Cutaneous Squamous Cell Carcinoma (cSCC) and its precursors, actinic keratosis and Bowen's disease, are the most common types of keratinocytic skin lesions (KSLs) which account for the majority of non-melanoma skin cancer lethality. Currently, clinical and histopathological criteria are used for the diagnosis, classification and therapeutic intervention of KSLs. However, discrepancies exist between the clinical presentations and histologic analyses of these lesions, making the diagnosis difficult. The identification of biomarkers as companion diagnostics for accurately stratifying KSL types is required to support the paradigm shift in current cancer care to personalized, precision medicine and ameliorate the negative impact of misdiagnoses or delayed diagnoses on patient outcome. Also, it is essential to elaborate on the poorly defined molecular modifications required for the initiation, development and progression of KSLs from normal keratinocytes. The development of non-invasive tools such as liquid biopsy based on molecular analysis of blood samples will complement existing clinical and histopathological parameters, leading to an improvement in patient outcomes.
The proposed project aims to exploit the extraordinary features of extracellular vesicles (EVs) to verify the hypothesis that circulating EVs could transport and transmit specific messenger RNAs (mRNAs) to recipient cells useful as predictive markers of progression from normal to proliferative lesions as well as from low risk, non-metastasizing forms to high risk, highly invasive carcinomas. We will also test the hypothesis that the overexpression of these mRNAs could be due to microRNAs (miRNAs) dysregulation. Finally, we will correlate mRNAs/miRNAs expression with anti-PD1 antibody therapy and patient outcome, proposing them as therapeutic markers.
Currently, the histopathological examination of biopsy is required to confirm a clinical diagnosis of skin lesions. Biopsy, is an invasive procedure that may lead to complications including anaphylaxis, bleeding, wound infection and pain and require a number of different specialized personnel for sample taking, processing and analysis with a turnaround of, at least, a few days [Azimi et al., 2018, 10.1002/prca.201800084]. Also, issues with sample collection and processing affect the diagnosis of these lesions, leading to a current lack of consistency in clinical outcomes due to interobserver disagreement. Imprecise diagnosis of KSLs also has a psychosocial burden on patients. Also, a biopsy may not always be appropriate, particularly if it is located in a cosmetically sensitive site such as the face.
Recently, tape stripping was evaluated as a non-invasive, easy and cheaper method for the collection of stratum corneum samples from the skin lesions [Azimi et al., 2018]. Using tape stripping, the upper layers of the stratum corneum are removed and skin samples stay adherent to the tapes. Unlike paraffin embedded specimens, these samples are prepared immediately following the collection, reducing the sample collection time and minimizing the chance of possible contaminations. Nevertheless, the amount of collected material is the major limiting factor. The ideal solution is to apply the concept of liquid biopsy also to this type of cancer as it is already ongoing for hematologic malignancies. From this point of view, the analysis of EVs-associated mRNA and/or miRNA represent an unevaluable tool, due to the extraordinary feature of these nanoparticles to protect their cargo, precisely including labile nucleic acids as RNAs, from the degradation operated by serum RNAses. The proposed project is aimed to develop this concept in the context of skin malignancies trying to identify putative markers of disease progression from low- to high risk of metastasizing cSCC as well as prognostic markers of anti-PD1 therapy outcome. The target we selected were chosen based on their overexpression in metastatic cSCC rather than on their mutational state thereby allowing their measurement by fast, widespread and cheap standard technique as Real Time RT-PCR rather than time-consuming, not largely accessible and expensive technique as NGS. The same rationale could be applied also to miRNA associated to the regulation thereof that could be quantified not only as EV-associated molecules but also as cfc-miRNAs due to the ability of miRNA/Ago1 complexes to avoid RNAase-mediated degradation.
The obtained results will be also the proof-of-concept that skin malignancies have the ability to subvert their microenvironment through the leakage of EVs and, if these EVs could reach the draining lymph node as well as the blood stream, to settle a favorable ground to the development of metastasis. On the other hand, these EVs could be exploited to detect the possible development of metastasis, thereby representing a promising tool in the early detection thereof.