for bi-directional associations between CLL and different cancers, and HCL and different cancers was highly significant. For CLL (also for HCL), the bi-directional risks with skin cancers and other immune-related cancers suggest the probable involvement of immune dysfunction. For ALL, treatment may contribute to risks of multiple SPCs. Increased risk of ALL after haematological neoplasms may indicate bone marrow dysfunction. These findings may help guide treatment decisions and prognostic assessment.

BackgroundMalignant melanoma (MM) patients are at increasing risk of developing second primary cancers (SPCs). We assessed mortality and risk of SPCs in MM patients with siblings or parents affected with same cancer compared with that of the general population.MethodsWe used the Swedish Family-Cancer Database to assess relative risks (RRs) and causes of death in SPCs until 2015 in patients with a MM diagnosis between 1958 and 2015. We identified 35 451patients with MM among whom 3212 received a subsequent diagnosis of SPC. RRs of SPCs after MM diagnosis were calculated stratifying over concordant family history of cancer in first-degree relatives.ResultsFamilial RRs were increased for second melanoma (RR = 19.28, 95% CI = 16.71 to 22.25), squamous cell skin cancer (RR = 7.58, 95% CI = 5.57 to 10.29), leukemia (RR = 5.69, 95% CI = 2.96 to 10.94), bladder (RR = 4.15, 95% CI = 2.50 to 6.89), ovarian (RR = 3.89, 95% CI = 1.46 to 10.37), kidney cancer (RR = 3.77, 95% CI = 1.57 to 9.06), cancer of unknown primary (RR = 3.67, 95% CI = 1.65 to 8.16), nervous system (RR = 2.88, 95% CI = 1.20 to 6.93), breast (RR = 2.34, 95% CI = 1.92 to 2.84), lung (RR = 2.24, 95% CI = 1.50 to 3.35), and prostate cancer (RR = 2.22, 95% CI = 1.89 to 2.61) with statistical significance. For all cancers, familial RR was in excess (2.09, 95% CI = 2.02 to 2.16 vs 1.78, 95% CI = 1.69 to 1.87; Ptrend ConclusionsSPCs appear higher with prior family history of cancer and contribute to mortality. SPC was the most common cause of death in patients with SPC and is almost uniformly the major contributing cause of death for all cancer sites. For improved survival in MM patients, prevention and early detection of SPCs would be important.

Oncolytic viruses provide a biologically multi-faceted treatment option for patients who cannot be cured with currently available treatment options. We constructed an oncolytic adenovirus, TILT-123, to support T-cell therapies and immune checkpoint inhibitors in solid tumors. Adenoviruses are immunogenic by nature, are easy to produce in large quantities, and can carry relatively large transgenes. They are the most commonly used gene therapy vectors and are well tolerated in patients. TILT-123 expresses two potent cytokines, tumor necrosis factor alpha and interleukin-2, to stimulate especially the T-cell compartment in the tumor microenvironment. Before entering clinical studies, the safety and biodistribution of TILT-123 was studied in Syrian hamsters and in mice. The results show that TILT-123 is safe in animals as monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. The virus treatment induces acute changes in circulating immune cell compartments, but the levels return to normal by the middle of the treatment period. The virus is rapidly cleared from healthy tissues, and it does not cause damage to vital organs. The results support the initiation of a phase 1 dose-escalation trial, where melanoma patients receiving a tumor-infiltrating lymphocyte therapy are treated with TILT-123 (NCT04217473).

Background It has been proposed that cancer is more common in some families than in others, but the hypothesis lacks population level support. We use a novel approach by studying any cancers in large three-generation families and thus are able to find risks even though penetrance is low. Methods Individuals in the nation-wide Swedish Family-Cancer Database were organised in three generations and the relative risk (RR) of cancer was calculated to the persons in the third generation by the numbers of patients with cancer in generations 1, 2 and 3. Results The RRs for any cancer in generation 3 increased by the numbers of affected relatives, reaching 1.61 when at least seven relatives were diagnosed. The median patient had two affected relatives, and 7.0% had five or more affected relatives with an RR of 1.46, which translated to an absolute risk of 21.5% compared with 14.7% in population by age 65 years. For prostate cancer, the RR was 2.85 with four or more affected family members with any cancer, and it increased to 14.42 with four or more concordant cancers in family members. RRs for prostate cancer were approximately equal (2.70 vs 2.85) if a man had one relative with prostate cancer or four or more relatives diagnosed with any cancer. Conclusions A strong family history of cancer, regardless of tumour type, increases cancer risk of family members and calls for mechanistic explanations. Our data provide tools for counselling of patients with cancer with both low and high familiar risks.

The epidemiology of metastases in neuroendocrine tumors (NETs) is virtually unknown. The present novel approach took use of two nationwide Swedish registers to assess the distribution of metastatic sites in comparison to adenocarcinoma. 7,334 patients with NET were identified from the Swedish Cancer Registry. Metastatic sites were identified from the National Patient and Cause of Death Registries. Sites of metastasis were investigated depending on the primary site of NET. The metastatic potential of NET was assessed. The liver was the most common site of metastasis (82% of patients with metastases), and the small intestine was the most common source of NET metastases. Of all patients with metastatic lung NETs, 66% had liver metastases, whereas the corresponding number for adenocarcinoma of lung was only 20%. The risk of metastasis was highest if the primary was in the small intestine or pancreatohepatobiliary tract, whereas it was lower with appendiceal and rectal NET. Men had more bone metastases compared to women. Patients with metastatic NET had worse prognosis if the primary site was unknown (11 months, 9% of NET patients) compared to those whose primary was known (19 months). The metastatic potential of NETs varies profoundly depending on the primary site. NETs show a clear preference to metastasize to the liver. Surveillance of liver metastases may enable earlier diagnosis and treatment. In liver metastases from NET, the small intestine should be suspected as the primary site, whereas the lung should be suspected in nervous system metastases of NET origin.

// Matias Riihimaki 1, 2 , Akseli Hemminki 3, 4 , Kristina Sundquist 2 , Jan Sundquist 2 , Kari Hemminki 1, 2 1 Division of Molecular Genetic Epidemiology, German Cancer Research Centre (DKFZ), Heidelberg, Germany 2 Center for Primary Health Care Research, Lund University, Malmo, Sweden 3 Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, Finland 4 Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland Correspondence to: Matias Riihimaki, email: matias.riihimaki@gmail.com Keywords: gastric cancer, metastasis, epidemiology Received: December 22, 2015 Accepted: June 16, 2016 Published: July 20, 2016 ABSTRACT Background: The epidemiology of metastatic gastric cancer is unexplored because cancer registries seldom cover metastatic involvement apart from “present or not”. We used a novel approach by utilizing Swedish registers to assess metastatic spread in gastric cancer. To our knowledge, this is the first nationwide description of metastases in gastric cancer. Results: The most common sites of metastasis were liver (in 48% of metastatic cancer patients), peritoneum (32%), lung (15%), and bone (12%). Metastases to the lung, nervous system, and bone were more frequent in cardia cancer and men, whereas non-cardia cancer more frequently metastasized within the peritoneum. Signet ring adenocarcinomas more frequently metastasized within the peritoneum, bone and ovaries, and less frequently to the lungs and liver compared with generic adenocarcinoma. The liver and the peritoneum were commonly single metastases while lung metastases occurred frequently together with liver metastases. The median survival in metastatic gastric cancer was 3 months, worst among those with bone and liver metastases (2 months). Methods: A total of 7,559 patients with gastric cancer were identified. Metastatic patterns and survival depending on sex, age, stage, anatomical location (cardia and non-cardia), and histological type were assessed. Conclusions: The patterns of metastasis differ notably depending on histological type. Cardia cancer exhibits a completely different metastatic behavior than non-cardia cancer. Awareness of the differing patterns may guide in tailored diagnosis of metastases. Survivors from cardia cancer would benefit from increased surveillance of extraperitoneal metastases. Bone metastases should be considered in patients with signet ring adenocarcinoma if symptoms emerge.

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.

Dendritic cell (DC) therapy is considered a promising immunotherapeutic approach for treatment of advanced cancer. However, the immunosuppressive tumor microenvironment leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to activate antitumor immune responses. Importantly, they can convert immunosuppression locally into a proinflammatory state. To improve the proper activation of transferred DCs, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is known to play an important role in the regulation of immune cells through its capacity to stimulate dendritic cells that leads to the activation of cytotoxic T cells. Therefore, we generated a novel virus Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 adenovirus (Ad3) for intravenous delivery. It features a human telomerase reverse transcriptase (hTERT) promoter for tumor specific replication and expresses human CD40L (hCD40L) under a cytomegalovirus (CMV) promoter for induction of antitumor efficacy. Of note, human and animal data have shown the ability of Ad3 to successfully reach tumors through the intravenous route. In syngeneic studies in an immunocompetent model, DC therapy with our murine CD40L-armed adenovirus showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T cells and induction of T-helper type 1 immune response. This synergistic effect was further evaluated in mice humanized with human peripheral blood mononuclear cells. Treatment with hCD40L-armed adenovirus and human DCs showed 100% survival in conjunction with tumor control. To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Citation Format: João Manuel Santos, Akseli Hemminki, Mikko Siurala, Otto Hemminki, Riikka Havunen, Victor Cervera-Carrascon, Suvi Sorsa, Hongjie Wang, Andre Lieber, Tanja de Gruijl, Anna Kanerva, Sadia Zafar. Oncolytic adenovirus 3 coding for CD40L as an enhancer of dendritic cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B07.

The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A15.

Background: Treatment of multiple tumors and metastasis remains a challenge for current therapeutic approaches in cancer treatment. Some responses in noninjected metastases were described in the middle of the last century after using local radiotherapy in breast cancer patients. This phenomenon was named abscopal effect. Nowadays, this effect is well known to be elicited by systemic adaptive immune responses against tumor cells. Of note, it has not been fully reproducible in the clinic mainly due to the suppressive microenvironment surrounding cancer cells. The systemic use of immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1, has shown promise in the treatment of many tumors, but a minority of patients respond. Oncolytic virotherapy is a promising strategy that has emerged in the last few decades. It is able to prime the host immune system against tumor epitopes, thus theoretically complementing aPD1 therapy in an appealing manner. The challenge with oncolytic immunotherapy is that not all metastases can usually be injected. Therefore, it is of importance to study if local treatment can induce distant responses. While our previous work has shown synergy between oncolytic adenovirus and aPD1, here we sought to establish if the synergy extends to noninjected tumors. Aim: In the present study we aimed to assess if local adenovirus injection can impact also noninjected tumors in animals receiving aPD1 therapy. Methods: We utilized a murine melanoma model (B16.OVA) in which each mouse has two tumors, where only one tumor receives local virotherapy and the secondary tumor is studied for possible systemic effects following local injection. Results: Improvement in the overall survival was seen in the group using both therapies (aPD-1 plus virus) compared to the single therapies alone. As well, secondary tumor growth was better controlled in that group. Further information, including the final survival curve, tumor growth and immunologic mechanism-of-action data, will be presented. Conclusion: These two cancer immunotherapeutics seem to be a promising dynamic approach that may increase the life expectancy and cure the patients suffering from metastatic cancer when translated to the clinic, besides minimizing considerably the toxicity due to the specificity of such approach, and finally improving patients’ quality of life during the treatment. Citation Format: Dafne C.A. Quixabeira, Víctor Cervera-Carrascón, Riikka Havunen, Mikko Siurala, Akseli Hemminki. Adenovirus coding for TNF-α and IL-2 proteins mediates abscopal effect in mice receiving anti-PD1 immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A08.

Checkpoint inhibitors (CPI) started a new golden era of immunotherapy for cancer treatment. Since approval of the first product in 2011, a number of these antibodies are now available for different indications. Hundreds of trials are ongoing, which could predict many further approvals soon. Unfortunately, only a minority of patients currently benefit from CPI treatment. One of the biggest limitations appears to be poor immune infiltration of many tumors, known as immune desert or excluded tumors. Oncolytic viruses are an interesting tool to use together with CPI to increase the frequency and the quality of patients’ responses. Besides their inherent ability to raise the immune alarm towards the tumor, resulting in recruitment of lymphocytes and other cells, they can be armed with transgenes to potentiate these effects. In a data-driven approach, adenoviruses expressing Tumor Necrosis Factor Alpha and Interleukin 2 were identified as the optimal approach for recruiting and activating T cells in tumors. In this approach, the regimen of administration must be considered as it can affect the outcome of the treatment. We used an aggressive and immunosuppressive solid tumor model (B16.OVA melanoma) to study the interaction between the above-mentioned viruses and anti-PD-1 and anti-PD-L1. After optimizing treatment regimes, control of every tumor led to complete responses in 100% of the animals when the viral platform and anti-PD-1 (or anti-PD-L1) were used together. This was a significant improvement when compared to them used as monotherapies (p-value Citation Format: Victor Cervera-Carrascon, Dafne Quixabeira, Eleonora Munaro, Joao M. Santos, Riikka Havunen, Mikko Siurala, Akseli Hemminki. Enabling checkpoint inhibitors with oncolytic viruses to deliver complete responses: A matter of timing [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B16.

Background Dendritic Cell (DC) therapy is considered as a promising immunotherapeutic approach for treatment of advanced cancer. However, the tumor microenvironment is highly immunosuppressive that leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to preferentially target and kill cancer cells. Therefore, to improve the therapeutic efficacy of DC therapy, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is well known to regulate immune responses through its capacity to stimulate dendritic cells that lead to the activation of cytotoxic T-cells. Methods In this study, we generated a novel virus Ad3-hTERT-CMV-hCD40L (Ad3-hCD40L), which is fully serotype 3 adenovirus. It features a human telomerase reverse transcriptase promoter for tumor specificity and expresses human CD40L (hCD40L) under a cytomegalovirus promoter for induction of antitumor immune responses. Animal experiments were implanted in immunocompetent and in humanized mice model. To further deeply dissect if Ad3-hCD40L can modulate tumor microenvironment, tumor histocultures derived from prostate patients were used. Results In syngeneic studies in animal models, DC therapy with Ad3-hCD40L showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T-cells and induction of T-helper type 1 immune response. Moreover, Ad3-hCD40L and human DCs showed 100 percent survival in conjunction with tumor control. Tumor histocultures treated with Ad3-hCD40L showed that virally expressed hCD40L in the tumor microenvironment leads to significant activation of dendritic cells and induction of Th1 immune response. Conclusion To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Legal entity responsible for the study Cancer Gene Therapy Group, University of Helsinki. Funding University of Helsinki. Disclosure V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.

Background During the last decade, a revived enthusiasm about T-cell related therapies emerged after promising clinical outcomes. Nevertheless, many patients (especially with solid tumours) still do not have adequate therapeutic options. The complexity of the tumour microenvironment is a likely factor acting in detriment of many of those therapies by multiple suppressive mechanisms. To tackle a complex mechanism, an oncolytic adenovirus 2 (Ad5/3-E2F-d24-hTNFa-IRES-hIL2, a.k.a. TILT-123) was engineered to enable T-cell therapies in those circumstances. Methods To study the efficacy of TILT-123 together with different T-cell related therapies (adoptive cell transfer, checkpoint inhibitors and CAR T cell therapy) different models were used including mouse, Syrian Hamster and patient derived in vivo models for different indications were tested. Results Antitumor efficacy analyses showed complete responses in all animals receiving a T-cell therapy or checkpoint inihibitor (aPD1 or aPDL-1) and the T-cell enabling virus. Further, other aspects such as safety, influence on different immune populations, abscopal effect, antitumor memory and ability to replace lympho-depleting chemotherapy and postconditioning with high-dose IL-2 treatment were studied. Conclusion The use of TILT-123 to enable T-cell therapies (including checkpoint-inhibiting antibodies) delivered encouraging preclinical results pointing to it as a valuable approach to increase the number of patients that benefit from T-cell therapies. Those results include not only cell based therapies but also checkpoint inhibitors, a kind of therapy that is making the difference in the field and becoming first-line treatment for an increasing number of indications. Because of the favourable preclinical studies, the first in human clinical trials with TILT-123 will start in the upcoming months. Legal entity responsible for the study The authors. Funding TILT Biotherapeutics. Disclosure V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics. R. Havunen: Full / Part-time employment: TILT Biotherapeutics. J.M. Santos: Full / Part-time employment: TILT Biotherapeutics. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics. All other authors have declared no conflicts of interest.

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.

Lymphodepleting preconditioning with high-dose chemotherapy is commonly used to increase the clinical efficacy of adoptive T cell therapy (ACT) strategies, however, with severe toxicity for patients. Conversely, oncolytic adenoviruses are safe and, when engineered to express interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha), they can achieve antitumor immunomodulatory effects similar to lymphodepletion. Therefore, we compare the safety and efficacy of such adenoviruses with a cyclophosphamide-and fludarabine- containing lymphodepleting regimen in the setting of ACT. Human adenovirus (Ad5/3-E2F-D24-hTNF-alpha-IRES-hIL-2; TILT-123) replication was studied using a Syrian hamster pancreatic tumor model (HapT1) infused with tumor- infiltrating lymphocytes (TILs). Using the oncolytic virus instead of lymphodepletion resulted in superior efficacy and survival. Immune cells responsive to TNF-alpha IL-2 were studied using an immunocompetent mouse melanoma model (B16. OVA) infused with ovalbumin-specific T (OT-I) cells. Here, the adenovirus approach improved tumor control together with increased intratumoral Th1 cytokine levels and infiltration of CD8+ T cells and CD86+ dendritic cells. Similar to humans, lymphodepleting preconditioning caused severe cytopenias, systemic inflammation, and damage to vital organs. Toxicity was minimal in adenovirus- and OT-Itreated mice. These findings demonstrate that ACT can be effectively facilitated by cytokine-coding adenovirus without requiring lymphodepletion, a rationale being clinically investigated.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.

Oncolytic immunotherapy with cytokine armed replication competent viruses is an emerging approach in cancer treatment. In a recent randomized trial, an increase in response rate was seen but the effect on overall survival is not known with any virus. To facilitate randomized trials, we performed a case–control study assessing the survival of 270 patients treated in an Advanced Therapy Access Program (ATAP), in comparison to matched concurrent controls from the same hospital. The overall survival of all virus treated patients was not increased over controls. However, when analysis was restricted to GMCSF-sensitive tumor types treated with GMSCF-coding viruses, a significant improvement in median survival was present (from 170 to 208 days, P = 0.0012, N = 148). An even larger difference was seen when analysis was restricted to good performance score patients (193 versus 292 days, P = 0.034, N = 90). The survival of ovarian cancer patients was especially promising as median survival nearly quadrupled (P = 0.0003, N = 37). These preliminary data lend support to initiation of randomized clinical trials with GMCSF-coding oncolytic adenoviruses.

Objectives Population-based data on metastatic sites and survival in site-specific metastases are lacking for lung cancer and for any cancer because most cancer registries do not record metastases. This study uses a novel population-based approach to identify metastases from both death certificates and national inpatient data to describe metastatic pathways in lung cancer patients. Materials and methods 17,431 deceased lung cancer patients diagnosed 2002–2010 were identified from the nationwide Swedish Cancer Registry, which is based on compulsory reports. The influence of age at diagnosis, sex, and histological subtype on metastatic spread was investigated. Survival in metastatic lung cancer was assessed by histology and metastatic site. Results The most frequent metastatic sites were the nervous system, bone, liver, respiratory system, and adrenal gland. Liver (35%) and nervous system (47%) metastases were common in patients with metastases from small cell lung cancer, and bone (39%) and respiratory system (22%) metastases in adenocarcinoma. Women (43% vs. 35%) and younger patients had more metastases to the nervous system. Median survival after diagnosis was 13 months for non-metastatic and five months for metastatic lung cancer. In this novel data, liver metastases conferred the worst prognosis (three months), especially for large cell histology. Bone metastases also featured poor survival, whereas survival in respiratory and nervous system metastases was better. Conclusion Metastatic sites and survival in metastatic lung cancer is influenced by sex, histological subtype, and age at diagnosis. Liver and bone metastases signal poor survival, compared with nervous system metastases.

Familial risks of breast cancer (BC) are well established but whether BC clusters with other, i.e. discordant, cancers is less certain but of interest for the identification of common genetic and possible environmental factors contributing to a general cancer susceptibility. We apply a novel approach to search for familial associations of BC with other (discordant) cancers based on the Swedish Family-Cancer Database. Relative risks (RRs) were calculated for BC in families with increasing numbers of patients with discordant cancer X, and conversely, familial RRs for cancer X in families with increasing numbers of BC patients. Joint p-values were calculated from independent analyses. The total number of familial BCs was 12,266, 14.9% with one first-degree relative with BC and 1.2% with at least 2 affected relatives. Ovarian and prostate cancers showed the strongest associations with BC (p-value