Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) ...against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR
+
.CIK). We set a patient-derived experimental platform. CAR
+
.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR
+
.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR
+
.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR
+
.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR
+
.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR
+
.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities.
We report first evidence of CAR
+
.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR
+
.CIK in clinical trials against high grade STS.
Summary
Background
Gap‐junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte–keratinocyte contact and loss of the intercellular ...junction’s integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions.
Objectives
The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen‐activated protein kinase (MAPK) inhibitor (MAPKi) treatment.
Methods
GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets.
Results
Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF‐mutated vs. BRAF‐wildtype (BRAFWT) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi‐resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR‐335‐5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR‐335‐5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR‐335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR‐335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages.
Conclusions
We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap‐junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.
What is already known about this topic?
GJB5 expression has never been studied in melanoma.
Although there is very limited knowledge about connexins in melanoma, these types of gap‐junction proteins have recently been linked with late stages of tumorigenesis and metastasis and are considered as tumour suppressors.
What does this study add?
This study establishes a significant association between BRAFV600E, metastases and GJB5 downregulation in melanoma.
GJB5 underexpression also correlates with worse patient overall survival. Therefore, the data support a prognostic role for GJB5 in cutaneous melanoma.
What is the translational message?
This study highlights the importance of monitoring the integrity of connexin and junctional proteins during melanomagenesis, providing novel therapeutic target options for melanoma treatment, as well as a novel prognostic biomarker to predict melanoma progression.
Linked Comment: J.E. Fromme and P. Zigrino. Br J Dermatol 2022; 186:13–14.
Plain language summary available online
Sentinel lymph node (SLN)-positive melanoma patients are a heterogeneous group of patients with survival rates ranging from ∼20 to over 80%. No data are reported concerning the role of histological ...regression on survival in stage III melanoma.
The study included 365 patients with positive SLN from two distinct hospitals. The model was developed on patients from 'AOU Città della Salute e della Scienza di Torino', and externally validated on patients from IRCCS of Candiolo. Survival analyses were carried out according to the presence of regression and adjusted for all other prognostic factors.
Among patients followed at 'AOU Città della Salute e della Scienza di Torino' (n=264), the median follow-up time to death or censoring (whatever two events occurred earlier) was 2.7 years since diagnosis (interquartile range: 1.3-5.8). In all, 79 patients died from melanoma and 11 from other causes. Histological regression (n=43) was associated with a better prognosis (sub-HR=0.34, CI 0.12-0.92), whereas the other factors above showed an inverse association. In the external validation, the concordance index was 0.97 at 1 year and decreased to 0.66 at 3 years and to 0.59 at 5 years. Adding histological regression in the prognostic model increased the discriminative ability to 0.75 at 3 years and to 0.62 at 5 years. Finally, using a cutoff of 20% for the risk of death led to a net re-classification improvement of 15 and 11% at 3 and 5 years after diagnosis, respectively.
Histological regression could lead to an improvement in prognostic prediction in patients with stage III-positive SLN melanoma.
Despite improvements in surgery and medical treatments, epithelial ovarian cancer (EOC) remains the most lethal gynaecological malignancy. Aim of this study is to investigate the preclinical ...immunotherapy activity of cytokine-induced killer lymphocytes (CIK) against epithelial ovarian cancers, focusing on platinum-resistant settings. We generated CIK ex vivo starting from human peripheral blood samples (PBMCs) collected from EOC patients. Their antitumor activity was tested in vitro and in vivo against platinum-resistant patient-derived ovarian cancer cells (pdOVCs) and a Patient Derived Xenograft (PDX), respectively. CIK were efficiently generated (48 fold median ex vivo expansion) from EOC patients; pdOVCs lines (n = 9) were successfully generated from metastatic ascites; the expression of CIK target molecules by pdOVC confirmed pre and post treatment in vitro with carboplatin. The results indicate that patient-derived CIK effectively killed autologous pdOVCs in vitro. Such intense activity was maintained against a subset of pdOVC that survived in vitro treatment with carboplatin. Moreover, CIK antitumor activity and tumor homing was confirmed in vivo within an EOC PDX model. Our preliminary data suggest that CIK are active in platinum resistant ovarian cancer models and should be therefore further investigated as a new therapeutic option in this extremely challenging setting.
Purpose
Pathological complete response (pCR) following neoadjuvant chemoradiotherapy or radiotherapy in locally advanced rectal cancer (LARC) is reached in approximately 15–30% of cases, therefore it ...would be useful to assess if pretreatment of
18
F-FDG PET/CT and/or MRI texture features can reliably predict response to neoadjuvant therapy in LARC.
Methods
Fifty-two patients were dichotomized as responder (pR+) or non-responder (pR-) according to their pathological tumor regression grade (TRG) as follows: 22 as pR+ (nine with TRG = 1, 13 with TRG = 2) and 30 as pR- (16 with TRG = 3, 13 with TRG = 4 and 1 with TRG = 5). First-order parameters and 21 second-order texture parameters derived from the Gray-Level Co-Occurrence matrix were extracted from semi-automatically segmented tumors on T2w MRI, ADC maps, and PET/CT acquisitions. The role of each texture feature in predicting pR+ was assessed with monoparametric and multiparametric models.
Results
In the mono-parametric approach, PET homogeneity reached the maximum AUC (0.77; sensitivity = 72.7% and specificity = 76.7%), while PET glycolytic volume and ADC dissimilarity reached the highest sensitivity (both 90.9%). In the multiparametric analysis, a logistic regression model containing six second-order texture features (five from PET and one from T2w MRI) yields the highest predictivity in distinguish between pR+ and pR- patients (AUC = 0.86; sensitivity = 86%, and specificity = 83% at the Youden index).
Conclusions
If preliminary results of this study are confirmed, pretreatment PET and MRI could be useful to personalize patient treatment, e.g., avoiding toxicity of neoadjuvant therapy in patients predicted pR-.
BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great ...variability in patient outcomes.
The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients’ clinical characteristics. Kaplan–Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression.
A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively).
These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.
•In melanoma the response to anti-BRAF targeted therapies is heterogeneous and influenced by several features.•The role of the BRAF VAF as provider of sensitivity to target therapies is debated.•We found that high BRAF VAFs are associated with patient age, melanoma thickness, non-brisk TILs and lymph node metastases.•We proved the independent prognostic value of high BRAF VAFs in melanoma patients treated with targeted therapies.•The quantitative evaluation of BRAF mutations allows stratifying melanoma patients to the BRAF/MEK targeted treatment.
Abstract Aims, patients and methods The umbilical melanoma is rare, and the surgical treatment can create difficulties for both radical excision and plastic reconstruction. Our aims are to present a ...case of primary melanoma of the umbilicus and to discuss the best surgical treatment, as well as review the relevant literature. Results Surgical excision of primary melanoma of the umbilicus must be carried out to reach the peritoneum. Sentinel lymph node biopsy must be carried as well as plastic reconstruction. Conclusion Despite the progress in new medical therapy for melanoma, suitable surgical excision is, at present, the only treatment able to improve patient prognosis. In this report we describe the surgical treatment and plastic reconstruction of a case of umbilical melanoma.
Vasculogenic mimicry, as previously described in aggressive melanoma, is characterized by the de novo generation of intratumoral patterned vascular channels, composed of PAS-positive basement ...membrane in the absence of endothelial cells, providing additional microcirculation, in support to the classic tumoral angiogenesis.
We investigated the immunohistochemical expression of two endothelial markers, CD31 and CD34, in tumoral cells of 60 melanomas (45 primary cutaneous and 15 metastatic) as possible evidence of vasculogenic mimicry. In addition we investigated the relationship between CD31 and CD34 expression and three pathological markers such as Clark's level, and skin ulceration, predictive of melanoma's aggressive behaviour, and mitotic index.
No cases of common melanocytic nevi immunoreacted with CD31 or CD34. Random CD31 immunoreactivity was present in 6% of Clark's level I/II, 50% of Clark's level III and 80% Clark's level IV/V. CD34 was negative in Clark's level I/II but randomly stained the 20% and 55% of level III and IV/V respectively. 66% (10/15) of metastatic melanomas were CD31 positive showing a canalicular immunostaining pattern, conversely CD34 expression was never found. 7/8 cutaneous ulcerated melanomas immunostained for CD31 and 4/8 for CD34. CD31 immunostained 88% high/intermediate MI, and 53% of low MI melanomas. CD34 decorated the 29% of high/intermediate and 38% of low MI melanomas.
CD31 and CD34 immunoreactivity closely parallel both the different morphologic steps of melanocytic tumor progression and the presence of histological parameters related to the aggressive behaviour. Their expression could be related to endothelial transdifferentiation of melanoma cells although a consequent functional role has not been demonstrated yet.
mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was ...identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/γIFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.
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