The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K₂EDTA (EDTA) tubes or cell-free DNA BCT^® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

The aim of this review was to explore the status of urine sampling as a liquid biopsy for noninvasive cancer research by reviewing used preanalytical parameters and protocols. We searched two main health sciences databases, PubMed and Web of Science. From all eligible publications (2010‐2022), information was extracted regarding: (a) study population characteristics, (b) cancer type, (c) urine preanalytics, (d) analyte class, (e) isolation method, (f) detection method, (g) comparator used, (h) biomarker type, (i) conclusion and (j) sensitivity and specificity. The search query identified 7835 records, of which 924 unique publications remained after screening the title, abstract and full text. Our analysis demonstrated that many publications did not report information about the preanalytical parameters of their urine samples, even though several other studies have shown the importance of standardization of sample handling. Interestingly, it was noted that urine is used for many cancer types and not just cancers originating from the urogenital tract. Many different types of relevant analytes have been shown to be found in urine. Additionally, future considerations and recommendations are discussed: (a) the heterogeneous nature of urine, (b) the need for standardized practice protocols and (c) the road toward the clinic. Urine is an emerging liquid biopsy with broad applicability in different analytes and several cancer types. However, standard practice protocols for sample handling and processing would help to elaborate the clinical utility of urine in cancer research, detection and disease monitoring.

The constitutive expression of CD70 has been described in various haematological and solid tumour types. In addition, the co-expression of its receptor in tumours has been demonstrated, mediating tumour cell proliferation. Although CD70 expression is a prerequisite to enrol patients in solid tumour clinical trials using anti-CD70 immunotherapy, there is currently no standardised test to evaluate CD70 expression. These differences in immunohistochemistry (IHC) protocols make it challenging to compare the expression levels that were obtained in different studies, pointing out the need for one uniform methodology. In this retrospective study, over 600 tumour samples from different solid and haematological malignancies were analysed while using one validated IHC method. CD70 and CD27 expression was demonstrated in a broad range of tumour types. In solid tumours, 43% demonstrated CD70 positivity with the highest degree in renal cell carcinoma (79.5%). Kaposi sarcoma showed no CD70 expression on the tumour cells. In lymphoma samples, 58% demonstrated CD70 positivity. Moreover, the co-expression of CD70 and CD27 was observed in 39% of lymphoma samples. These findings highlight the need to further explore anti-CD70 therapies in a broad range of CD70 expressing tumour types and in doing so, implementing one standardised protocol to define CD70 overexpression to use it as a diagnostic tool.

Cancer-associated fibroblasts (CAFs) are involved in the proliferative and invasive behavior of colorectal cancer (CRC). Nonetheless, CAFs represent a heterogeneous population with both cancer-promoting and cancer-restraining actions, lacking specific markers to target them. Expression of the immune checkpoint molecule CD70 is normally limited to cells of the lymphoid lineage. Instead, tumor cells hijack CD70 to facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs). The aim of this study was to explore CD70 expression patterns in CRC, not merely focusing on the tumor cells, but also taking the tumor stromal cells into account. We have analyzed the prognostic value of CD70 expression by immunohistochemistry in CRC specimens and its relationship with well-known fibroblast markers and Tregs. In addition, in vitro experiments were conducted to unravel the role of CD70-positive CAFs on migration and immune escape. We reveal prominent expression of CD70 on a specific subset of CAFs in invasive CRC specimens. Cancer cells show almost no expression of CD70. The presence of CD70-positive CAFs proved to be an independent adverse prognostic marker. Functionally, CD70-positive CAFs stimulated migration and significantly increased the frequency of naturally occurring Tregs. In conclusion, we have identified the expression of CD70 on CAFs as a novel prognostic marker for CRC. We have found evidence of a cross talk between CD70⁺ CAFs and naturally occurring Tregs, paving the way towards immune escape. As such, this study provides a strong rationale for the exploration of CD70-targeting antibodies in CRC.

Prognosis of glioblastoma remains dismal, underscoring the need for novel therapies. Immunotherapy is generating promising results, but requires combination strategies to unlock its full potential. We investigated the immunomodulatory capacities of poly(I:C) on primary human glioblastoma cells and its combinatorial potential with programmed death ligand (PD-L) blockade. In our experiments, poly(I:C) stimulated expression of both PD-L1 and PD-L2 on glioblastoma cells, and a pro-inflammatory secretome, including type I interferons (IFN) and chemokines CXCL9, CXCL10, CCL4 and CCL5. IFN-β was partially responsible for the elevated PD-1 ligand expression on these cells. Moreover, real-time PCR and chloroquine-mediated blocking experiments indicated that poly(I:C) triggered Toll-like receptor 3 to elicit its effect. Cocultures of poly(I:C)-treated glioblastoma cells with peripheral blood mononuclear cells enhanced lymphocytic activation (CD69, IFN-γ) and cytotoxic capacity (CD107a, granzyme B). Additional PD-L1 blockade further propagated immune activation. Besides activating immunity, poly(I:C)-treated glioblastoma cells also doubled the attraction of CD8⁺ T cells, and to a lesser extent CD4⁺ T cells, via a mechanism which included CXCR3 and CCR5 ligands. Our results indicate that by triggering glioblastoma cells, poly(I:C) primes the tumor microenvironment for an immune response. Secreted cytokines allow for immune activation while chemokines attract CD8⁺ T cells to the front, which are postulated as a prerequisite for effective PD-1/PD-L1 blockade. Accordingly, additional blockade of the concurrently elevated tumoral PD-L1 further reinforces the immune activation. In conclusion, our data proposes poly(I:C) treatment combined with PD-L1 blockade to invigorate the immune checkpoint inhibition response in glioblastoma.