Melanoma Brain Research Articles

FSMP-03. INVESTIGATING CO-OPTED ASTROCYTIC METABOLISM IN MELANOMA BRAIN METASTASIS

Melanoma, an aggressive form of skin cancer, frequently metastasizes to the brain. While peripheral melanoma is largely treatable, MBM fail to respond to current therapeutics and is a clear unmet clinical need. Initial clinical symptoms of Melanoma Brain Metastases (MBM) typically include headaches, seizures and other neurological deficits, suggesting that MBM disrupt normal brain functions. One of the major cell types that melanoma encounter and interact with during brain metastasis are astrocytes. Astrocytes, the most abundant cell in the brain, interact with neurons and the vasculature, provide trophic and energetic support to neurons, and regulate local blood flow. Metabolic pathways in astrocytes, particularly the glutamate-glutamine cycle, are essential for the recycling and resupply of neurotransmitters needed to maintain the excitation/inhibition balance. We propose that MBM co-opt astrocytic metabolism, fueling MBM growth, and deplete metabolic intermediates crucial for neuronal activity leading to altered neurologic function. We begin to unravel the metabolic interactions between astrocytes and MBM using novel modeling platforms with genetic and pharmacological tools to manipulate the tumor microenvironment. This project investigates the contribution of astrocytic metabolism to MBM growth. We intend on dissecting the distinct metabolic needs of metastatic brain melanoma in the CNS microenvironment and the subsequent neurological consequences. Completion of this project will provide a platform to study MBM and interaction with the local brain microenvironment. Inhibiting metabolic interactions between melanoma and glial cells may provide new avenue for therapeutic targeting of MBM.

Primary Brain Melanoma in a Pediatric Patient: A Case Report

Patient: Male, 17-year-oldFinal Diagnosis: Primary malignant melanoma of brainSymptoms: Tonic-clonic seizureMedication: —Clinical Procedure: Resection of brain tumorSpecialty: Neurosurgery • Pediatrics and Neonatology • RadiologyObjective:Rare diseaseBackground:Primary malignant melanoma of the brain is a challenging radiological diagnosis and a high index of suspicion is required about patients with the condition. In the pediatric population, only a few cases have been reported in the literature. The purpose of this report was to describe the expected imaging characteristics and the importance of a multidisciplinary approach in the diagnosis of this rare entity.Case Report:A 17-year-old Hispanic male who presented with new-onset tonic-clonic seizures had no focal neurologic deficits on physical examination. An initial computed tomography scan showed a hyperdense, right frontal, parafalcine mass. Brain magnetic resonance imaging was performed and revealed a T1 hyperintense and T2 hypointense, right-frontal-lobe, extra-axial mass with foci of susceptibility. Resection of the mass revealed a lesion that had a dark, pigmented macroscopic appearance. Histopathologic analysis confirmed that it was a primary intracranial malignant melanoma after no primary site was identified on dermatologic and ophthalmologic evaluations.Conclusions:Diagnosing a primary intracranial melanoma with imaging alone is virtually impossible if clinical data and findings from a thorough physical examination are unavailable. Intracranial primary malignant melanoma remains a complex radiological diagnosis that relies on the exclusion of other potentially more common entities and an optimal multidisciplinary approach.

Visualizing Pericyte Mimicry of Angiotropic Melanoma by Direct Labeling of the Angioarchitecture.

In addition to intravascular dissemination, angiotropic melanoma cells have the propensity to spread along the external surface of blood vessels in a pericytic location, or pericytic mimicry. Such continuous migration without intravasation has been termed "extravascular migratory metastasis" or EVMM. In order to visualize this mechanism of tumor propagation, we used a murine brain melanoma model utilizing green fluorescent human melanoma cells and red fluorescent lectin-tagged murine vessels. This model allows the direct microscopic visualization and mapping of the interaction of melanoma cells with the brain vasculature. In this chapter, we describe the methodology of lectin perfusion to label the entire angioarchitecture in conjunction with confocal microscopy imaging to study the pericyte mimicry of the angiotropic GFP+ melanoma cells.

MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies.

Stemness and apoptosis may highlight the dichotomy between regeneration and demise in the complex pathway proceeding from ontogenesis to the end of life. In the last few years, the concept has emerged that the same microRNAs (miRNAs) can be concurrently implicated in both apoptosis-related mechanisms and cell differentiation. Whether the differentiation process gives rise to the architecture of brain areas, any long-lasting perturbation of miRNA expression can be related to the occurrence of neurodevelopmental/neuropathological conditions. Moreover, as a consequence of neural stem cell (NSC) transformation to cancer stem cells (CSCs), the fine modulation of distinct miRNAs becomes necessary. This event implies controlling the expression of pro/anti-apoptotic target genes, which is crucial for the management of neural/neural crest-derived CSCs in brain tumors, neuroblastoma, and melanoma. From a translational point of view, the current progress on the emerging miRNA-based neuropathology therapeutic applications and antitumor strategies will be disclosed and their advantages and shortcomings discussed.

Molecular profiling of melanoma brain metastases compared to primary cutaneous melanoma and to extracranial metastases.

Background: Brain metastases are a significant cause of mortality and morbidity for patients with melanoma. We hypothesize that the development of brain metastases may be explained by molecular heterogeneity between primary cutaneous melanoma (PCM) or extracranial (ECM) and brain (MBM) melanoma metastases.Materials and Methods: We compared next-generation sequencing, tumor mutational burden (TMB), and immunohistochemical staining for PD-L1 expression, among 132 MBM, 745 PCM, and 1190 ECM.Results: The most common genetic alterations among MBM included: BRAF (52.4%), NRAS (26.6%), CDKN2A (23.3%), NF1 (18.9%), TP53 (18%), ARID2 (13.8%), SETD2 (11.9%), and PBRM1 (7.5%). Four genes were found with higher frequency among MBM compared to PCM or ECM: BRAF (52.4% v 40.4% v 40.9%), SETD2 (11.9% v 1.9% v 3.9%), PBRM1 (7.5% v 1.6% v 2.6%), and DICER1 (4.4% v 0.6% v 0.4%). MBM showed higher TMB (p = .04) and higher PD-L1 expression (p = .002), compared to PCM. PD-L1 expression was slightly higher among MBM compared to ECM (p = .042), but there was no difference between TMB (p = .21).Conclusions: Our findings suggest a unique molecular profile for MBM, including higher rates of BRAF mutations, higher TMB and higher PD-L1 expression, and also implicate chromatin remodeling in the pathogenesis of MBM.

Abstract 4440: Bempegaldesleukin (NKTR-214), a CD122 preferential IL-2 pathway agonist, augments the in situ vaccine response to radiation of an extracranial tumor in a murine melanoma model, conferring response at non-radiated tumor sites in the brain

Abstract Brain metastases develop in many patients with advanced metastatic cancers, and this challenging clinical scenario demands improved treatment approaches. We previously observed that an in situ vaccination (ISV) regimen combining local radiation (RT) and intratumoral (IT) injection of the radiated tumor site with interleukin-2 (IL2) resulted in enhanced response to immune checkpoint inhibition (ICI: αCTLA4 Ab) in immunologically cold murine models of melanoma. This ISV treatment resulted in complete response (CR) at both the ISV-targeted tumor and at distant extracranial tumor sites in most mice. However, in a model of melanoma brain metastases, this same ISV provided only a modest improvement in survival with all mice succumbing to progressive tumor burden in the brain (Sriramaneni et al., 2020 AACR submitted abstract). Bempegaldesleukin (NKTR-214, Nektar Therapeutics) is a PEGylated derivative of IL2 that can be administered systemically with minimal toxicity, has a longer half-life (~20 hours), and preferentially activates cytotoxic (CD8+) T cells and NK cells over regulatory T-cells within the tumor microenvironment. We hypothesized that these qualities of NKTR-214 could enhance immune response and efficacy of ISV against brain melanoma metastases, and therefore we tested in combination with local RT targeting an extracranial tumor site. Immunologically cold B78 melanoma tumor cells were implanted into the right flank and the right striatum of the brain in syngeneic C57BL/6 mice. Right flank tumors (150-200 mm3) were irradiated (12 Gy) on treatment day 1. NKTR-214 was administered intravenously on treatment days 6, 15, 24. NKTR-214 combined with RT significantly extended survival in these mice, compared to RT or NKTR-214 alone (p=0.008). Twenty percent of these mice treated with right flank RT + NKTR-214 exhibited durable CR at all tumor sites, whereas no CR was observed in mice treated with RT or NKTR-214 alone. Using this same regimen, we treated mice bearing a right flank B78 melanoma tumor (targeted by local RT) and non-radiated B78 melanoma tumors on the left flank and in the brain. In these mice, we compared the immune response in the brain and left flank tumors following treatment with right flank RT + systemic NKTR-214. Analysis of these tumors by microscopy at treatment day 15 demonstrated significantly increased CD8+ T cell infiltrate and an increased ratio of CD8+ T cells: FOXP3+ regulatory T cells in both the left flank and brain tumors compared to untreated control mice. In contrast to our prior observations with RT + IT-IL2, the combination of RT + NKTR-214 triggered a comparable immune response in both the brain and left flank tumors with respect to the degree of CD8+ T cell infiltration and the ratio of CD8+ effector: FOXP3+ regulatory T cells. These results suggest that a combination of RT + NKTR-214 may elicit an ISV effect that is capable of conferring enhanced anti-tumor immune response against brain metastases, and could potentially abrogate the need for direct brain radiation or surgery. Citation Format: Paul A. Clark, Raghava N. Sriramaneni, Alexander Pieper, Amber M. Bates, Bryce R. Anderson, Wonjong Jin, Justin C. Jagodinsky, Alexander L. Rakhmilevich, Zachary S. Morris. Bempegaldesleukin (NKTR-214), a CD122 preferential IL-2 pathway agonist, augments the in situ vaccine response to radiation of an extracranial tumor in a murine melanoma model, conferring response at non-radiated tumor sites in the brain [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4440.

Abstract 2256: Low dose brain radiotherapy enhances the efficacy of an extracranial in situ vaccine regimen against melanoma brain metastases in a pre-clinical murine model

Abstract Brain metastases develop in >60% of advanced melanoma patients, resulting in considerable morbidity and mortality. We previously showed that a regimen combining radiation (RT) and intra-tumoral (IT) injection of immunocytokine (IC: αGD2 antibody fused to IL2) resulted in enhanced response to immune checkpoint inhibition (ICI: αCTLA4 Ab) in mice bearing immunologically cold murine melanoma tumors. Here, we used the immunologically cold B78 murine melanoma model to investigate the efficacy of this in situ vaccine (ISV) regimen (RT + IT-IC + ICI) in mice bearing melanoma tumors in the brain and at extracranial sites. B78 (GD2+) cells were implanted into the right and left flanks and the right striatum of the brain in syngeneic C57BL/6 mice. Right flank tumors (150-200 mm3) were treated following a previously optimized ISV regimen [RT (12Gy, day 1), IT-IC (days 6-10) and αCTLA4 (days 3, 6, 9)]. ISV eliminated both flank tumors [22/22 complete response (CR)] compared to αCTLA-4 alone (0/8 CR). However, ISV had only modest effect in prolonging survival in mice with a B78 brain melanoma tumor and all mice succumbed to progressive brain tumor burden. Comparing tumors from the left flank and brain of mice treated with ISV to a right flank tumor, qPCR demonstrated no difference in the expression of Mhc-1 or of various checkpoint receptors/ligands (including Ctla4, Tigit, Pd-L1, Lag3). Immunohistochemistry and flow cytometry at day 15 after ISV demonstrated decreased regulatory T cells (Tregs; CD4+, FOXP3+, CD25+) and a higher ratio of CD8+ T cells:Tregs in the left flank tumor compared to the brain tumor. In contrast, these levels did not differ between left flank and brain tumors in untreated mice. Following ISV, CD8+ T cell:Treg ratio at both the left flank and brain tumor sites significantly increased compared to these respective tumor sites in untreated mice. Multiplex profiling of 32 cytokines demonstrated indistinguishable cytokine expression between untreated tumors in the left flank and brain but showed a distinct pattern of response at these locations following right flank ISV with reduced activation of pro-inflammatory cytokines in the brain melanoma tumor. Given the capacity of low dose RT to temporarily deplete Tregs and increase production of pro-inflammatory cytokines, we hypothesized that low dose whole brain (WB) RT (4 Gy x 1) would enhance ISV response of melanoma brain tumors. Addition of WBRT at day 15 after ISV significantly increased survival of mice with a melanoma tumor in the brain compared to ISV or WBRT alone with 2/11 exhibiting durable CR compared to 0/10 with either ISV or WBRT alone. Interestingly, WBRT administered concurrently on day 1 with ISV had no survival benefit compared to ISV alone - suggesting a critical immunomodulatory role for WBRT at day 15. With WBRT + ISV, we observed an increased ratio of CD8+:FOXP3+ T cells in melanoma brain tumors compared to ISV alone. These results suggest that low dose brain RT may enhance the response to an extracranial ISV at immunologically cold brain metastases. Citation Format: Raghava N. Sriramaneni, Paul A. Clark, Amber M. Bates, Bryce R. Anderson, Wonjong Jin, Justin C. Jagodinsky, Alexander L. Rakhmilevich, Zachary S. Morris. Low dose brain radiotherapy enhances the efficacy of an extracranial in situ vaccine regimen against melanoma brain metastases in a pre-clinical murine model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2256.

Verification of 5-Aminolevurinic Radiodynamic Therapy Using a Murine Melanoma Brain Metastasis Model.

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, (“radiodynamic therapy (RDT)”), instead of with 5-ALA and laser beams (“photodynamic therapy”), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.

Treatment monitoring of immunotherapy and targeted therapy using FET PET in patients with melanoma and lung cancer brain metastases: Initial experiences.

e13525 Background: Due to the lack of specificity of contrast-enhanced (CE) MRI, the differentiation of progression from pseudoprogression (PsP) following immunotherapy using checkpoint inhibitors (IT) or targeted therapy (TT) may be challenging, especially when IT or TT is applied in combination with radiotherapy (RT). Similarly, for response assessment of RT plus IT or targeted therapy (TT), the use of CE MRI alone may also be difficult. For problem solving, the integration of advanced imaging methods may add valuable information. Here, we evaluated the value of amino acid PET using O-(2-[18F]fluoroethyl)-L-tyrosine (FET) in comparison to CE MRI for these important clinical situations in patients with brain metastases (BM) secondary to malignant melanoma (MM) and non-small cell lung cancer (NSCLC). Methods: From 2015-2018, we retrospectively identified 31 patients with 74 BM secondary to MM (n = 20 with 42 BM) and NSCLC (n = 11 with 32 BM) who underwent 52 FET PET scans during the course of disease. All patients had RT prior to IT or TT initiation (61%) or RT concurrent to IT or TT (39%). In 13 patients, FET PET was performed for treatment response assessment of IT or TT using baseline and follow-up scans (median time between scans, 4.2 months). In the remaining 18 patients, FET PET was used for the differentiation of progression from PsP related to RT plus IT or TT. In all BM, metabolic activity on FET PET was evaluated by calculation of tumor/brain ratios. FET PET imaging findings were compared to CE MRI and correlated to the clinical follow-up or neuropathological findings after neuroimaging. Results: In 4 of 13 patients (31%), FET PET provided additional information for treatment response evaluation beyond the information provided by CE MRI alone. Furthermore, responding patients on FET PET had a median stable clinical follow-up of 10 months. In 10 of 18 patients (56%) with CE MRI findings suggesting progression, FET PET detected PsP. In 9 of these 10 patients, PsP was confirmed by a median stable clinical follow-up of 11 months. Conclusions: FET PET may add valuable information for treatment monitoring in individual BM patients undergoing RT in combination with IT or TT.

Regeneration Enhances Metastasis: A Novel Role for Neurovascular Signaling in Promoting Melanoma Brain Metastasis.

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions in vitro displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both in vitro and in vivo expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.

Human melanoma brain metastases cell line MUG-Mel1, isolated clones and their detailed characterization

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Susceptibility‐weighted imaging in malignant melanoma brain metastasis

The value of cerebral susceptibility-weighted imaging (SWI) in malignant melanoma (MM) patients remains controversial and the effect of melanin on SWI is not well understood. To systematically analyze the spectrum of intracerebral findings in MM brain metastases (BM) on SWI and to determine the diagnostic value of SWI. Retrospective. In all, 100 patients with melanoma BM (69 having received radiotherapy [RT] and 31 RT-naïve) and a control group of 100 melanoma patients without BM were included. For detailed analysis of signal characteristics, 175 metastases were studied. Gradient echo SWI sequence at 1.5, 3.0, and 9.4 T. Signal characteristics from melanotic and amelanotic BMs on SWI with a focus on blooming artifacts were analyzed, as well as the presence and longitudinal dynamics of isolated SWI blooming artifacts in patients with and without BM. Chi-squared and Student's t-test were used for contingency table measures and group data of signal and clinical characteristics, respectively. Melanotic and amelanotic metastases did not show significant differences of SWI blooming artifacts (38% vs. 43%, P = 0.61). Most metastases without an initial SWI artifact developed a signal dropout during follow-up (80%; 65/81). Isolated SWI artifacts were detected more frequently in patients with BM (20 vs. 9, P = 0.03), of which the majority were found in patients who had received RT (17 vs. 3, P = 0.08). None of these isolated SWI blooming artifacts turned into overt metastases over time (median follow-up: 8.5 months). Similar findings persisted as remnants of successfully treated metastases (88%; 7/8). We conclude that SWI provides little additional diagnostic benefit over standard T1 -weighted imaging, as melanin content alone does not cause diagnostically relevant SWI blooming. Signal transition of SWI may rather indicate secondary phenomena like microbleeding and/or metal scavenging. Our results suggest that isolated SWI artifacts do not constitute vital tumor tissue but represent unspecific microbleedings, RT-related parenchymal changes or posttherapeutic remnants of former metastatic lesions. 3 Technical Efficacy Stage: 5 J. Magn. Reson. Imaging 2019;50:1251-1259.

Multiple massive intratumoral hemorrhages of metastatic brain melanoma after ventriculoperitoneal shunt

A 50-year-old male presented with sudden-onset headache, diplopia, and unsteadiness due to a posterior fossa hemorrhagic melanoma causing hydrocephalus. Computed tomography of the brain showed a 2.3 cm × 2.0 cm hemorrhagic tumor in the cerebellar vermis and a concomitant fourth ventricular hemorrhage. He underwent removal of the tumor and then ventriculoperitoneal shunt insertion for hydrocephalus. Because of his widespread disease, the patient died 2 weeks later from multiple massive intratumoral hemorrhages. To avoid this unfortunate consequence, we discuss the mechanism and suggest methods for treatment improvement. The variable we could control carefully was the opening pressure of the shunt and the flow of cerebrospinal fluid drainage. Setting a higher opening pressure and changing the pressure more slowly should be considered. To achieve this goal, the use of a programmable valve and anti-siphon system should strongly be recommended in these high-risk populations with extremely high bleeding tendency.

A case of long-term disease control in a patient with disseminated melanoma with synchronous detection of multiple brain metastases

The article presents clinical trials data of melanoma with brain metastasis and a clinical case of long-term survival of a patient with disseminated skin melanoma and synchronous multiple metastatic brain damage by controlling a tumor disease using modern antitumor treatment, including targeted therapy, immunotherapy, radiation therapy, radiosurgery and neurosurgery (at the stage of diagnosis). The total duration of life after detection of the disease is 22 months, with an expected 2-3 months.

Abstract 5329: Telomerase reverse transcriptase promoter methylation in metastatic melanoma

Abstract Aberrant methylation of a particular region of the Telomerase Reverse Transcriptase (TERT) promoter, termed the upstream of transcription start site (UTSS), is associated with increased TERT expression and may be useful for differentiating malignant tumors, such as childhood brain tumors, prostate cancer, gastric cancer and melanoma, from their benign/low-grade histologic mimics. However, it is unclear whether UTSS methylation is actually responsible for transcription or is only associated with it. The purpose of this study was to test the hypothesis that UTSS methylation is an activating mechanism for transcription of this gene in melanoma. Since the majority of melanomas express TERT mRNA, we predicted that i) the majority of these samples would have some detectable aberration in the TERT promoter that might account for expression and ii) UTSS methylation would preferentially occur in samples which lacked other recurrent TERT promoter aberrations (point mutation or rearrangement). To test this prediction, we used a cohort of 57 adult melanoma samples to catalog the presence of UTSS methylation (by high-throughput bisulfite-sequencing), promoter mutation (by Sanger sequencing) and/or promoter rearrangement (by fluorescence in situ hybridization) in each case. 5 cases of atypical Spitz tumors and congenital nevi served as negative controls, since these do not express TERT mRNA and were therefore not expected to have such aberrations. As predicted, 9 of the 10 melanoma samples which lacked promoter mutations or rearrangement had a high level of UTSS methylation. Unexpectedly, UTSS methylation sometimes overlapped with the presence of point mutations (9 of 40 cases) or promoter rearrangement (3 of 5 cases). None of the negative control samples expressed TERT mRNA or harbored any promoter aberrations. We next predicted that, in samples with methylated UTSS, the actively transcribed TERT allele(s) would harbor the methylated UTSS while the transcriptionally silent allele(s) would not. Since methylation analysis of clinical samples is complicated by the presence of unmethylated UTSS from contaminating normal tissue, we tested this prediction in two relatively pure cancer cell lines. Although only one TERT allele was transcribed in each cell line, as measured by digital droplet PCR, we found that in each case both the transcriptionally active and transcriptionally silent alleles had methylated UTSS. These results suggest that UTSS methylation is not sufficient for TERT upregulation, although it may still be useful as a biomarker for diagnosing melanoma and other cancers. Future studies will be aimed to determine if methylation of other regions of the TERT promoter marks the active allele in samples lacking mutations or rearrangement. Citation Format: Sumit Borah, Seung J. Lee, Yiping Fan, Raymond L. Barnhill, Reinhard Dummer, John M. Kirkwood, Armita Bahrami. Telomerase reverse transcriptase promoter methylation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5329.

Methylation among members of the PI3K/AKT/mTOR pathway in melanoma brain and extracerebral metastases.

e21515Background: Around 75% of stage IV melanoma patients develop brain metastases during the course of their disease. In recent years, effective therapy options became available for treatment of ...

Safety and efficacy of combining pembrolizmumab and dose escalation/fraction de-escalation SRS for melanoma and NSCLC brain metastasis: Preliminary results from arm a of a prospective pilot trial.

e21542Background: Brain metastases occur in 15–40% of patients (pts) with systemic cancers, such as lung and melanoma. The standard of care for brain metastases is radiation therapy. Methods to min...

Abstract P3-07-08: Temozolomide as a targeted therapy strategy for triple negative breast cancer

Abstract BACKGROUND: Approximately 15% of newly diagnosed breast cancers are classified as triple negative (TNBC). TNBCs are considered more aggressive and have a worse prognosis as no targeted therapies are currently available. These tumors are routinely treated with chemotherapy agents with only modest proven efficacy. Temozolomide (TMZ) is an oral chemotherapy agent commonly used for the treatment of brain tumors and melanoma. TMZ is an alkylating agent, and its therapeutic benefit depends on its ability to alkylate/methylate DNA, most commonly at the N-7 or O-6 positions of guanine residues. This process leads to DNA damage and subsequently triggers cell death. Cells that express the enzyme O6-Methylguanine-DNA Methyltransferase (MGMT) are able to repair damage caused by TMZ. Tumors that lack expression of MGMT, owing to methylation of the gene promoter, demonstrate a better response to TMZ treatment as a result of synthetic lethality. It was first reported in 2012 that TNBCs were more likely to be MGMT methylated, which was confirmed by another group that reported up to 64% of wild-type BRCA1 TNBC exhibited MGMT gene methylation. In 2013 it was found that basal-like breast cancers were more likely to be MGMT methylated and linked to larger tumor size. Together these findings suggest that a sub-population of TNBCs lack MGMT expression, due to promoter methylation. Currently, TMZ is not a treatment option for breast cancers given the modest efficacy of TMZ noted in breast cancer clinical trials; however, most of these trials have focused on using this agent to either treat or prevent brain metastases, due to TMZs ability to cross the blood-brain barrier. Importantly, none of these trials investigated MGMT expression or specifically TNBC populations. We hypothesize that TMZ may be a viable and efficacious treatment option for TNBCs that lack MGMT expression, due to promoter methylation. METHODS: We analyzed 12 archival specimens and 4 TNBC cell lines (HTB132, HTB26, HTB126 and HCC1806) for MGMT expression using a qRT-PCR clinical assay available from Calgary Laboratory Services. Additionally, we also looked at MGMT protein expression in the cell lines using Western Blot analysis to confirm the qRT-PCR results. Finally, we performed an in vitro assay with TNBC cell lines to determine cytotoxicity of TMZ. RESULTS: Analysis of the archival specimens found that 33% of samples analyzed had MGMT promoter methylation by qRT-PCR. Additionally, we found that HTB26 and HTB126 cell lines showed MGMT promoter methylation by qRT-pCR analysis. Western Blot analysis confirmed lack of MGMT expression in these two cell lines, and also identified another cell line (HCC1806) lacking MGMT protein that was classified as unmethylated by the qRT-PCR clinical assay. Moreover, our in vitro assay found that two cell lines (HTB26 and HCC1806) showed a noticeable response to treatment with TMZ. Interestingly, HTB126 did not show response to TMZ, suggesting that there may be another putative resistance pathway. CONCLUSIONS: Preliminary findings suggest that TMZ may be a viable targeted treatment option for TNBCs. Currently, we are investigating drug response using in vivo mouse models, as well as investigating synergistic combination therapy options. Citation Format: Kornaga EN, Gratton K, Shi Q, Yang A, Nixon NA, Roldan Urgoiti G, Morris DG. Temozolomide as a targeted therapy strategy for triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-08.

Advanced Survival in Patients with Multiple Irradiations for Brain Melanoma Metastases and Associated Abscopal Effect

Brain metastases are often detected in melanoma patients. Central nervous system (CNS) involvement is usually associated with poor prognosis. We describe a case of a female patient with multiple melanoma brain metastases who survived for 49 months after detection of CNS involvement and the first radiosurgery. She underwent several courses of irradiation (five radiosurgeries, stereotactic hypofractionated radiotherapy, and whole brain radiotherapy), chemotherapy (started in 12 months after the first radiosurgery), and targeted therapy (started 30 months after the first radiosurgery). The clear abscopal effect in the form of disappearance of intradermal lesions was observed after the second radiosurgery without any systemic treatment. We believe that the application of combination of different treatment modalities can significantly prolong survival in certain patients with melanoma brain metastases, and can be recommended even in case of poor prognosis.

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL

Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.