Intradermal Tumors Research Articles

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Host-functionalization of macrin nanoparticles to enable drug loading and control tumor-associated macrophage phenotype.

Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified β-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of β-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug's effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages targeted with R848-loaded macrins, relative to vehicle controls, validating the desired pro-inflammatory reprogramming of TAMs in vivo by cell-targeted drug delivery. Finally, in an intradermal MC38 tumor model, cyclodextrin-modified macrin NPs loaded with immunostimulatory drugs significantly reduced tumor growth. Therefore, efficient and effective repolarization of tumor-associated macrophages to an M1-like phenotype-via drug-loaded macrins-inhibits tumor growth and may be useful as an adjuvant to existing immune checkpoint therapies.

A RARE CASE REPORT ON TUBULAR APOCRINE ADENOMA

Tubular apocrine adenoma, the apocrine variant of papillary tubulopapillary hidradenoma, is a rare well-circumscribed, intradermal benign tumor, with tubular structures showing apocrine differentiation. It usually presents as a slow-growing well-circumscribed nodule. The most common site of involvement is the scalp, although it has also been reported to occur at other sites like face, neck, axilla, trunk, upper and lower extremities. The present study describes a case of 76-year-old female who presented with swelling on back. An excisional biopsy was performed which on histopathological examination shows multiple irregular-shaped tubular structures lined by two layers of epithelial cells focally. On histopathology, the case was diagnosed as tubular apocrine adenoma.

Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors

BackgroundImmunologically cold tumors with an ‘immune desert’ phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can...

Grade of Primary Cutaneous Leiomyosarcoma Dictates Risk for Metastatic Spread and Disease-Specific Mortality.

Primary cutaneous leiomyosarcoma (cLMS), a rare, typically intradermal tumor, has previously been reported to exhibit an indolent course of disease with zero-to-low risk of local recurrence or distant metastasis. This study seeks to evaluate recurrence and survival of cLMS patients through study of its clinicopathologic and treatment characteristics. All patients included underwent resection of primary cLMS at this institution between 2006 and 2019. A retrospective cohort study analysis of clinicopathologic characteristics, treatment, recurrence, and overall survival was performed. Data was assessed through descriptive statistics and outcome measures assessed by Cox proportional models and log-rank tests. Eighty-eight patients with cLMS were evaluated. The majority were men (n = 68, 77%) and Caucasian (n = 85, 97%), with median age at diagnosis of 66years (range 20-96). 65% of tumors were located on the extremities, with a median size of 1.3cm (range .3-15). Assessment revealed low (n = 41, 47%), intermediate (n = 29, 33%), and high (n = 18, 20%) grade tumors, demonstrating extension into subcutaneous tissue in 38/60 (60%), with 3 patients exhibiting extension into muscle (3%). All underwent resection as primary treatment with median 1cm margins (range .5-2). With median follow-up of 27.5months (IQR 8-51; range 1-131), no low-grade cases had recurrence or death while there was a recurrence rate of 19.1% (9/47) and death rate of 8.5% (4/47) in intermediate- to high-grade cases. Primary tumor resection of cLMS provides excellent local control for low-grade tumors as no low-grade cases experienced recurrence. For patients with intermediate- to high-grade tumors, there is potential for local recurrence, distant metastasis, and death, and therefore surveillance following treatment is encouraged.

Trichoblastoma of the Lower Eyelid

Purpose: We report an unusual trichoblastoma of the lower eyelid.Case summary: A 74-year-old male presented with an asymptomatic 0.6 × 0.4-cm left lower eyelid tumor that had developed 3 years prior. On histological examination, a 0.3 × 0.3-cm intradermal tumor that was not connected to the epidermis was observed. The tumor was multilobular, and was surrounded by fibrous stroma and connective tissue. The tumor lobules consisted of basaloid cells, and a few keratinizing cysts were evident. These features are consistent with a trichoblastoma. We observed no recurrence at 1 year after complete resection.Conclusions: Trichoblastoma (a rare, benign skin tumor) is histologically similar to basal cell carcinoma but the prognosis is excellent after complete resection. Trichoblastoma should be considered during the differential diagnosis of basal cell carcinoma of the eyelids.

EPIDERMAL INCLUSION CYST - A REPORT & REVIEW ON ITS DIAGNOSTIC APPROACH

Epidermoid cysts are common benign intradermal or subcutaneous tumors. Their prevalence is 7% in head and neck patient and 1.6% within the oral cavity. We present the case of a 30 year old male with a solitary epidermoid cyst on the right supra-orbital region which was asymptomatic but aesthetics being his primary concern. Clinical examination was performed and decided upon initial investigation with FNAC followed by surgical enucleation of the lesion. Histopathological findings of the excised lesion revealed that it was an epidermoid cyst. From the surgical point of view, they have a very good prognosis and plus it is a nonaggressive lesion. Even Though this is the classical site for occurrence of epidermoid cysts the removal of these cysts is of great concern as it can cause social stigma, aesthetic and functional impairment.

Abstract 4182: Intratumoral treatment with VAX014 facilitates in situ immunization leading to systemic antitumor responses that enhance systemic immune checkpoint blockade

Abstract Intratumorally (i.t.) delivered immunotherapies stimulate in situ immunization by promoting local tumor inflammation, immune cell recruitment, and the generation of systemic antitumor lymphocytic responses. Clinical studies indicate combining i.t. immunotherapy with systemic immune checkpoint blockade (ICB) enhances durable complete response rates (CRR) in treated and distal untreated tumors (abscopal effect), although the number of i.t. therapies tested in this setting remains is limited. The purpose of this study was to evaluate and characterize the immunotherapeutic activity of VAX014, a novel clinical-stage bacterial minicell-based oncolytic agent, following i.t. delivery in two syngeneic murine solid tumor models. The nonimmunogenic B16F10 melanoma and immunogenic MB49 urothelial carcinoma models were used to establish single agent activity of VAX014 in mice bearing a single intradermal (i.d.) tumor, followed by an evaluation of synergy between VAX014 and ICB combinations (CTLA-4 ± PD-1) in mice bearing dual bilateral i.d. B16F10 tumors where only one tumor is treated with VAX014.Weekly i.t. treatment with VAX014 led to significant CD8+ T cell mediated antitumor activity in single i.d. tumor models. A significant reduction in tumor growth rate was observed in all mice (p< 0.0001, 38/38 for B16F10; p< 0.0001, 12/12 for MB49) which ultimately resulted in a durable CRR in 58.0% and 100% in the B16F10 and MB49 models, respectively. B16F10 tumors actively responding to i.t. treatment with VAX014 had a significant increase in CD8+ T cells (p= 0.0004) with a concomitant decrease in CD11b+ myeloid cells (p= 0.039) even after a single i.t. dose of VAX014. VAX014-mediated antitumor activity was specific to the treated tumor type as determined by cytotoxic T lymphocyte assay against the treated tumor cell line versus haplotype matched control target cells (p= 0.0015). Using a bilateral variation of each model, VAX014 exhibited a significant abscopal effect in the immunogenic MB49 model (22.0% CRR and ↓ tumor growth rate in untreated tumors, p= 0.009), an effect that was less pronounced in the nonimmunogenic B16F10 model. Addition of systemic CTLA-4 ICB significantly enhanced abscopal activity in the bilateral B16F10 model whereas PD-1 ICB did not. Remarkably, tripartite combination of VAX014 with CTLA-4/PD-1 ICB resulted in a significant reduction in treated (p< 0.0001) and distal untreated B16F10 tumors (p< 0.0012) as well as an enhanced survival advantage versus CTLA-4/PD-1 ICB combination (p= 0.03). Survival was associated with an improved CRR of both tumors (50.0% vs. 20.0%).In conclusion, these preclinical data provide scientific rationale and set the foundation for i.t. treatment with VAX014 in patients with injectable tumors refractory to standard treatment options and may be particularly effective when combined with systemic ICB. Citation Format: Shingo Tsuji, Katherine A. Reil, Kinsey L. Nelson, Kathleen L. McGuire, Matthew J. Giacalone. Intratumoral treatment with VAX014 facilitates in situ immunization leading to systemic antitumor responses that enhance systemic immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4182.

Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer.

Emerging clinical evidence indicates that the combination of local administration of immunotherapy with systemic immune-checkpoint blockade targeting the PD-1/PD-L1 pathway improves response rates in select solid tumor indications; however, limited clinical experience with this approach exists in advanced bladder cancer patients. VAX014 is a novel bacterial minicell-based, integrin-targeted oncolytic agent undergoing clinical investigation for intravesical (IVE) treatment of nonmuscle-invasive bladder cancer. Here, we demonstrated that the antitumor activity of VAX014 following IVE administration was dependent upon CD4+ and CD8+ T cells in two syngeneic orthotopic bladder tumor models (MB49 and MBT-2). PD-L1 upregulation was found to be an acquired immune-resistance mechanism in the MB49 model, and the combination of VAX014 with systemic PD-L1 blockade resulted in a significant improvement in bladder tumor clearance rates and development of protective antitumor immunologic memory. Combination treatment also led to enhanced systemic antitumor immune responses capable of clearing distal intradermal tumors and controlling pulmonary metastasis. Distal tumors actively responding to combination therapy demonstrated a phenotypic shift from regulatory T cell to Th1 in intratumoral CD4+ T cells, which was accompanied by a higher percentage of activated CD8+ T cells and higher IFNγ. Finally, VAX014's target integrins α3β1 and α5β1 were overexpressed in tumor biopsies from advanced-stage bladder cancer patients, as well as in both the MB49 and MBT-2 orthotopic mouse models of bladder cancer. These collective findings provide a rationale for the clinical investigation of VAX014 and systemic PD-1/PD-L1 blockade in advanced-stage bladder cancer.

Сongenital infantile fibrosarcoma with a non-canonical TPM3-NTRK1 fusion transcript: a case report and a literature review

Infantile fibrosarcoma (IFS) is a rare malignant soft tissue tumor characterized by local invasion, a low rate of distant metastasis (1–13%), and manifestation during the first years of life. Overall survival rates range from 89 to 94%, event-free survival rates – from 81 to 84%. Classic IFS is characterized by t(12;15)(p13;q25) translocation resulting in the formation of the ETV6-NTRK3 fusion transcript. However, over the past few years, there have been numerous reports of IFS-like tumors with non-canonical genetic aberrations (BRAF, NTRK1, MET genes) whose prognosis is less predictable. Here we report a rare case of congenital IFS with involvement of subcutaneous fat, indolent course and a non-canonical TPM3-NTRK1 fusion transcript identified in the soft tissue and intradermal tumor components with different histological features. The patient’s parents gave their consent to the use of their child’s data, including photographs, for research purposes and in publications. The literature review explores modern algorithms for the diagnosis and treatment of IFS in children, including the use of new therapies, such as tropomyosin receptor kinase inhibitors, as well as algorithms for detecting NTRK1, NTRK2, NTRK3 gene rearrangements in solid neoplasms in general, and soft tissue tumors in particular.

Clinicopathological study of cutaneous adnexal cyst with some unusual presentation

Background: Cutaneous cysts are slow-growing benign intradermal or subcutaneous tumors that occur commonly in the hair-bearing areas of the body. Sometimes they occur in non-hair-bearing areas due to traumatic implantation of the epidermis. The present study was conducted to determine the prevalence of various cutaneous cysts in our hospital set up, to record an unusual presentation and clinico-histopathological correlation. Material and methods: This is a hospital-based retrospective analysis of cutaneous cysts conducted for 6 months. All skin biopsies diagnosed under the category of the cutaneous cyst were included in the study. Histopathology slides stained with Hematoxylin and eosin were analyzed. Results: A total of 185 cases of cutaneous cysts were included in the study. The most frequent cutaneous cyst was an epidermal cyst (75.13%) followed by a dermoid cyst (18.38%). Epidermal cysts occurred at unusual locations of the palm, lower extremity, and breast. Unusual locations of the dermoid cyst were the floor of the mouth, gluteal region, scalp, and back. Two cases of scrotal epidermal cysts were associated with idiopathic calcinosis of the scrotum. In one case each proliferating trichilemmal cyst and apocrine hidrocystoma occurred over the unusual location of the back while cysticercosis occurred at an unusual location of the scalp. Inflammatory changes were found in 27 cases (14.59%) of cutaneous cysts. An overall clinicopathological correlation was seen in 91.89% (170/185) cases of cutaneous cysts Conclusions: Unusual clinical presentation of cutaneous cysts can pose a diagnostic dilemma. Definitive diagnosis in such cases is possible by histopathological analysis.

Porocarcinoma of the Eyelid Presenting as an Umbilicated Lesion.

A 29-year-old woman presented with a 2.5-mm umbilicated flesh-colored nodule of the right upper eyelid margin that was considered to represent a basal cell carcinoma, keratoacanthoma or molluscum contagiosum (A). Excisional biopsy displayed an intradermal tumor comprised of jaggedly infiltrating cords of cells characterized by enlarged hyperchromatic nuclei and small amounts of eosinophilic cytoplasm (B, C) with ductal differentiation (arrow) (D). Tumor was present on the surgical margins and was cleared by Mohs micrographic surgery. Histologic diagnosis is usually based on morphology but may be confirmed by immunohistochemistry. Immunostains showed characteristic focal reactivity for cytokeratins 19 and 7 (E, F), CD117 (G), and epithelial membrane antigen (H). Ki67 displayed a brisk proliferation rate. No distant metastases were detected by MRI examinations of the face, neck, and lung over 10 months follow-up.Porocarcinomas are unusual cutaneous tumors developing from eccrine sweat glands. Rarely occurring on the eyelid, the 6 previously reported cases all displayed larger lesions than the current case without distinguishing characteristics. None recurred, compared with lesions elsewhere on the integument showing 20% local recurrence, 20% lymph node involvement, and 10% metastasis to solid organs. The better prognosis for eyelid lesions may reflect earlier detection and smaller size.

Metronomic Photodynamic Therapy Using an Implantable Electronic Device, a Preclinical Experiment for Introducing Phototherapy into Cancer Treatments in Internal Organs

Introduction: In a modern aging society, people expect high-quality cancer treatment but don't want to impair their quality of life. These trends made us imagine the needs of a novel cancer treatment system. We intend to develop a less invasive, low-cost cancer treatment system by using an implantable electronic device and introducing photodynamic therapy (PDT) into the treatment of malignancies in internal organs, including HBP area. Methods: We combined wirelessly powered fingernail-size LED chip, and tissue adhesive biomaterials to develop suture-free, tissue-adhesive, wirelessly powered LED devices. As a preclinical experiment, we investigated the anti-tumor effect of low-power and long-term photodynamic therapy, termed metronomic PDT, by using this device for the mouse cancer model. The device was implanted subcutaneously beneath intradermal tumors on the back of mice, and the mice could move freely in the cage placed on the antenna board that enables continuous illumination on the tumor. During the ten days of treatment, a photosensitizer (photofrin) was administered intravenously for two times. Results: The mice receiving mPDT showed significant growth suppression of the tumor when compared to the control mice. There was no adverse reaction in the surrounding normal tissues of the treatment group. Also, this experiment showed the effectiveness of green light, which has yet to be used clinically in PDT for the treatment of solid tumors. Conclusions: Metronomic PDT using implantable optoelectronic devices can be applied safely into the treatment of HBP malignancies as a low cost and less invasive local treatment method.

Metronomic photodynamic therapy using an implantable LED device and orally administered 5-aminolevulinic acid

Metronomic photodynamic therapy (mPDT) is a form of PDT that induces cancer cell death by intermittent continuous irradiation with a relatively weak power of light for a long duration (several days). We previously developed a wirelessly powered, fully implantable LED device and reported a significant anti-tumor effect of mPDT. Considering application in clinical practice, the method used for repeated administrations of photosensitizers required for mPDT should not have a high patient burden such as the burden of transvenous administration. Therefore, in this study, we selected 5-aminolevulinic acid (ALA), which can be administered orally, as a photosensitizer, and we studied the antitumor effects of mPDT. In mice with intradermal tumors that were orally administered ALA (200 mg/kg daily for 5 days), the tumor in each mouse was simultaneously irradiated (8 h/day for 5 days) using a wirelessly powered implantable green LED device (532 nm, 0.05 mW). Tumor growth in the mPDT-treated mice was suppressed by about half compared to that in untreated mice. The results showed that mPDT using the wirelessly powered implantable LED device exerted an antitumor effect even with the use of orally administered ALA, and this treatment scheme can reduce the burden of photosensitizer administration for a patient.

A Fully-Human Armored BCMA CAR Boosts Function of CD4+ CAR-T Cells and Resists TGF-β Suppression in Pre-Clinical Models of Multiple Myeloma

The B-cell maturation antigen (BCMA) is an immunotherapy target selectively expressed on multiple myeloma cells (MM). Despite recent success of experimental BCMA CAR-T cell therapy, clinical remissions in MM are often short, in part due to low persistence of the BCMA CAR-T cells. Additionally, immunosuppressive factors, notably TGF-β, are known to be elevated in the peripheral blood and tumor microenvironment in the bone marrow of MM patients, potentially contributing to the lack of durability of CAR-T cell therapy. We aimed to develop a fully-human BCMA CAR with long-term persistence and functional resistance to the suppressive effects of TGF-β. Initially, two fully human single chain variable fragments (scFv) specific for BCMA, derived by biopanning of a yeast display human scFv library, were characterized in a CAR format. Each of the two scFvs was cloned into a lentiviral vector CAR backbone, comprised of the CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ signaling domain, and termed BCMA1 and BCMA2, respectively. BCMA2 and BCMA1 T cells, generated by lentiviral vector transduction of primary human T cells, exhibited high CAR expression at multiplicities of infection 10 to 40 (BCMA1: 64-81%; BCMA2: 84-94%), and consistently demonstrated potent cytotoxicity in an overnight co-culture with BCMA+ MM cell lines RPMI-8226 and MM1.S, but not the BCMA- 293T cells. BCMA2 CAR showed robust proliferative capacity in response to repeated, long-term exposure to MM1.S target cells, concordant with the sustained CD4 T-cell subset and high IL-2 production during the course of repeated exposure to target cells. This is in contrast to BCMA1, that showed precipitous decrease in CD4+ T-cell subset upon co-culture with MM cells, resulting in a CAR T-cell population dominated by CD8+ T-cells. Furthermore, the BCMA2 demonstrated prolonged potency in clearing tumor cells compared to BCMA1, even after continuous 20-day exposure to target cells. This was further confirmed in a mouse intradermal RPMI-8226 xenograft tumor model, in which infusion of BCMA2 T-cells resulted in the rapid and complete eradication of tumors, while BCMA1 showed a slower decline in tumor burden. To further improve the efficacy of the BCMA2, specifically within the TGF-β-rich immunosuppressive tumor microenvironment, we developed an armored BCMA2 CAR variant, which co-expresses the dominant negative TGF-β RII bicistronically via a 2A sequence (BCMA2-TbnegCAR). In a 10 day-long co-culture assay with MM1.S targets in the presence of spiked 10 ng/ml TGF-β, BCMA2-TbnegCAR retained high proliferative capacity and potent cytotoxicity, while the unarmored BCMA2 had diminished proliferation and substantially reduced cytokine and granzyme B production. Consistently, in the in vivo intradermal tumor model that utilizes RPMI-8226, which is a MM cell line that endogenously produces TGF-β, we observed that BCMA2-TbnegCAR treatment resulted in higher T-cell counts in the tumors and an earlier decrease of tumor burden compared to infusion with BCMA2, further demonstrating the increased efficacy and potency of the BCMA2-TbnegCAR. In conclusion, we have designed and characterized a new fully-human BCMA2-TbnegCAR, with a novel scFv that exhibits robust proliferative capacity and persistent cytotoxicity, and remains functionally resistant to the immunosuppressive effects of TGF-β. This novel BCMA CAR can potentially improve the effectiveness and durability of the current BCMA CAR-T cell therapy. Disclosures Alabanza: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Vu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Wu:Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

AVIDIO as a novel oncolytic immunotherapy platform for the treatment of colorectal cancer.

e15210 Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.

Tissue-resident memory CD8+ T cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells

Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors. However, whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear. Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs. Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs. These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope. Moreover, analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival. This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs, thereby strengthening anti-tumor immunity.

149 Histologic, immunohistochemical, and molecular features of melanocytic BAP1-mutated atypical intradermal tumors (MBAIT)

Germline BAP1 mutations predispose patients to cutaneous and uveal melanoma, mesothelioma, kidney cancer, and basal cell carcinoma. Patients with BAP1 cancer predisposition syndrome often develop melanocytic BAP1-mutated atypical intradermal tumors (MBAITs) starting at a young age, which resemble atypical Spitz tumors histologically and are characterized by loss of BAP1 protein. Conservative re-excision is often recommended for these lesions, but their malignant potential remains uncertain. This study investigates the clinical, histologic, immunohistochemical and molecular features of 10 MBAITs. Study cases were collected from three patients who ranged in age from 7 to 34. Lesions were biopsied from the head and neck (N=7, 70%), upper extremities (N=2, 20%), and back (N=1, 10%). The percentage of melanocytes demonstrating loss of BAP1 nuclear expression ranged from 50-100% with 7 cases demonstrating more prominent loss of BAP1 expression in the deeper aspect of the lesion. The percentage of melanocytes expressing p16 ranged from 25% to 100%. Ki-67 nuclear positivity was 5% or less. There was loss of HMB45 expression in melanocytes with increasing dermal depth in 2 cases (immunostain not performed in 8 cases). FISH analysis for one MBAIT detected signals within normal limits for RREB1, cMYC, CDKN2A, and CCND1. In conclusion, MBAIT tumors demonstrate a reassuring immunohistochemical phenotype in the majority of cases including low-Ki67 positivity, loss of HMB45 expression with increasing dermal depth, and retention of p16 expression in a subset of the lesional cells. FISH analysis was also reassuring in 1 case. Further reassuring is that none of the lesions demonstrated local recurrence or distant metastasis with 9 of 10 lesions having at least 1 year follow-up. Whole exome sequencing is currently being performed for the MBAITs in this study to determine if they harbor genetic alterations that may suggest more aggressive behavior.

NIR laser pointer for in vivo photothermal therapy of murine LM3 tumor using intratumoral China ink as a photothermal agent.

The photothermal effect is one of the most promising photonic procedures currently under development to successfully treat several clinical disorders, none the least some kinds of cancer. At present, this field is undergoing a renewed interest due to advances in both photothermal materials and better-suited light sources. However, scientific studies in this area are sometimes hampered by the relative unavailability of state-of-art materials or the complexity of setting up a dedicated optical facility. Here, we present a simple and affordable approach to do research in the photothermal field that relies on a commercial NIR laser pointer and a readily available everyday pigment: China ink. A proof-of-concept study is presented in which mice bearing intradermal LM3 mammary adenocarcinoma tumors were successfully treated in vivo employing China ink and the laser pointer. TUNEL and Ki-67 post-treatment tissue assessment clearly indicates the deleterious action of the photothermal treatment on the tumor. Therefore, the feasibility of this simple approach has been demonstrated, which may inspire other groups to implement simple procedures to further explore the photothermal effect.

Randomized phase II trial of autologous dendritic cell vaccines versus autologous tumor cell vaccines in metastatic melanoma: 5-year follow up and additional analyses

BackgroundDespite improved survival following checkpoint inhibitors, there is still a potential role for anti-cancer therapeutic vaccines. Because of biological heterogeneity and neoantigens resulting from each patient’s mutanome, autologous tumor may be the best source of tumor-associated antigens (TAA) for vaccines. Ex vivo loading of autologous dendritic cells with TAA may be associated with superior clinical outcome compared to injecting irradiated autologous tumor cells. We conducted a randomized phase II trial to compare autologous tumor cell vaccines (TCV) and autologous dendritic cell vaccines (DCV) loaded with autologous TAA.MethodsShort-term autologous tumor cell lines were established from metastatic tumor. Vaccines were admixed with 500 micrograms of GM-CSF and injected weekly for 3 weeks, then at weeks 8, 12,16, 20, and 24. The primary endpoint was overall survival. Secondary objectives were identification of adverse events, and results of delayed type hypersensitivity (DTH) reactions to intradermal tumor cell injections.ResultsForty-two patients were randomized. All were followed from randomization until death or for five years; none were lost to follow-up. DCV was associated with longer survival: median 43.4 versus 20.5 months (95% CI, 18.6 to > 60 versus 9.3 to 32.3 months) and a 70% reduction in the risk of death (hazard ratio = 0.304, p = 0.0053, 95% CI, 0.131 to 0.702). Tumor DTH reactions were neither prognostic nor predictive. The most common treatment-related adverse events were mild to moderate local injection site reactions and flu-like symptoms; but grade 2 treatment-related adverse events were more frequent with TCV. Serum marker analyses at week-0 and week-4 showed that serum markers were similar at baseline in each arm, but differed after vaccination.ConclusionsThis is the only human clinical trial comparing DCV and TCV as platforms for autologous TAA presentation. DCV was associated with minimal toxicity and long-term survival in patients with metastatic melanoma. DTH to autologous tumor cells was neither prognostic for survival nor predictive of benefit for either vaccine.Trial registrationClinical trials.gov NCT00948480 retrospectively registered 28 July 2009.