KRAS: A Druggable Target in Colon Cancer Patients

Mutations in KRAS are among the most frequent aberrations in cancer, including colon cancer. KRAS direct targeting is daunting due to KRAS protein resistance to small molecule inhibition. Moreover, its elevated affinity to cellular guanosine triphosphate (GTP) has made the design of specific drugs challenging. Indeed, KRAS was considered ‘undruggable’. KRASG12C is the most commonly mutated variant of KRAS in non-small cell lung cancer. Currently, the achievements obtained with covalent inhibitors of this variant have given the possibility to assess the best therapeutic approach to KRAS-driven tumors. Mutation-related biochemical assets and the tissue of origin are expected to influence responses to treatment. Further attempts to obtain mutant-specific KRAS (KRASG12C) switch-II covalent inhibitors are ongoing and the results are promising. Drugs targeted to block KRAS effector pathways could be combined with direct KRAS inhibitors, immunotherapy or T cell-targeting approaches in KRAS-mutant tumors. The development of valuable combination regimens will be essential against potential mechanisms of resistance that may arise during treatment

Afatinib therapy in case of EGFR G724S emergence as resistance mechanism to osimertinib

Osimertinib is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) used both as the first-line treatment of EGFR-mutated non-small cell lung cancer patients and in second-line after T790M-positive disease progression to first- or second-generation TKIs. Unfortunately, patients unavoidably experience disease progression to osimertinib and the current research is focused on resistance mechanisms and the relative therapeutic strategy. We report the case of a patient with advanced EGFR-mutated (exon 19 deletion and T790M-positive) non-small cell lung cancer who developed disease progression to osimertinib characterized by the loss of T790M concurrently with the emergence of G724S EGFR mutation, which was tackled by subsequent afatinib treatment. Next-generation sequencing molecular study of rebiopsy at time of progression to osimertinib revealed the persistence of EGFR exon 19 deletion, loss of T790M with a new G724S EGFR mutation; other concomitant mechanisms were excluded. Retrospective analysis of cell-free DNA revealed the emergence of G724S EGFR mutation four months before the radiologically-proven disease progression. The patient, after chemotherapy, was treated with afatinib with clinical and radiological benefit. Our case report contributes to increase the knowledge on acquired resistance mechanisms to osimertinib treatment, and it shows, for the first time, the efficacy of afatinib in the case of T790M loss and emergence of G724S EGFR mutation. Anti-Cancer Drugs 32: 758-762 Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved

Emergence of a HER2-amplified clone during disease progression in an ALK-rearranged NSCLC patient treated with ALK-inhibitors: A case report

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) are the standard treatment for advanced ALK-positive non-small cell lung cancer (NSCLC) allowing survivals up to 5 years. However, duration of responses is limited by the almost certain occurrence of drug resistance. Here, we report a case of a never smoker, 59-year-old female with metastatic ALK-positive adenocarcinoma, solid and signet ring patterns, who developed resistance to alectinib, a second-generation ALK-TKI, mediated by HER2 gene amplification. The patient received 22 months of crizotinib as first-line and subsequently 1-year of alectinib therapy. A study of resistance mechanism was performed with next generation sequencing (NGS) on tissue re-biopsy. A HER2-amplified emerging clone was identified by NGS in a liver metastasis and confirmed by fluorescent in situ hybridization (FISH) analysis. The resistant clone was detectable 2 months before disease progression in plasma cell-free DNA (cfDNA) using digital droplet PCR (ddPCR) copy number variation (CNV) assay and it was retrospectively traced in rare cells of the lung primary by FISH. To our best knowledge, this is first evidence of HER2 gene amplification as a resistance mechanism to ALK-TKI in a NSCLC. Future strategies against oncogene-addicted NSCLC might benefit of combined drug treatments, such as ALK and HER2 inhibition

Monitoring cfDNA in plasma and in other liquid biopsies of advanced EGFR mutated NSCLC patients: A pilot study and a review of the literature

In order to study alternatives at the tissue biopsy to study EGFR status in NSCLC patients, we evaluated three different liquid biopsy platforms (plasma, urine and exhaled breath condensate, EBC). We also reviewed the literature of the cfDNA biological sources other than plasma and compared our results with it about the sensitivity to EGFR mutation determination. Twenty-two EGFR T790M-mutated NSCLC patients in progression to first-line treatment were enrolled and candidate to osimertinib. Plasma, urine and EBC samples were collected at baseline and every two months until progression. Molecular analysis of cfDNA was performed by ddPCR and compared to tissue results. At progression NGS analysis was performed. The EGFR activating mutation detection reached a sensitivity of 58 and 11% and for the T790M mutation of 45 and 10%, in plasma and urine samples, respectively. Any DNA content was recovered from EBC samples. Considering the plasma monitoring study, the worst survival was associated with positive shedding status; both plasma and urine molecular progression anticipated the radiological worsening. Our results confirmed the role of plasma liquid biopsy in testing EGFR mutational status, but unfortunately, did not evidence any improvement from the combination with alternative sources, as urine and EBC

Correlations between tumor-infiltrating and circulating lymphocyte subpopulations in advanced renal cancer patients treated with nivolumab

Background: In clinical trials with immunotherapy, histological features such as tumor-infiltrating lymphocytes (TILs) are investigated as potential predictive biomarkers, with the limit of an outdated parameter for a typically dynamic element. Methods: This explorative study compared, in metastatic renal cell carcinoma (mRCC) patients, basal pathological data about TILs on diagnostic histological specimens with circulating lymphocyte subpopulations measured before and during therapy with nivolumab. Results: Of 11 mRCC patients, 5 had low presence of TILs (L-TILs), 3 moderate amount (M-TILs) and 3 high number (H-TILs). Overall, 8 patients had low intratumoral pathological CD4+/CD8+ ratio (LIPR) ≤1 and 3 cases high intratumoral pathological ratio (HIPR) ≥2. Of 8 patients with LIPR, only 2 matched with low circulating CD4+/CD8+ ratio (LCR) ≤1; 5 had high circulating ratio (HCR) ≥2. All 3 cases with HIPR (≥2) conversely had LCR (≤1). Circulating CD4+/CD8+ ratio remained unchanged during therapy (mean-0.12 in 8 weeks). The respective percentage values of CD4+ and CD8+ circulating T cells also remained stable (variation 0%); the absolute value of CD4+ was more likely to increase (mean +46.3/mm3); the level of CD8+ tended to slightly decrease (mean-6.5/mm3). No correlation of lymphocyte subpopulations with treatment outcome was found. Of note, we did not evidence correspondence between histopathological and circulating findings in terms of T-lymphocyte subpopulations, also suggesting the inconsistency of circulating data in terms of relative variations. Conclusions: Considering the likely high dynamism of TILs, rebiopsy before therapy might be proposed to assess the utility of TILs characterization for predictive purpose. (www.actabiomedica.it)

High levels of Notch intracellular cleaved domain are associated with stemness and reduced bevacizumab efficacy in patients with advanced colon cancer

δ-like ligand 4 (DLL4)-Notch signaling is associated with tumor resistance to anti-vascular endothelial growth factor (VEGF) therapy. Furthermore, Notch signaling is critical for the maintenance of colon cancer stem cells (CSCs), which are relevant in drug resistance and tumor angiogenesis. CD44 is a transmembrane glycoprotein and is considered a putative marker of CSCs. To assess the association of Notch intracellular cleaved domain (NICD), DLL4 and CD44 expression with the efficacy of anti‑angiogenic drugs, a series of samples derived from patients with advanced colon cancer enrolled in prospective clinical trials were analyzed. Histological samples from 51 primary tumors that originated from patients treated with bevacizumab‑based first‑line chemotherapy were analyzed by immunohistochemistry for NICD, DLL4 and CD44 expression, and CD31 for microvessel count. The expression levels of genes relevant for angiogenesis [angiopoietin (ANGPT)1, ANGPT2, fibroblast growth factor (FGF)1, FGF2, epidermal growth factor, placental growth factor, VEGFA and DLL4] were detected by reverse transcription-quantitative PCR using RNA extracte

Sexual dimorphism in cancer.

The incidence of many types of cancer arising in organs with non-reproductive functions is significantly higher in male populations than in female populations, with associated differences in survival. Occupational and/or behavioural factors are well-known underlying determinants. However, cellular and molecular differences between the two sexes are also likely to be important. In this Opinion article, we focus on the complex interplay that sex hormones and sex chromosomes can have in intrinsic control of cancer-initiating cell populations, the tumour microenvironment and systemic determinants of cancer development, such as the immune system and metabolism. A better appreciation of these differences between the two sexes could be of substantial value for cancer prevention as well as treatment

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF.

AIMS: The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year. METHODS AND RESULTS: GARFIELD-AF is an ongoing, global observational study of adults with newly diagnosed NVAF. Two-year outcomes of 17 162 patients prospectively enrolled in GARFIELD-AF were analysed in light of baseline characteristics, risk profiles for stroke/systemic embolism (SE), and antithrombotic therapy. The mean (standard deviation) age was 69.8 (11.4) years, 43.8% were women, and the mean CHA2DS2-VASc score was 3.3 (1.6); 60.8% of patients were prescribed anticoagulant therapy with/without antiplatelet (AP) therapy, 27.4% AP monotherapy, and 11.8% no antithrombotic therapy. At 2-year follow-up, all-cause mortality, stroke/SE, and major bleeding had occurred at a rate (95% confidence interval) of 3.83 (3.62; 4.05), 1.25 (1.13; 1.38), and 0.70 (0.62; 0.81) per 100 person-years, respectively. Rates for all three major events were highest during the first 4 months. Congestive heart failure, acute coronary syndromes, sudden/unwitnessed death, malignancy, respiratory failure, and infection/sepsis accounted for 65% of all known causes of death and strokes for <10%. Anticoagulant treatment was associated with a 35% lower risk of death. CONCLUSION: The most frequent of the three major outcome measures was death, whose most common causes are not known to be significantly influenced by anticoagulation. This suggests that a more comprehensive approach to the management of NVAF may be needed to improve outcome. This could include, in addition to anticoagulation, interventions targeting modifiable, cause-specific risk factors for death. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01090362