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Simple Summary: Recurrent non-medullary thyroid cancer (NMTC) is difficult to treat and therapy options are limited. Of the available compounds, serine/threonine kinase (STK) inhibitors are currently widely used. However, this form of targeted therapy is not always effective and additional response-related biological information may improve both accuracy and efficacy. Using in-depth STK activity profiling, in this study, we aimed to determine whether benign and malignant thyroid tumors can be differentiated based on these profiles. In addition, we analyzed the impact of the BRAF V600E-specific inhibitor dabrafenib, as well as the generic RAF inhibitors sorafenib and regorafenib, in a subgroup of BRAF V600E and non-BRAF V600E papillary thyroid carcinomas. We demonstrate that STK activity profiling can differentiate benign from malignant thyroid tumors. Furthermore, the BRAF V600E-specific inhibitor dabrafenib can distinguish BRAF V600E from non-BRAF V600E papillary thyroid carcinomas. We conclude that STK activity profiling is beneficial when the goal is to differentiate benign from malignant thyroid tumors. In addition, this approach aids in the selection of likely effective (novel) kinase inhibitors for treatment of recurrent thyroid and other cancers. Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers. [ABSTRACT FROM AUTHOR]

Abstract Background Previous studies indicate that alterations in Human Leukocyte Antigen (HLA) class I expression are frequent in colorectal tumors. This would suggest serious limitations for immunotherapy-based strategies involving T-cell recognition. Distinct patterns of HLA surface expression might conceal different immune escape mechanisms employed by the tumors and are worth further study. Method We applied four-color multiparameter flow cytometry (FCM), using a large panel of alloantigen-specific anti-HLA-A and -B monoclonal antibodies, to study membranous expression of individual HLA alleles in freshly isolated colorectal cancer cell suspensions from 21 patients. Results Alterations in HLA class I phenotype were observed in 8 (38%) of the 21 tumors and comprised loss of a single A or B alleles in 4 cases, and loss of all four A and B alleles in the other 4 cases. Seven of these 8 tumors were located on the right side of the colon, and those showing loss of both HLA-A and -B membranous expression were all of the MSI-H phenotype. Conclusion FCM allows the discrimination of complex phenotypes related to the expression of HLA class I. The different patterns of HLA class I expression might underlie different tumor behavior and influence the success rate of immunotherapy.

Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere-forming, Aldefluor, and side-population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness-related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/β-catenin activation were evaluated. We show that different cancer stem cell populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly-proliferating populations, overexpress SOX2, and KLF4 stemness-related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β-catenin signaling as evidenced by increased nuclear b-catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor-positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side-population phenotype is correlated with ABCG2 drug-efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. [ABSTRACT FROM AUTHOR]