Cancer stem cells (CSCs) have been identified and characterized in both hematopoietic and solid tumors. Their existence was first predicted by Virchow and Cohnheim in the 1870s. Later, many studies showed that CSCs can be identified and isolated by their expression of specific cell markers. The significance of CSCs with respect to tumor biology and anti-cancer treatment lies in their ability to maintain quiescence with very slow proliferation, indefinite self-renewal, differentiation, and trans-differentiation such as epithelial–mesenchymal transition (EMT) and its reverse process mesenchymal–epithelial transition (MET). The ability for detachment, migration, extra- and intravasation, invasion and thereby of completing all necessary steps of the metastatic cascade highlights their significance for metastasis. CSCs comprise the cancer cell populations responsible for tumor growth, resistance to therapies and cancer metastasis. In this review, the history of the CSC theory, their identification and characterization and their biology are described. The contribution of the CSC ability to undergo EMT for cancer metastasis is discussed. Recently, novel strategies for drug development have focused on the elimination of the CSCs specifically. The unique functional and molecular properties of CSCs are discussed as possible therapeutic vulnerabilities for the development of novel anti-metastasis treatments. Prospectively, this may provide precise personalized anti-cancer treatments with improved therapeutic efficiency with fewer side effects and leading to better prognosis.

Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic patterns of HPV⁺/p16⁻ or HPV⁻/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using a QuantiGene-Molecular-Profiling-Histology (QG-MPH) assay for identifying transcriptionally active HPV. Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16 ^INK4a mRNA by QG-MPH. p16 ^INK4a mRNA expression was compared with p16 protein expression via immunohistochemistry (p16 IHC). Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. Concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9% of DNA⁺ cases, demonstrating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA⁻/p16 IHC⁻ group (p = 0.001). Specifically, the OS rate was 57.1% (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcomes for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant patterns and the advantages for incorporating the molecular mRNA profiling by QG-MPH to p16 IHC. In conclusion, QG-MPH quantification of E7 and p16 ^INK4a mRNA more precisely identifies truly HPV-driven from non-HPV-driven HNSCC, compared to HPV DNA testing alone or with p16 IHC, which reduces misclassification and provides valuable implications for improved prognosis prediction and therapeutic decision-making.

Background Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic pat-terns of HPV⁺/p16⁻ or HPV⁻/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using mRNA profiling by QuantiGene Molecu-lar-Profiling-Histology (QG-MPH) assay for more precise etiological diagnosis. Methods Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16ᴵᴺᴷ⁴ᵃ mRNA using QG-MPH. p16ᴵᴺᴷ⁴ᵃ mRNA expression was then compared with p16 protein expression by immunohistochemistry (p16 IHC). Results Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. The concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9 % of DNA⁺ cases, demon-strating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA⁻/p16 IHC⁻ group (p = 0.001). Specifi-cally, the OS rate was 57.1 % (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcome for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant categories and the advantages for incorpo-rating the molecular mRNA profiling by QG-MPH to p16 IHC in making therapeutic decision and predicting patient outcomes in HNSCC. Conclusion QG-MPH quantification of E7 and p16ᴵᴺᴷ⁴ᵃ mRNA is more precise in identifying truly HPV-driven HNSCC than HPV DNA testing alone or with p16 IHC, enabling improved prognosis prediction and therapeutic deci-sion-making.