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Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10−4 ng/μL (seven PCR assays positive), corresponding to 37–42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.

Confirmed diagnosis of alveolar echinococcosis (AE) is based on pathological criteria and molecular evidence. This parasite-borne disease, caused by the cestode Echinococcus multilocularis, sparingly involves humans as a dead-end host. In humans, the parasite mainly colonizes the liver but can colonize any organ and cause atypical forms, often difficult to characterize clinically. Moreover, molecular methods may be suitable to make the diagnosis of AE in cases of atypical forms, extra-hepatic localizations, or immunosuppressed patients. The aim of this study was to determine the most relevant published PCR techniques, for diagnosis of AE in patients and adopt the best strategy for molecular diagnosis depending on the nature of the tested sample. In this study, we evaluated nine end-point PCR assays and one real-time PCR assay (qPCR), targeting mitochondrial genes, using a total of 89 frozen or formalin-fixed paraffin-embedded (FFPE) samples from either 48 AE or 9 cystic echinococcosis patients. Targeted fragment-genes ranged from 84 to 529 bp. Six PCR assays were able to amplify the DNA of 100% of the frozen AE-samples and for one PCR, 69.8% of the FFPE AE-samples. The 16S rrnL PCR (84 bp) was positive in PCR for 77% of the AE samples and in qPCR for 86.5%. The sensitivity of the PCR assays was higher for fresh samples and FFPE samples stored for less than 5 years. The qPCR assay further increased sensitivity for the tested samples, confirming the need for the development of an Echinococcus spp. qPCR to improve the molecular diagnosis of echinococcoses.

Stratification of the prognosis of pancreatic cancer (PDAC) patients treated by surgery is based solely on clinical variables, such as tumor stage and node status. The development of biomarkers of relapse is needed, especially to drive administration of adjuvant therapy in this at-risk population. Our study evaluates the prognostic performance of a CD3- and CD8-based immune score. CD3, CD8 and Foxp3 expression were evaluated on whole slides in two retrospective PDAC cohorts totaling 334 patients. For this study, we developed an immune score to estimate CD3 and CD8 infiltration in both tumor core and invasive margin using computer-guided analysis with QuPath software. Variables were combined in a dichotomous immune score. The association between immune and clinical scores, and both PFS and OS was investigated. We observed that a dichotomous immune score predicts both PFS and OS of localized PDAC. By univariate and multivariate analysis, immune score, tumor grade, adjuvant therapy, lymph node status, and adjuvant chemotherapy administration were associated with PFS and OS. We subsequently associated the PDAC immune score and clinical variables in a combined score. This combined score predicted patient outcomes independently of adjuvant or neoadjuvant treatment, and improved patient prognostic prediction compared to clinical variables or immune score alone. [ABSTRACT FROM AUTHOR]

The natural history of cervical cancer is closely linked to that of high‐risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high‐grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening. [ABSTRACT FROM AUTHOR]

Background: HER2-targeted therapies have recently emerged as an option in the management of metastatic colorectal cancer (mCRC) overexpressing HER2. However, data regarding HER2 status in primary CRC and its corresponding liver metastases are limited, potentially influencing clinical decisions. Therefore, the aim of this study was to compare the HER2 status in primary CRC and paired liver metastases. Methods: Patients with mCRC who were operated from their primary colorectal cancer and their corresponding synchronous or metachronous liver metastases, in the digestive surgery department of Besançon University Hospital, between April 1999 and October 2021, were included. Tissue microarrays were constructed from matched primary CRC and liver metastastic tissue samples. HER2 status was assessed by immunohistochemistry and in situ hybridization according to Valtorta's criteria. Results: A series of 108 paired primary CRC and liver metastases, including a series of multiple liver metastases originating from the same patients (n = 24), were assessed. Among the primary CRC, 89 (82.4%), 17 (15.8%) and 2 (1.8%) cases were scored 0, 1 + and 2 + respectively. In liver metastases, 99 (91.7%), 7 (6.5%) and 2 (1.8%) were scored 0, 1 + and 2, respectively. Overall, there was a 19% discrepancy rate in HER2 status between primary CRC and metastases, which increased to 21% in cases with multiple synchronous or metachronous liver metastases in a given patient. No significant difference was found between metachronous and synchronous metastases regarding the HER2 status (p = 0.237). Conclusions: Our study highlights the temporal and spatial heterogeneity of HER2 status between primary CRC and corresponding liver metastases. These findings raise the question of a sequential evaluation of the HER2 status during disease progression, to provide the most suitable treatment strategy. [ABSTRACT FROM AUTHOR]

Œsogastric cancers can now benefit from a personalized approach targeting the tumor and its microenvironment, mainly relying on tissue biomarkers determination. In the context of immunotherapy, TPS or CPS score assessment and MMR status are mandatory and EBV status might be useful. These parameters can lead to the use of immune check point inhibitors (anti-PD-1 and PD-L1) which recently revolutionized the therapeutic management of oesogastric cancers. Tumor targeting necessarily encompasses HER2 status which impact on trastuzumab use. In addition, other promising targets are available such as claudin 18, FGFR2 and NTRK, which might, for some of them, belong to the future clinical practice. The aim of this article is to describe the validated and promising oesogastric cancer biomarkers, and their assessment. We also highlight the importance of the management of the biopsy material in this era of precision medicine. [ABSTRACT FROM AUTHOR]