New pan-ALK inhibitor-resistant EML4::ALK mutations detected by liquid biopsy in lung cancer patients.

ALK and ROS1 fusions are effectively targeted by tyrosine kinase inhibitors (TKIs), however patients inevitably relapse after an initial response, often due to kinase domain mutations. We investigated circulating DNA from TKI-relapsed NSCLC patients by deep-sequencing. New EML4::ALK substitutions, L1198R, C1237Y and L1196P, were identified in the plasma of NSCLC ALK patients and characterized in a Ba/F3 cell model. Variants C1237Y and L1196P demonstrated pan-inhibitor resistance across 5 clinical and 2 investigational TKIs.

A Liquid Biopsy Signature for the Detection of Patients With Early-Onset Colorectal Cancer.

BACKGROUND & AIMS: Early-onset colorectal cancer (EOCRC) is a distinct clinical and molecular entity with poor survival outcomes compared with late-onset CRC. Although the incidence of EOCRC is rising, current CRC screening strategies have several limitations in diagnostic performance for EOCRC. In view of this clinical challenge, novel and robust biomarkers for detection of EOCRC are necessary. The aim of this study was to develop a circulating micro RNA (miRNA) signature for the diagnosis of patients with EOCRC. METHODS: A systematic discovery approach by analyzing a large, publicly available, noncoding RNA expression profiling dataset (GSE115513) was used. A panel of miRNAs was identified, which was subsequently validated in blood samples from patients with EOCRC in 2 independent cohorts (n = 149) compared with controls (n = 110) and pre/postoperative plasma specimens (n = 22) using quantitative reverse-transcription polymerase chain reaction assays. RESULTS: In the discovery phase, 4 miRNAs were found to be expressed in blood samples. A combination signature of these 4 miRNAs (miR-193a-5p, miR-210, miR-513a-5p, and miR-628-3p) yielded an area under the curve of 0.92 (95% confidence interval, 0.85-0.96) for identification of EOCRC in the training cohort. The miRNA panel performance was then confirmed in an independent validation cohort (area under the curve, 0.88; 95% confidence interval, 0.82-0.93). Moreover, the miRNA panel robustly identified patients with early-stage EOCRC (P < .001). The decreased expression of miRNAs in postsurgery plasma specimens indicated their tumor specificity. CONCLUSIONS: Our novel miRNA signature for the diagnosis of EOCRC has the potential to identify patients with EOCRC with high accuracy for clinical application in the noninvasive diagnosis of EOCRC.

New Advances in Liquid Biopsy Technologies for Anaplastic Lymphoma Kinase (ALK)-Positive Cancer.

Cancer cells are characterized by high genetic instability, that favors tumor relapse. The identification of the genetic causes of relapse can direct next-line therapeutic choices. As tumor tissue rebiopsy at disease progression is not always feasible, noninvasive alternative methods are being explored. Liquid biopsy is emerging as a non-invasive, easy and repeatable tool to identify specific molecular alterations and monitor disease response during treatment. The dynamic follow-up provided by this analysis can provide useful predictive information and allow prompt therapeutic actions, tailored to the genetic profile of the recurring disease, several months before radiographic relapse. Oncogenic fusion genes are particularly suited for this type of analysis. Anaplastic Lymphoma Kinase (ALK) is the dominant driver oncogene in several tumors, including Anaplastic Large-Cell Lymphoma (ALCL), Non-Small Cell Lung Cancer (NSCLC) and others. Here we review recent findings in liquid biopsy technologies, including ctDNA, CTCs, exosomes, and other markers that can be investigated from plasma samples, in ALK-positive cancers.

Synergistic Drug Combinations Prevent Resistance in ALK+ Anaplastic Large Cell Lymphoma.

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30-40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.

Enhanced anti-cancer activity of andrographis with oligomeric proanthocyanidins through activation of metabolic and ferroptosis pathways in colorectal cancer.

The high degree of morbidity and mortality in colorectal cancer (CRC) patients is largely due to the development of chemoresistance against conventional chemotherapeutic drugs. In view of the accumulating evidence that various dietary botanicals offer a safe, inexpensive and multi-targeted treatment option, herein, we hypothesized that a combination of Andrographis paniculata and Oligomeric Proanthocyanidins (OPCs) might interact together with regard to anti-tumorigenic activity in CRC. As a result, we demonstrated the enhanced anti-cancer activity between these two botanical extracts in terms of their ability to inhibit cancer cell growth, suppress colony formation and induce apoptosis. Furthermore, we validated these findings in subcutaneous xenograft model and in patient derived primary epithelial 3D organoids. Transcriptomic profiling identified involvement of metabolic pathways and ferroptosis-associated genes, including HMOX1, GCLC and GCLM, that may be responsible for the increased anti-tumorigenic activity by the two compounds. Collectively, our study provides novel evidence in support of the combinatorial use of andrographis and OPCs as a potential therapeutic option, perhaps as an adjunctive treatment to classical drugs, in patients with colorectal cancer.

Non-coding RNA biomarkers in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, which is usually diagnosed at an advanced stage. The late disease diagnosis, the limited availability of effective therapeutic interventions and lack of robust diagnostic biomarkers, are some of the primary reasons for the dismal 5-year survival rates (∼8%) in patients with PDAC. The pancreatic cancer develops through accumulation of a series of genomic and epigenomic alterations which lead to the transformation of normal pancreatic epithelium into an invasive carcinoma - a process that can take up to 15-20 years to develop, from the occurrence of first initiating mutational event. These facts highlight a unique window of opportunity for the earlier detection of PDAC, which could allow timely disease interception and improvement in the overall survival outcomes in patients suffering from this fatal malignancy. Non-coding RNAs (ncRNAs) have been recognized to play a central role in PDAC pathogenesis and are emerging as attractive candidates for biomarker development in various cancers, including PDAC. More specifically, the ncRNAs play a pivotal role in PDAC biology as they affect tumor growth, migration, and invasion by regulating cellular processes including cell cycle, apoptosis, and epithelial-mesenchymal transition. In this review, we focus on three types of well-established ncRNAs - microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) - and discuss their potential as diagnostic, prognostic and predictive biomarkers in PDAC.

IL10RA modulates crizotinib sensitivity in NPM1-ALK+ anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor-relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance-specific relapse.

Lorlatinib Treatment Elicits Multiple On- and Off-Target Mechanisms of Resistance in ALK-Driven Cancer.

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib.