Liquid biopsy in colorectal cancer: Onward and upward.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide. In recent years, liquid biopsy has emerged as one of the most interesting areas of research in oncology, leading to innovative trials and practical changes in all aspects of CRC management. RNAs and cell free DNA (cfDNA) methylation are emerging as promising biomarkers for early diagnosis. Post-surgical circulating tumour DNA (ctDNA) can aid in evaluating minimal residual disease and personalising adjuvant treatment. In rectal cancer, ctDNA could improve response assessment to neoadjuvant therapy and risk stratification, especially in the era of organ-preservation trials. In the advanced setting, ctDNA analysis offers the opportunity to monitor treatment response and identify driver and resistance mutations more comprehensively than traditional tissue analysis, providing prognostic and predictive information. The aim of this review is to provide a detailed overview of the clinical applications and future perspectives of liquid biopsy in CRC.

Sequence self-selection by cyclic phase separation.

The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the dense phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the previously most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for auto-catalytic self-replicating oligonucleotides.

Synergetic collision and space separation in microfluidic chip for efficient affinity-discriminated molecular selection.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

How to improve metastatic pancreatic ductal adenocarcinoma patients' selection: Between clinical trials and the real-world.

As underlined in the minireview by Blomstrand et al, given the poor prognosis and the paucity of data on a therapeutic sequence in pancreatic ductal adenocarcinoma (PDAC), additional randomized controlled trials and real-world evidence studies addressing current and novel regimens are needed. The real-world outcomes of first-line chemotherapy regimens such as FOLFIRINOX and gemcitabine/nab-paclitaxel are thoroughly reviewed and seem to be largely generalizable in a real-world context. Regarding second-line chemotherapy, the key question about the optimal sequence of regimens remains uncertain. Precisely in this setting, it is therefore useful to encourage the implementation of clinical studies that may contribute to the scarcity of data available up to now. We report our experience with a small group of patients treated with second-line liposomal irinotecan (nal-IRI) plus 5-fluorouracil and leucovorin. To improve the treatment of patients affected by PDAC, it is useful to identify subgroups of patients who may benefit from target treatments (e.g., BRCA mutant) and it is also important to focus on any prognostic factors that may affect the survival and treatment of these patients.

Functions of intrinsically disordered proteins through evolutionary lenses.

Protein sequences are the result of an evolutionary process that involves the balancing act of experimenting with novel mutations and selecting out those that have an undesirable functional outcome. In the case of globular proteins, the function relies on a well-defined conformation, therefore, there is a strong evolutionary pressure to preserve the structure. However, different evolutionary rules might apply for the group of intrinsically disordered regions and proteins (IDR/IDPs) that exist as an ensemble of fluctuating conformations. The function of IDRs can directly originate from their disordered state or arise through different types of molecular recognition processes. There is an amazing variety of ways IDRs can carry out their functions, and this is also reflected in their evolutionary properties. In this chapter we give an overview of the different types of evolutionary behavior of disordered proteins and associated functions in normal and disease settings.

Implementing anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer: challenges and future perspectives.

Epidermal growth factor receptor (EGFR) inhibitors are valuable therapeutics in metastatic colorectal cancer (mCRC). Anti-EGFR monoclonal antibodies (MoAbs), such as cetuximab or panitumumab, in combination with chemotherapy are effective treatment options for patients with RAS and BRAF wild-type mCRC. Nevertheless, several issues are still open concerning the optimal use of anti-EGFR drugs in the continuum of care of mCRC. Novel approaches for increasing the efficacy of anti-EGFR therapies include better molecular selection of EGFR-dependent mCRC, intensification of chemotherapy, combination of anti-EGFR MoAbs and immune checkpoint inhibitors, and reintroduction of EGFR blockade or 'rechallenge' in selected patients who have previously responded to anti-EGFR MoAb therapy. An extensive translational research program was conducted in the Cetuximab After Progression in KRAS wIld-type colorectal cancer patients-Gruppo Oncologico dell' Italia Meridionale (CAPRI-GOIM) study with the aims of determining which subgroups of patients could benefit from the continuous inhibition of EGFR, from evaluating the role of liquid biopsy-based and its concordance with tissue-based molecular testing, and from investigating novel potential mechanisms of resistance to anti-EGFR therapies. In this review, we summarize the translational and clinical findings of the CAPRI-GOIM program in the context of the current knowledge of therapeutic strategies and of ongoing research on more appropriate uses of anti-EGFR therapies in RAS and BRAF wild-type mCRC patients.

Genotyping of Low β -ODAP Grass Pea (Lathyrus sativus L. ) Germplasm with EST-SSR Markers

Abstract Grass pea (Lathyrus sativus L.) is an important protein source in arid regions as both human and animal food. Despite its significance, the use of grass pea is limited by the presence of β-N-oxalyl-L-a,b-diaminopropionic acid (β-ODAP) which can cause neurological disorders. Breeding studies in grass pea have therefore focused on developing high-yielding varieties with low β-ODAP content. However, the narrow range of genetic diversity and the restricted genomic tools in grass pea have slowed progress in such breeding. The present investigation was conducted to explore the genetic diversity of low β-ODAP germplasm consisting of 22 accessions with 31 EST-SSR markers. The molecular analyses revealed a total of 133 alleles ranging from 142 to 330 bp with a mean number of alleles per locus of 4.29. The mean polymorphic information content (PIC) value was calculated as 0.49, and the EST-SSRs in loci S5, S6 and S116 were of the most informative PICs. A dendrogram based on Nei's genetic distance matrix revealed that breeding lines were grouped in two main clusters. Genetic distances were higher between GP6/GP11, GP4/GP11 and GP5/GP8 accessions which could be further used in crop improvement studies for developing wider genetic diversity.

Efficacy and safety of buparlisib, a PI3K inhibitor, in patients with malignancies harboring a PI3K pathway activation: a phase 2, open-label, single-arm study.

Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation plays a key role in tumorigenesis and has been associated with poor prognosis and resistance to multiple therapies in various cancers. Results: There were 146 patients enrolled; common tumor types were colorectal, sarcoma, and ovarian. Tumors had PI3K pathway alterations and a median of four mutations with tissue-specific patterns of mutation burden (lowest: sarcoma [2.5]; highest: esophagus, germ cell tumor, skin non-melanoma, vaginal [7]). The number of prior therapies did not correlate with the number of genetic alterations (Pearson r = -0.037). The clinical benefit rate was 15.1% (n = 22). An additional patient had an unconfirmed complete response. The most common adverse events were fatigue, nausea, hyperglycemia, decreased appetite, and diarrhea. Patient and Methods: In this phase 2, open-label, single-arm study, patients with solid or hematologic malignancies with PI3K pathway activation and progression on or after standard treatment received buparlisib (100 mg once daily). The primary endpoint was clinical benefit rate per local investigator assessment (response or stable disease at ≥16 weeks). Conclusions: Buparlisib was well tolerated, however efficacy was limited despite selection of PI3K pathway aberrations. Future studies may provide insight into buparlisib efficacy by refining the molecular selection of different tumor types.

Phase II Study of Tivantinib and Cetuximab in Patients With KRAS Wild-type Metastatic Colorectal Cancer With Acquired Resistance to EGFR Inhibitors and Emergence of MET Overexpression: Lesson Learned for Future Trials With EGFR/MET Dual Inhibition.

BACKGROUND: MET overexpression/amplification has been associated with resistance to anti- epidermal growth factor receptor therapies in patients with metastatic colorectal cancer (mCRC). Combining tivantinib, an inhibitor of the MET receptor tyrosine kinase, and cetuximab may be effective in patients with epidermal growth factor receptor-resistant MET-high mCRC. PATIENTS AND METHODS: This multicenter, single-arm, Simon 2-stage, phase II study enrolled patients with MET-high, KRAS wild-type mCRC, who were treated with ≥ 1 prior systemic therapy, with at least stable disease on the last treatment regimen containing cetuximab or panitumumab. Patients were enrolled if they presented tumor progression on cetuximab or panitumumab within 3 months before enrollment. Patients received tivantinib (360 mg twice daily) plus cetuximab (500 mg intravenously every 2 weeks). The primary endpoint was objective response rate; secondary endpoints included progression-free survival, overall survival, and safety. The treatment would be considered effective if ≥ 5 confirmed partial responses were observed among 41 patients. RESULTS: In total, 41 patients were evaluated, 4 patients (9.8%) achieved an objective response, the median progression-free survival was 2.6 months (95% confidence interval, 1.9-4.2 months), and the median overall survival was 9.2 months (95% confidence interval, 7.1-15.1 months). Among 13 patients with tested MET amplification, 2 responding patients had MET amplification compared with none of the nonresponding patients. The most common grade ≥ 3 treatment-emergent adverse events were neutropenia (14.6%), skin toxicity (12.2%), and fatigue (9.8%). CONCLUSION: Although the study did not meet its primary endpoint, efficacy results suggest some activity of the tested combination, with almost 10% of patients achieving objective response in a difficult-to-treat setting. Treatment-emergent adverse events were consistent with the known safety profile of tivantinib and cetuximab.

TRPA1 is selected as a semi-conserved channel during vertebrate evolution due to its involvement in spermatogenesis.

TRPA1 is a non-selective cation channel originated in invertebrates. The genomic locus containing TRPA1 gene remains highly conserved and retained in all vertebrates. TRPA1 gene is evolutionarily selected, yet maintained as a highly diverged protein. Throughout the vertebrate evolution, the extracellular loops of TRPA1 become most diverged indicating that TRPA1 may be involved in detecting large spectrum and uncertain stimulus which is critical for adaptive benefit. We tested the expression of TRPA1 in mature sperm from different vertebrates. This is the first report demonstrating that TRPA1 is expressed endogenously in mature spermatozoa of multiple species representing entire vertebrate phyla. However, its specific localization within sperm remains species-specific. Accordingly, we report that in rodents TRPA1 expression correlates with different stages of spermatogenesis. We propose that presence of endogenous TRPA1 in testes and in mature sperm provides reproductive benefit.

The Use of EGFR Tyrosine Kinase Inhibitors in EGFR Wild-Type Non-Small-Cell Lung Cancer.

The objective response rate and progression-free survival observed with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients with metastatic epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) are modest. The adverse events associated with EGFR TKIs are manageable but they must be considered in the context of the limited efficacy. The development of anti-PD-1 immunotherapy as second-line therapy has reduced the role of EGFR TKIs in EGFR wild-type NSCLC. Recently, there has been increased recognition of the benefit of the earlier integration of palliative care and symptom management, and this is reasonable alternative to treatment with an EGFR TKI for many patients. My practice pattern for patients with EGFR wild-type NSCLC is platinum-based chemotherapy as first-line therapy, immunotherapy as second-line therapy, and single-agent chemotherapy as third-line therapy for patients with preserved performance status who want to pursue further therapy. Only a small proportion of patients are eligible for fourth-line therapy, and I prefer to enroll them in clinical trials rather than use EGFR TKIs. I suspect that the use of EGFR TKIs in clinical use and as a comparator arm for clinical trials will continue to decline over the next several years.