Exploring novel circulating biomarkers for liver cancer through extracellular vesicle characterization with infrared spectroscopy and plasmonics.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with cirrhosis being a major risk factor. Traditional blood markers like alpha-fetoprotein (AFP) demonstrate limited efficacy in distinguishing between HCC and cirrhosis, underscoring the need for more effective diagnostic methodologies. In this context, extracellular vesicles (EVs) have emerged as promising candidates; however, their practical diagnostic application is restricted by the current lack of label-free methods to accurately profile their molecular content. To address this gap, our study explores the potential of mid-infrared (mid-IR) spectroscopy, both alone and in combination with plasmonic nanostructures, to detect and characterize circulating EVs. RESULTS: EVs were extracted from HCC and cirrhotic patients. Mid-IR spectroscopy in the Attenuated Total Reflection (ATR) mode was utilized to identify potential signatures for patient classification, highlighting significant changes in the Amide I-II region (1475-1700 cm-1). This signature demonstrated diagnostic performance comparable to AFP and surpassed it when the two markers were combined. Further investigations utilized a plasmonic metasurface suitable for ultrasensitive spectroscopy within this spectral range. This device consists of two sets of parallel rod-shaped gold nanoantennas (NAs); the longer NAs produced an intense near-field amplification in the Amide I-II bands, while the shorter NAs were utilized to provide a sharp reflectivity edge at 1800-2200 cm-1 for EV mass-sensing. A clinically relevant subpopulation of EVs was targeted by conjugating NAs with an antibody specific to Epithelial Cell Adhesion Molecule (EpCAM). This methodology enabled the detection of variations in the quantity of EpCAM-presenting EVs and revealed changes in the Amide I-II lineshape. SIGNIFICANCE: The presented results can positively impact the development of novel laboratory methods for the label-free characterization of EVs, based on the combination between mid-IR spectroscopy and plasmonics. Additionally, data obtained by using HCC and cirrhotic subjects as a model system, suggest that this approach could be adapted for monitoring these conditions.

PCR analysis in archival postmortem tissues.

BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.

Molecular analyses of sentinel lymph nodes: an open question.

AIMS: To detect micrometastases in the sentinel lymph nodes (SLN) of melanoma patients the authors analysed 52 lymph nodes (47 SLNs and five non-sentinel) and 17 corresponding primary skin melanomas using reverse transcriptase-polymerase chain reaction assays in paraffin-embedded tissues to detect the mRNAs of tyrosinase, MAGE1, MAGE3, MIA, MART-1 and mRNA coding for telomerase catalytic component. RESULTS: Our data show that the use of molecular markers for melanoma micrometastases detection in SLN is still in a very preliminary stage. In comparing the molecular analysis results with the pathological staging we did not find any evident correlation with the expression of the analysed genes in SLN. There are no data for judging the prognostic significance of the detection of circulating tumour cells in patients without clinically recognizable metastases. Despite progress in the field with simultaneous detection of several markers it was assumed that tyrosinase mRNA remains the best target for the detection of metastatic melanoma cells.

Catalytic subunit of telomerase expression is related to RNA component expression.

Telomere length is maintained by the ribonucleoprotein enzyme telomerase. The RNA component of telomerase (hTR) is widespread, and only the expression of the mRNA encoding the catalytic protein subunit (hTRT) is correlated with telomerase activity. We have studied the level of expression of hTR and hTRT in four different models of neoplastic and preneoplastic lesions using the RT-PCR method on RNA extracted from paraffin-embedded human tissues after microdissection. The expression at the mRNA level was compared with the enzymatic activity. Our results suggest that there may be a reciprocal control at the transcriptional level of the expression of hTRT and hTR which in turn is associated with tumor progression.