The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application

Chemotherapy for human solid tumors in clinical practice is far from satisfactory. Despite the discovery and synthesis of hundreds of thousands of anticancer compounds targeting various crucial units in cancer cell proliferation and metabolism, the fundamental problem is the lack of targeting delivery of these compounds selectively into solid tumor tissue to maintain an effective concentration level for a certain length of time for drug-tumor interaction to execute anticancer activities. The enhanced permeability and retention effect (EPR effect) describes a universal pathophysiological phenomenon and mechanism in which macromolecular compounds such as albumin and other polymer-conjugated drugs beyond certain sizes (above 40 kDa) can progressively accumulate in the tumor vascularized area and thus achieve targeting delivery and retention of anticancer compounds into solid tumor tissue. Targeting therapy via the EPR effect in clinical practice is not always successful since the strength of the EPR effect varies depending on the type and location of tumors, status of blood perfusion in tumors, and the physical-chemical properties of macromolecular anticancer agents. This review highlights the significance of the concept and mechanism of the EPR effect and discusses methods for better utilizing the EPR effect in developing smarter macromolecular nanomedicine to achieve a satisfactory outcome in clinical applications.

Prevalence of inferred clonal hematopoiesis (CH) detected on comprehensive genomic profiling (CGP) of solid tumor tissue or circulating tumor DNA (ctDNA).

3009 Background: The increased use of ctDNA CGP has paralleled increased detection and interest in CH, which can confound CGP results from ctDNA or tissue, and can be associated with hematologic and cardiovascular morbidity. However, paired-depth sequencing of white blood cells (WBC) for confirmation of CH is not widely available. We here study the prevalence of inferred CH (iCH), which refers to incidental detection on routine clinical CGP of variants attributable to CH due to their known CH association and their negligible prevalence in solid tumors. Methods: A database of clinical CGP results was reviewed, including two 324-gene NGS panels for tumor tissue (FoundationOne CDx) and plasma ctDNA (FoundationOne Liquid CDx). Analysis was limited to NSCLC, breast, prostate, colorectal, and pancreatic cancers. iCH was defined as any pathogenic mutation in ASXL1, DNMT3A, and TET2, and prespecified mutations in JAK2, SF3B1, U2AF1, MYD88, IDH2, MPL, CBL. Variant allelic frequency (VAF) > 2% was considered clinically significant and VAF > 10% was considered high risk. Results: 100,905 total cases were studied; median age was 65 for tissue CGP and 68 for ctDNA. iCH was more commonly detected in ctDNA (1468/2891, 51%) than in tissue (9416/97993, 10%). Among cases with any iCH detected, multiple iCH mutations were seen more commonly in ctDNA (640/2891, 22%) than in tissue (987/98014, 1%). Focusing on clinically significant iCH ( > 2% VAF), prevalence remained higher in ctDNA (22%, 637) than in tissue (8%, 7878), while the higher sensitivity of ctDNA testing identified more low level iCH (< 2% VAF, 40% in ctDNA, 2% in tissue). Across cancer types, iCH > 2% was consistently more common in ctDNA (Table). As expected, prevalence of iCH > 2% increased with age (continuous variable, p < 0.001). High risk iCH ( > 10% VAF) was seen in 4% of total cases (most commonly ASXL1, TET2, DNMT3A); 1% of all cases had multiple clinically significant iCH variants ( > 2% VAF). Focusing on a subset of 439 cases with both tissue and ctDNA results (median 1.5 months between samples), 290 iCH mutations were detected in ctDNA (median VAF 1%) but only 38 in tissue (median VAF 9%). Conclusions: Inferred CH is common on somatic CGP of cancer patients, with a high prevalence in ctDNA likely due to the deeper sequencing depth and WBC contamination. For the minority of patients with high VAF iCH, further research is needed to understand whether this might be representative of an occult hematologic condition deserving of further evaluation.[Table: see text]

THE INTRA-ASSAY PRECISION AND TIME STABILITY OF QUANTITATIVE βIII-TUBULIN EXPRESSION ASSESSMENT IN SOLID TUMOR TISSUE

Introduction. The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks.Objective. Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue.Materials and methods. Method validation was carried out by quantitative estimation of βIII-tubulin (TUBB3) expression in single-cell suspensions of non-small-cell lung cancer obtained from surgical tumor samples. Primary antibodies to TUBB3 (ab7751) and secondary DyLight 650-conjugated antibodies (ab98729) were used for immunofluorescent staining. The «Navios» flow cytometer (Beckman Coulter) was used to measure the fluorescence. The validation parameters were assessed by the coefficient of variation calculated as the ratio of standard deviation of TUBB3 level to its mean value.Results. Two parameters were analyzed: intra-assay precision and time stability of the results of the TUBB3 expression assessment. It was demonstrated that the mean coefficients of variation of the marker expression level in the tumor tissue did not exceed 20 % for both parameters. According to recommendations on the analytical validation of methods based on flow cytometry, it proves the validity of the method for these parameters.Conclusions. The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors. The practical value of the time stability of immunofluorescence stain during 24 h storage of a stained cells suspension in the dark at 4 °C was highlighted. It shows the possibility of adjusting the time interval between completion of the analytical study part and flow cytometer measurement.

Liquid Biopsy in Colorectal Carcinoma: Clinical Applications and Challenges

Colorectal carcinoma (CRC) is characterized by wide intratumor heterogeneity with general genomic instability and there is a need for improved diagnostic, prognostic, and therapeutic tools. The liquid biopsy provides a noninvasive route of sample collection for analysis of circulating tumor cells (CTCs) and genomic material, including cell-free DNA (cfDNA), as a complementary biopsy to the solid tumor tissue. The solid biopsy is critical for molecular characterization and diagnosis at the time of collection. The liquid biopsy has the advantage of longitudinal molecular characterization of the disease, which is crucial for precision medicine and patient-oriented treatment. In this review, we provide an overview of CRC and the different methodologies for the detection of CTCs and cfDNA, followed by a discussion on the potential clinical utility of the liquid biopsy in CRC patient care, and lastly, current challenges in the field.

The Inflammatory Milieu of Adamantinomatous Craniopharyngioma and Its Implications for Treatment

Pediatric Adamantinomatous Craniopharyngiomas (ACPs) are histologically benign brain tumors that often follow an aggressive clinical course. Their suprasellar location leaves them in close proximity to critical neurological and vascular structures and often results in significant neuroendocrine morbidity. Current treatment paradigms, involving surgical resection and radiotherapy, confer significant morbidity to patients and there is an obvious need to discover effective and safe alternative treatments. Recent years have witnessed significant efforts to fully detail the genomic, transcriptomic and proteomic make-up of these tumors, in an attempt to identify potential therapeutic targets. These studies have resulted in ever mounting evidence that inflammatory processes and the immune response play a critical role in the pathogenesis of both the solid and cystic portion of ACPs. Several inflammatory and immune markers have been identified in both the cyst fluid and solid tumor tissue of ACP. Due to the existence of effective agents that target them, IL-6 and immune checkpoint inhibitors seem to present the most likely immediate candidates for clinical trials of targeted immune-related therapy in ACP. If effective, such agents may result in a paradigm shift in treatment that ultimately reduces morbidity and results in better outcomes for our patients.