Pharmacokinetics and safety of LEAD-452, an EGFR-specific 4-1BB-agonistic trimerbody in non-human primates.

LEAD-452 is a humanized bispecific EGFR-targeted 4-1BB-agonistic trimerbody with a unique trimeric configuration compared to other 4-1BB-specific antibodies that are currently in development. Indeed, enhanced tumor-specific costimulation and very remarkable safety and efficacy profiles have been observed in mouse models. Here, we conducted for the first time a preclinical pharmacokinetic and toxicity study in non-human primates (NHP) (Macaca fascicularis). LEAD-452 exhibits comparable binding affinity for human and macaque targets, indicating its pharmacological significance for safety testing across species. The NHP were administered LEAD-452 in a series of ascending doses, ranging from 0.1 mg/kg to 10 mg/kg, and repeated doses up to 20 mg/kg. The administration of LEAD-452 was found to be clinically well tolerated, with no major related adverse effects observed. Furthermore, there have been no reported cases of liver toxicity, thrombocytopenia, and neutropenia, which are commonly associated with treatments using conventional anti-4-1BB IgG-based antibodies. In addition, neither IgM nor IgG-based anti-drug antibodies were detected in serum samples from NHP during the study, regardless of the dose of LEAD-452 administered. These results support the clinical development of LEAD-452 for the treatment of solid tumors.

pTERT C250T mutation: A potential biomarker of poor prognosis in metastatic melanoma.

Melanoma is the most aggressive form of skin cancer and the leading cause of death from cutaneous tumors. Several studies have associated alterations in the TERT promoter region (pTERT) with gene overexpression, aggressiveness and poor prognosis of the disease. The aim of this study was to clarify the role of pTERT molecular status in paired samples of primary melanoma and metastasis using tissue and plasma to establish a correlation with disease progression and survival. A total of 88 FFPE tissue samples from 53 patients with advanced melanoma were analyzed. Of these, 35 had paired samples. We also examined cfDNA samples from plasma of 25 patients. We detected a good correlation between primary tumors and metastases in pTERT mutation and methylation status. We were also able to identify pTERT mutations in plasma samples that correlated with mutational status in tissue samples. Interestingly, the C250T mutation was associated with worse survival and higher TERT mRNA expression, compared to the other most common mutation: C228T. In addition, hyper-methylation of the promoter region seems to be related to the progression of pTERT wild type (WT) patients. These results suggest that TERT gene alterations plays an important role during tumor progression, with the detection of the C250T mutation in tissue and plasma as a potential biomarker of poor prognosis in patients with advanced melanoma.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response.

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Correlation Between Endoglin and Malignant Phenotype in Human Melanoma Cells: Analysis of hsa-mir-214 and hsa-mir-370 in Cells and Their Extracellular Vesicles.

Endoglin (CD105) is an auxiliary receptor of transforming growth factor (TGF)-ß family members that is expressed in human melanomas. It is heterogeneously expressed by primary and metastatic melanoma cells, and endoglin targeting as a therapeutic strategy for melanoma tumors is currently been explored. However, its involvement in tumor development and malignancy is not fully understood. Here, we find that endoglin expression correlates with malignancy of primary melanomas and cultured melanoma cell lines. Next, we have analyzed the effect of ectopic endoglin expression on two miRNAs (hsa-mir-214 and hsa-mir-370), both involved in melanoma tumor progression and endoglin regulation. We show that compared with control cells, overexpression of endoglin in the WM-164 melanoma cell line induces; (i) a significant increase of hsa-mir-214 levels in small extracellular vesicles (EVs) as well as an increased trend in cells; and (ii) significantly lower levels of hsa-mir-370 in the EVs fractions, whereas no significant differences were found in cells. As hsa-mir-214 and hsa-mir-370 are not just involved in melanoma tumor progression, but they can also target endoglin-expressing endothelial cells in the tumor vasculature, these results suggest a complex and differential regulatory mechanism involving the intracellular and extracellular signaling of hsa-mir-214 and hsa-mir-370 in melanoma development and progression.

Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics.

Long-range communication between tumor cells and the lymphatic vasculature defines competency for metastasis in different cancer types, particularly in melanoma. Nevertheless, the discovery of selective blockers of lymphovascular niches has been compromised by the paucity of experimental systems for whole-body analyses of tumor progression. Here, we exploit immunocompetent and immunodeficient mouse models for live imaging of Vegfr3-driven neolymphangiogenesis, as a versatile platform for drug screening in vivo. Spatiotemporal analyses of autochthonous melanomas and patient-derived xenografts identified double-stranded RNA mimics (dsRNA nanoplexes) as potent inhibitors of neolymphangiogenesis, metastasis, and post-surgical disease relapse. Mechanistically, dsRNA nanoplexes were found to exert a rapid dual action in tumor cells and in their associated lymphatic vasculature, involving the transcriptional repression of the lymphatic drivers Midkine and Vegfr3, respectively. This suppressive function was mediated by a cell-autonomous type I interferon signaling and was not shared by FDA-approved antimelanoma treatments. These results reveal an alternative strategy for targeting the tumor cell-lymphatic crosstalk and underscore the power of Vegfr3-lymphoreporters for pharmacological testing in otherwise aggressive cancers.

Periocular Cutaneous Soft-Tissue Giant Cell Tumor in a Pediatric Patient.

ABSTRACT: Giant cell tumor of soft tissue (GCTST) is a rare neoplasm genetically unrelated but histopathologically indistinguishable to its osseous counterpart. Histologically, GCTST is characterized as a multinodular proliferation of bland histiocytoid mononuclear cells intermixed with osteoclast-like giant cells. GCTST most commonly presents as a soft-tissue mass located in the extremities of middle-aged adults. In this report, we describe a case of a dermal GCTST arising in the periocular region of a 3-year-old girl. This is the youngest patient diagnosed with GCTST reported in the literature and is also singular because of its anatomic location: Only a handful of head and neck GCTSTs have been reported to date. Furthermore, GCTST most often presents as a superficial or deep soft-tissue mass and much less commonly as a dermal-based skin tumor, as was our case. On microscopic examination, the resected lesion demonstrated classical features including numerous osteoclast-like giant cells embedded in a background of mononuclear ovoid cells which displayed brisk mitotic activity and were surrounded by variable stromal hemorrhage. Tumor cells presented a vaguely fascicular arrangement. Immunohistochemical profile demonstrated positivity for smooth muscle actin and CD68 and negativity for desmin, myo-D1, S100, HMB-45, Melan-A, p16, and NKIC3. The unusual characteristics of this case emphasize the clinicopathologic heterogeneity of GCTST.

An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity.

PURPOSE: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. EXPERIMENTAL DESIGN: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. RESULTS: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. CONCLUSIONS: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1.

BACKGROUND: FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. METHODS: FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. RESULTS: FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44-4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models. CONCLUSIONS: FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Differences in cutaneous melanoma survival between the 7th and 8th edition of the American Joint Committee on Cancer (AJCC). A multicentric population-based study.

BACKGROUND: The 8th edition of the AJCC manual for melanoma includes many changes leading to major substage migrations, which could lead to important clinical reassessments. OBJECTIVES: To evaluate the differences and prognostic value of the 8th AJCC classification in comparison with the 7th edition. METHODS: Clinical and histopathological data were retrieved from five melanoma referral centers including 7815 melanoma patients diagnosed between January 1998 and December 2018. All patients were reclassified and compared using the 7th and 8th classifications of the AJCC. Sankey plots were used to evaluate the migration of patients between the different versions. The primary outcome was overall survival (OS), and curves based on the Kaplan-Meier method were used to investigate survival differences between the 7th and 8th editions. RESULTS: The number of patients classified as stages IB, IIIA, and IIIB decreased while the patients classified as stages IA and IIIC increased notably. Migration analysis showed that many patients in group I were understaged whereas a significant percentage of patients in group III were upstaged. Indirect OS analysis showed a loss in the linearity in the AJCC 8th edition and the groups tended to overlap. Direct OS analysis between groups and versions of the AJCC showed a better prognosis within the new stage III patients, with no effect on those in stages I and II. CONCLUSION: The 8th AJCC edition represents an important change in the classification of patients. We observe that the main migratory changes occur in stage I and III, that severity linearity is lost and groups overlap, and that a more advanced stage does not mean a worse prognosis.

Three Cases of Clear Cell Hidradenoma With "Benign" Lymph Node Involvement.

ABSTRACT: The malignant counterpart of cutaneous clear cell hidradenoma (CCH), hidradenocarcinoma, is an aggressive neoplasm that may have a fatal outcome. However, some cases of benign looking CCH with isolated lymph node involvement and excellent prognosis have been described. "CCH-like neoplasm of uncertain malignant potential" or "atypical hidradenoma" have been proposed as designations for these lesions. We report 3 cases of CCH with lymph node involvement. Ages ranged from 29 to 51 years old. All cases involved the inguinal lymph nodes: 2 of them presented with an isolated lymph node lesion, and the third case had lymph node and cutaneous involvement following the resection of a previous cutaneous lesion. Imaging studies showed no systemic involvement. None of the lesions exhibited histopathologic features of malignancy. All neoplasms were well circumscribed, had cystic spaces, did not display atypia or necrosis, and had less than 4 mitoses per high power field. No recurrence has been observed at follow-up after resection in all cases. All published cases of CCH with lymph node involvement so far affected a single lymph node in the axillary or inguinal regions, lacked features of malignancy, and had excellent long-term prognosis. Some cases previously reported as hidradenocarcinoma probably fit into this category. Our series adds more evidence to this rare phenomenon of "benign metastasis." Aggressive treatment should be avoided in these cases, and a long-term follow-up is warranted.

Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state.

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.

Melanoma cutáneo asociado a la mutación del gen BRAF en dos hospitales de la ciudad de Guatemala / Cutaneous melanoma associated with the mutation of the BRAF gene in two hospitals from Guatemala City

Los estudios de melanoma en Guatemala han sido pocos y únicamente se ha evaluado el aspecto clínico e histológico. El objetivo del presente estudio fue determinar la proporción de casos de melanoma cutáneo por morfología, inmunohistoquímica y mutación del gen BRAF en pacientes con diagnóstico de melanoma en dos centros de referencia, Instituto de Cancerología e Instituto de Dermatología en Guatemala. El estudio es de tipo descriptivo, retrospectivo, transversal. El tipo de muestreo es no probabilístico, con una muestra por conveniencia de 100 casos de tejidos de piel de pacientes, caracterizados por edad, sexo y localización del tumor. Los estudios moleculares incluyeron la determinación de la mutación de la proteína BRAF, por la técnica de PCR-RT. Los resultados muestran que el sexo más afectado es el femenino (54 %). El grupo etario con mayor número de casos es entre 56-75 años (44 %). El tipo histológico predominante es el melanoma lentiginoso acral (59 %) y la localización más frecuente es en miembro inferior (71 %). No se encontraron casos de melanoma lentigo maligno. La mutación del gen BRAF se encontró en el 6 % de los casos, lo que representa un dato importante para el pronóstico y tratamiento del paciente. Por ser uno de los primeros estudios que incluyen el factor molecular, abre paso a una línea de investigación que permita dar continuidad a los pacientes con melanoma en Guatemala, lo que permitirá determinar factores pronóstico y predictivos, así como tratamientos de los casos en estudio.

Studies of melanoma in Guatemala have been few and only the clinical and histological aspects have been evaluated. The objective of this study was to determine the proportion of cases of cutaneous melanoma by morphology, immunohistochemistry and mutation of the BRAF gene in patients diagnosed with Melanoma in two important reference centers, Institute of Cancerology (Incan) and Institute of Dermatology of Guatemala (Inderma). The study is descriptive, retrospective, transversal. The type of sampling is non-probabilistic, with a convenience sample calculation of 100 cases of patient skin biopsies, characterized by age, sex and tumor anatomic location. Molecular studies included the determination of the BRAF protein mutation by means of the RT-PCR technique. Results show that the most affected sex is the female (54 %). Age group with the highest number of cases is between 56 and 75 years old (44 % of cases). The histological type that predominated is acral lentiginous melanoma (59 %) and the most frequent location is in the lower limb (71 %). No cases of malignant lentigo melanoma were found in the cases studied. The BRAF gene mutation was found in 6 % of the cases, which represents an important data for the prognosis and treatment of the patient. In addition, being one of the first studies that include the molecular factor, it opens the way to a line of research that allows patients with melanoma to continue in Guatemala. This would allow to determine prognostic and predictive factors, as well as treatments of the cases under study.

Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF V600E mutation.

Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.

p62/SQSTM1 Fuels Melanoma Progression by Opposing mRNA Decay of a Selective Set of Pro-metastatic Factors.

Modulators of mRNA stability are not well understood in melanoma, an aggressive tumor with complex changes in the transcriptome. Here we report the ability of p62/SQSTM1 to extend mRNA half-life of a spectrum of pro-metastatic factors. These include FERMT2 and other transcripts with no previous links to melanoma. Transcriptomic, proteomic, and interactomic analyses, combined with validation in clinical biopsies and mouse models, identified a selected set of RNA-binding proteins (RBPs) recruited by p62, with IGF2BP1 as a key partner. This p62-RBP interaction distinguishes melanoma from other tumors where p62 controls autophagy or oxidative stress. The relevance of these data is emphasized by follow-up analyses of patient prognosis revealing p62 and FERMT2 as adverse determinants of disease-free survival.

Mycosis fungoides progression could be regulated by microRNAs.

Differentiating early mycosis fungoides (MF) from inflammatory dermatitis is a challenge. We compare the differential expression profile of early-stage MF samples and benign inflammatory dermatoses using microRNA (miRNA) arrays. 114 miRNAs were found to be dysregulated between these entities. The seven most differentially expressed miRNAs between these two conditions were further analyzed using RT-PCR in two series comprising 38 samples of early MFs and 18 samples of inflammatory dermatitis. A series of 51 paraffin-embedded samples belonging to paired stages of 16 MF patients was also analyzed. MiRNAs 26a, 222, 181a and 146a were differentially expressed between tumoral and inflammatory conditions. Two of these miRNAs (miRNA-181a and miRNA-146a) were significantly deregulated between early and advanced MF stages. Bioinformatic analysis showed FOXP3 expression to be regulated by these miRNAs. Immunohistochemistry revealed the level of FOXP3 expression to be lower in tumoral MFs than in plaque lesions in paraffin-embedded tissue. A functional study confirmed that both miRNAs diminished FOXP3 expression when overexpressed in CTCL cells. The data presented here suggest that the analysis of a restricted number of miRNAs (26a, 222, 181a and 146a) could be sufficient to differentiate tumoral from reactive conditions. Moreover, these miRNAs seem to be involved in MF progression.