RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the ...regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs.
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•Unlike p110α, p110γ, and p110δ PI3K isoforms, RAS is unable to interact with p110β•The RHO family GTPases RAC1 and CDC42 directly bind and activate p110β via its RBD•GPCRs couple to PI3K via Dock180/Elmo1-mediated RAC activation and binding to p110β•Mice with RBD mutant p110β are resistant to experimental lung fibrosis
RAS proteins bind and activate multiple PI3K isoforms. The p110β isoform of PI3K, however, is instead activated by the binding of RHO family members RAC and CDC42. Blocking this interaction blunts GPCR-mediated cellular chemotaxis and confers resistance to lung fibrosis.
Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high ...sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid–based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget screening assays covering two to six single-nucleotide variants each. Limit of detection, limit of blank, and interassay accuracy were determined. Assay performance and suitability for screening in cfDNA were validated with plasma samples from a clinically fully characterized cohort of pancreatic cancer patients and healthy controls. Limits of detection for single-target assays were between 0.0015% and 0.069% variant allele fraction, and between 0.022% and 0.16% for multitarget assays. Dilution linearity and interassay accuracy were excellent throughout (r2 > 0.99). Multitarget assay screening of cfDNA extracted from pancreatic cancer patients with unknown KRAS mutational status correctly identified single-nucleotide variants in 45 of 45 (100%) of tumor-derived cell-free DNA–positive samples. In summary, we herein present and clinically validate generic single-target and discriminatory multitarget ddPCR assays for KRAS and NRAS hot spot mutations with broad applicability for clinical and translational research.
c‐Ros oncogene 1, receptor tyrosine kinase (ROS1) genomic rearrangements have been reported previously in rare cases of colorectal cancer (CRC), yet little is known about the frequency, molecular ...characteristics, and therapeutic vulnerabilities of ROS1‐driven CRC. We analyzed a clinical dataset of 40 589 patients with CRC for ROS1 genomic rearrangements and their associated genomic characteristics (Foundation Medicine, Inc FMI). We moreover report the disease course and treatment response of an index patient with ROS1‐rearranged metastatic CRC. ROS1 genomic rearrangements were identified in 34 (0.08%) CRC samples. GOPC‐ROS1 was the most common ROS1 fusion identified (11 samples), followed by TTC28‐ROS1 (3 samples). Four novel 5′ gene partners of ROS1 were identified (MCM9, SRPK1, EPHA6, P4HA1). Contrary to previous reports on fusion‐positive CRC, ROS1‐rearrangements were found exclusively in microsatellite stable (MSS) CRCs. KRAS mutations were significantly less abundant in ROS1‐rearranged vs ROS1 wild type cases. The index patient presented with chemotherapy‐refractory metastatic right‐sided colon cancer harboring GOPC‐ROS1. Molecularly targeted treatment with crizotinib induced a rapid and sustained partial response. After 15 months on crizotinib disseminated tumor progression occurred and KRAS Q61H emerged in tissue and liquid biopsies. ROS1 rearrangements define a small, yet therapeutically actionable molecular subgroup of MSS CRC. In summary, the high prevalence of GOPC‐ROS1 and noncanonical ROS1 fusions pose diagnostic challenges. We advocate NGS‐based comprehensive molecular profiling of MSS CRCs that are wild type for RAS and BRAF and patient enrollment in precision trials.
What's new?
The frequency, molecular characteristics, and therapeutic vulnerabilities of ROS1‐driven colorectal cancer remain largely unknown. Using a clinical dataset of 40 589 colorectal cancer patients, here the authors characterize the rare but highly actionable molecular colorectal cancer subgroup harboring genomic rearrangements of the receptor tyrosine kinase ROS1. They show that GOPC‐ROS1 is the most common ROS1 fusion and that unlike previously‐described fusion oncogenes, ROS1 rearrangements occur exclusively in microsatellite‐stable colorectal cancers. Moreover, they report the treatment course of an index patient, underlining the diagnostic challenge and therapeutic potential of ROS1 targeting in colorectal cancer.
Novel biomarkers and molecular monitoring tools hold potential to improve outcome for patients following resection of pancreatic ductal adenocarcinoma (PDAC). We hypothesized that the combined ...longitudinal analysis of mutated cell-free plasma KRAS (cfKRAS
) and CA 19-9 during adjuvant treatment and follow-up might more accurately predict disease course than hitherto available parameters.
Between 07/2015 and 10/2018, we collected 134 plasma samples from 25 patients after R0/R1-resection of PDAC during adjuvant chemotherapy and post-treatment surveillance at our institution. Highly sensitive discriminatory multi-target ddPCR assays were employed to screen plasma samples for cfKRAS
. cfKRAS
and CA 19-9 dynamics were correlated with recurrence-free survival (RFS) and overall survival (OS). Patients were followed-up until 01/2020.
Out of 25 enrolled patients, 76% had undergone R0 resection and 48% of resected PDACs were pN0. 17/25 (68%) of patients underwent adjuvant chemotherapy. Median follow-up was 22.0 months, with 19 out of 25 (76%) patients relapsing during study period. Median RFS was 10.0 months, median OS was 22.0 months. Out of clinicopathologic variables, only postoperative CA 19-9 levels and administration of adjuvant chemotherapy correlated with survival endpoints. cfKRAS
was detected in 12/25 (48%) of patients, and detection of high levels inversely correlated with survival endpoint. Integration of cfKRAS
and CA 19-9 levels outperformed either individual marker. cfKRAS
outperformed CA 19-9 as dynamic marker since increase during adjuvant chemotherapy and follow-up was highly predictive of early relapse and poor OS.
Integrated analysis of cfKRAS
and CA 19-9 levels is a promising approach for molecular monitoring of patients following resection of PDAC. Larger prospective studies are needed to further develop this approach and dissect each marker's specific potential.
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Circulating tumor cells (CTC) in the blood are hypothesized as the means of systemic tumor spread. Blood obtained from healthy donors ...and patients with PDAC was therefore subject to size-based CTC-isolation (ISET). We additionally compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in pancreatic CTC and corresponding tumors, and evaluated their significance as prognostic markers. Samples from 68 individuals (58 PDAC patients, 10 healthy donors) were analyzed; CTCs were present in patients with UICC stage IA-IV tumors and none of the controls (p < 0.001). Patients with >3 CTC/ml had a trend for worse median overall survival (OS) than patients with 0.3-3 CTC/ml (P = 0.12). Surprisingly, CTCs harbored various KRAS mutations in codon 12 and 13. Patients with a KRAS
mutation in their CTC (n = 14) had a trend to better median OS (24.5 months) compared to patients with other (10 months), or no detectable KRAS mutations (8 months; P = 0.04). KRAS mutations in CTC and corresponding tumor were discordant in 11 of 26 "tumor-CTC-pairs" (42%), while 15 (58%) had a matching mutation; survival was similar in both groups (P = 0.36). Genetic characterization, including mutations such as KRAS, may prove useful for prognosis and understanding of tumor biology.
Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and will become the second most common cause of cancer-associated mortality by 2030. The poor prognosis arises from a lack of ...sensitive biomarkers, limited therapeutic options, and the astonishingly high recurrence rate after surgery of 60–80%. The factors driving this recurrence, however, remain enigmatic. Therefore, we generated patient-derived organoids (PDOs) from early- and late-recurrent PDAC patients. Cellular identity of PDOs was confirmed by qPCR, ddPCR, and IHC analyses. This is the first study investigating the metabolism in PDOs of different, clinically significant PDAC entities by untargeted GC/MS profiling. Partial least square discriminant analysis unveiled global alterations between the two sample groups. We identified nine metabolites to be increased in early recurrent PDOs in comparison to late recurrent PDOs. More than four-times increased were fumarate, malate, glutamate, aspartate, and glutamine. Hence, α-keto acids were elevated in PDO-conditioned medium derived from early recurrent patients. We therefore speculate that an increased anaplerotic metabolism fuels the Krebs-cycle and a corresponding higher accessibility to energy fastens the recurrence in PDAC patients. Therein, a therapeutic intervention could delay PDAC recurrence and prolong survival of affected patients or could serve as biomarker to predict recurrence in the future.
To evaluate the role of ablative radiotherapy doses in the treatment of hilar or intrahepatic cholangiocarcinoma (CCC) using stereotactic body radiotherapy (SBRT).
Consecutive patients treated from ...2007 to 2016 with CCC were evaluated. Local control and toxicities were assessed every 3 months according to the Response Evaluation Criteria In Solid Tumors (RECIST) and the Common Terminology Criteria for Adverse Events v4.0, respectively. Overall survival (OS), local control (LC) and progression free survival were calculated from SBRT.
Thirty seven patients with 43 lesions were retrospectively evaluated. The median dose delivered was 45 Gy (range 25-66 Gy) in 3-12 fractions, corresponding to a median equivalent dose in 2 Gy fractions (EQD2
) of 56 (range 25-85) Gy. The median follow up was 24 months. The OS at 1 year was 56% with a median OS of 14 (95% CI: 7.8-20.2) months from start of SBRT and 22 (95% CI: 17.5-26.5) months from diagnosis. Eight lesions progressed locally. The local control rate (LC) at 1 year was 78%. The median progression free survival was 9 months (95% CI 2.8-15.2) 21 patients progressed in the liver but out of field and 15 progressed distantly. SBRT was well tolerated. Three patients (9%) developed a Grade III bleeding. Seven patients developed a cholangitis, one due to progression and the other because of a stent dysfunction 2-21(median 8) months from SBRT.
In patients with locally advanced cholangiocarcinoma, SBRT is a local treatment option with an acceptable toxicity profile which warrants further investigation in prospective trials.
Abstract
Hypoxia is prominent in solid tumors and a recognized driver of malignancy. Thus far, targeting tumor hypoxia has remained unsuccessful. Myo-inositol trispyrophosphate (ITPP) is a ...re-oxygenating compound without apparent toxicity. In preclinical models, ITPP potentiates the efficacy of subsequent chemotherapy through vascular normalization. Here, we report the results of an unrandomized, open-labeled, 3 + 3 dose-escalation phase Ib study (NCT02528526) including 28 patients with advanced primary hepatopancreatobiliary malignancies and liver metastases of colorectal cancer receiving nine 8h-infusions of ITPP over three weeks across eight dose levels (1'866-14'500 mg/m
2
/dose), followed by standard chemotherapy. Primary objectives are assessment of the safety and tolerability and establishment of the maximum tolerated dose, while secondary objectives include assessment of pharmacokinetics, antitumor activity via radiological evaluation and assessment of circulatory tumor-specific and angiogenic markers. The maximum tolerated dose is 12,390 mg/m
2
, and ITPP treatment results in 32 treatment-related toxicities (mostly hypercalcemia) that require little or no intervention. 52% of patients have morphological disease stabilization under ITPP monotherapy. Following subsequent chemotherapy, 10% show partial responses while 60% have stable disease. Decreases in angiogenic markers are noted in ∼60% of patients after ITPP and tend to correlate with responses and survival after chemotherapy.
Colorectal cancers (CRCs) remain one of the most common and challenging neoplasia in the Western world. The response rate of immunotherapeutic treatment approaches in a subset of advanced CRCs is ...remarkable and has sustainably changed treatment regimens. Unfortunately, currently available immunotherapeutics only displayed significant antitumoral activity – in terms of progression free survival (PFS) and objective response rate (ORR) – in microsatellite instability-high (MSI-H)/DNA mismatch repair deficient (dMMR) CRCs. Subsequently, these remarkable results had led to the US Food and Drug Administration’s approval of both immune checkpoint inhibitors (ICIs) pembrolizumab and nivolumab in the treatment of advanced MSI-H/dMMR CRCs. However, in microsatellite stable (MSS)/DNA mismatch repair proficient (pMMR) CRCs, ICIs have clearly failed to meet their expectations and are therefore not considered effective. As the vast majority of CRCs display a molecular MSS/pMMR profile, current treatment approaches endeavor to improve tumor immunogenicity that consecutively leads to increased proinflammatory cytokine levels as well as tumor infiltrating T-cells, which in turn may be targeted by various immunotherapeutic agents. Therefore, ongoing studies are investigating novel synergistic therapy modalities and approaches to overcome a “cold” to “hot” tumor conversion in MSS/pMMR CRCs. In this review, we summarize the efficacy and possible immune-related adverse events as well as novel therapeutic approaches of ICIs in the treatment of MSI-H/dMMR and MSS/pMMR CRCs.
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