Multifaceted Insights into Innovative Approaches to Treating Colorectal Cancer Metastasis: From Emerging Biological Factors to Radiomics.

We extend our appreciation to the authors who have made substantial contributions to the Special Issue focusing on "Colorectal Cancer Metastasis" [...].

KRAS p.G12C Mutation in Metastatic Colorectal Cancer: Prognostic Implications and Advancements in Targeted Therapies.

KRAS is frequently mutated in tumors. It is mutated in approximately 30% of all cancer cases and in nearly 50% of cases of metastatic colorectal cancer (CRC), which is the third leading cause of cancer-related deaths worldwide. Recent advancements in understanding CRC biology and genetics have highlighted the significance of KRAS mutations in the progression of CRC. The KRAS gene encodes a small GTPase (Guanosine TriPhosphatases) that plays a key role in signaling pathways associated with important proteins involved in amplifying growth factor and receptor signals. Mutations in KRAS are frequently observed in codons 12 and 13, and these mutations have oncogenic properties. Abnormal activation of KRAS proteins strongly stimulates signals associated with various cancer-related processes in CRC, including cell proliferation, migration and neoangiogenesis. In this review, we explore the distinct prognostic implications of KRAS mutations. Specifically, the KRAS p.G12C mutation is associated with a worse prognosis in metastatic CRC. The correlation between structure, conformation and mutations is visually presented to emphasize how alterations in individual amino acids at the same position in a single protein can unexpectedly exhibit complex involvement in cancer. Last, KRAS p.G12C is discussed as an emerging and promising therapeutic target in metastatic CRC, providing a concise overview of available clinical data regarding the use of new inhibitors.

The prognostic role of p53 mutations in metastatic colorectal cancer: A systematic review and meta-analysis.

INTRODUCTION: P53 is one of the most frequently mutated genes in colorectal cancer (CRC). The present study was undertaken to provide a solid estimate of the prognostic value of p53 mutations in metastatic CRC patients. METHODS: This meta-analysis was done in accordance to the Preferred Reporting Item For Systematic Reviews and Meta-Analysis 2020 guidelines. Studies in English published in the last ten years were searched through PubMed and Google Scholar. Final selection criteria were: 1) association with overall survival, 2) presence of Hazard Ratios (HRs) with 95% Confidence Intervals (CIs). The articles were evaluated for quality and risk of bias using the Newcastle-Ottawa Scale and QUIPS tool, respectively. The meta-analysis was conducted with random-effects model according to the Hartung-Knapp-Sidik-Jonkman method and results were depicted in classical Forest plots. Studies heterogeneity was determined by I2 and Tau2 statistics. The relationship between p53 mutation and clinic-pathological variables was examined using the χ2 test. RESULTS: Nine articles met the eligibility criteria and went to the final analysis. Sample size ranged from 51 to 1043 patients. All studies were retrospective. The Newcastle Ottawa Scale score was > 6 in all studies, QUIPS risk of bias was low in 6, moderate in 3 studies. Only three studies analysed the entire p53 gene coding region. The DNA sequencing technological platforms varied from Sanger to NGS sequencing techniques. The p53 mutational frequencies ranged from 35.0 % to 73.0 %. A strong association (p < 0.0001) emerged between p53 alteration and left-sided CRC. The final pooled HR (p53 mutated vs p53 wild-type tumors) for overall survival was 1.30 (95 % CI: 0.75-2.25) at random-effects model. CONCLUSIONS: The available evidence does not support a prognostic role for p53 in metastatic CRC patients. Prospective studies, with larger sample sizes and consistent and harmonized methodology, are needed to explore the prognostic role of p53 in metastatic CRC patients.

Oligo-Metastatic Cancers: Putative Biomarkers, Emerging Challenges and New Perspectives.

Some cancer patients display a less aggressive form of metastatic disease, characterized by a low tumor burden and involving a smaller number of sites, which is referred to as "oligometastatic disease" (OMD). This review discusses new biomarkers, as well as methodological challenges and perspectives characterizing OMD. Recent studies have revealed that specific microRNA profiles, chromosome patterns, driver gene mutations (ERBB2, PBRM1, SETD2, KRAS, PIK3CA, SMAD4), polymorphisms (TCF7L2), and levels of immune cell infiltration into metastases, depending on the tumor type, are associated with an oligometastatic behavior. This suggests that OMD could be a distinct disease with specific biological and molecular characteristics. Therefore, the heterogeneity of initial tumor burden and inclusion of OMD patients in clinical trials pose a crucial methodological question that requires responses in the near future. Additionally, a solid understanding of the molecular and biological features of OMD will be necessary to support and complete the clinical staging systems, enabling a better distinction of metastatic behavior and tailored treatments.

Hypertension, type 2 diabetes, obesity, and p53 mutations negatively correlate with metastatic colorectal cancer patients' survival.

Introduction: We studied the predictive and prognostic influences of hypertension (HT), type 2 diabetes (T2D), weight, and p53 mutations in metastatic colorectal cancer (CRC) patients. Patients and methods: T2D was diagnosed according to the ADA criteria. HT was classified according to the ACC/AHA guidelines. BMI (body-mass index) was calculated and classified according to the WHO criteria. TruSigt™Oncology 500 kit was applied to construct the genomic libraries for Next Generation Sequencing (NGS) analysis. The Illumina NovaSeq 6000 technological platform and the Illumina TruSight Oncology 500 bioinformatics pipeline were applied to analyze results. Overall survival (OS) was calculated through Kaplan-Meier curves. Univariate and multivariate analyses were performed to assess the relationships between clinical and/or molecular covariates. Associations between HT, T2D, BMI, p53, and clinical variables were evaluated by the χ2 test. P < 0.05 were considered statistically significant. Results: Two-hundred-forty-four patients were enrolled. One-hundred-twenty (49.2%), 110 (45.1%), and 50 (20.5%) patients were affected by overweight, HT, and T2D, respectively. DC (disease control) was achieved more frequently in patients without T2D (83.1%) compared to the diabetic ones (16.9%) (P = 0.0246). DC, KRAS mutational status, T2D, BMI, and concomitant presence of T2D, BMI, and HT associated with survival (P < 0.05). At multivariate analysis, age (≥65 vs. <65 years), response to first-line chemotherapy (DC vs. no DC), and concomitant presence of T2D, BMI, and HT (HR: 4.56; 95% CI: 2.40-8.67; P = 0.0217) emerged as independent prognostic variables. P53 was mutated in 31/53 analyzed cases (60.4%). The most frequent gene variants were p.Arg175His and p.Cys135Tyr. High BMI (>25 kg/m2) associated with occurrence of p53 mutations (P < 0.0001). P53 mutated patients presented a worse prognosis compared to the wild-type ones (HR: 3.21; 95% CI: 1.43-7.23; P = 0.0047). Conclusion: Diabetic, hypertensive and overweight metastatic CRC patients are a negative prognostic subgroup deserving specific therapeutic strategies. P53 mutations associate with prognosis and BMI unrevealing complex and unexplored connections between metabolism and cancer occurrence.

Genetic landscape of colorectal cancer patients manifesting tumor shrinkage during SARS-Cov-2 infection.

We previously described three patients affected by metastatic colorectal cancer (mCRC) who experienced spontaneous tumour shrinkage during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Thereafter, the patients were closely monitored and no systemic treatments were applied. Here, we report follow-up clinical information about these patients as well as genetic characterization of their primary tumours through the TruSigt™Oncology 500 Next Generation Sequencing test targeting 523 cancer-relevant genes. An Illumina NovaSeq 6000 platform was used to perform sequencing. Time-to-progression was 23 and 2 months, respectively, in Patients 2 and 3 while it was not reached in Patient 1. Patients 1 and 2 had the greatest anti-SARS-CoV-2 IgG titres. Assessment of genetic landscapes evidenced common mutation in BARD1 gene (p.Val507Met) in Patients 1 and 2. Although our report is descriptive in its nature, we suggest that complex and unexplored interactions between genetic background and components of the immune response to SARS-CoV-2 infection could be responsible of unexpected rare mCRC shrinkage.

Metastatic colorectal cancer and type 2 diabetes: prognostic and genetic interactions.

The present study was undertaken to analyze prognostic and genetic interactions between type 2 diabetes and metastatic colorectal cancer. Patients' survival was depicted through the Kaplan-Meier product limit method. Prognostic factors were examined through the Cox proportional-hazards regression model, and associations between diabetes and clinical-pathologic variables were evaluated by the χ2 test. In total, 203 metastatic colorectal cancer patients were enrolled. Lymph nodes (P = 0.0004) and distant organs (> 2 distant sites, P = 0.0451) were more frequently involved in diabetic patients compared with those without diabetes. Diabetes had an independent statistically significant negative prognostic value for survival. Highly selected patients with cancer and/or diabetes as their only illness(es) were divided into three groups: (a) seven oligo-metastatic patients without diabetes, (b) 10 poly-metastatic patients without diabetes, and (c) 12 poly-metastatic diabetic patients. These groups of patients were genetically characterized through the Illumina NovaSeq 6000 (San Diego, CA, USA) platform and TruSigt™Oncology 500 kit, focusing on genes involved in diabetes and colorectal cancer. Gene variants associated with diabetes and cancer were more frequent in patients in group 3. We found that type 2 diabetes is a negative prognostic factor for survival in colorectal cancer. Diabetes-associated gene variants could concur with malignancy, providing a rational basis for innovative models of tumor progression and therapy.

Vitamin D deficiency and tumor aggressiveness in gastroenteropancreatic neuroendocrine tumors.

PURPOSE: Data regarding vitamin D status in patients affected by gastroenteropancreatic (GEP) neuroendocrine tumor (NET) are limited and often showing contrasting results. The aim of the study was to evaluate the incidence of vitamin D deficiency (<20 ng/mL) in GEP-NET patients and compare the 25-hydroxyvitamin D (25(OH)D) levels with clinicopathological parameters and clinical outcome. METHODS: A retrospective cross-sectional study including 75 low grade (G1-G2) GEP-NETs and 123 healthy controls matched for age, sex, and body mass index, was performed. RESULTS: GEP-NET patients had significantly lower 25(OH)D levels compared to controls (17.9 ± 7.8 vs 24.2 ± 7.7 ng/mL, p < 0.0001). Ileal NETs were associated to lower 25(OH)D levels compared to other primary tumor sites (p = 0.049) and small bowel resection posed a significant increased risk of severe vitamin D deficiency (OR = 2.81, 95% CI = 1.25-3.37, p = 0.018). No correlation with somatostatin analogs treatment was found. 25(OH)D levels were significantly lower in G2 compared to G1 GEP-NETs (15.6 ± 7.8 vs 19.9 ± 7.4 ng/mL, p = 0.016) and in patients with progressive disease (12.6 ± 5.7 ng/mL) compared to those with stable disease (mean 21.5 ± 8.2 ng/mL, p = 0.001) or tumor free after surgery (19.6 ± 7.3 ng/mL, p = 0.002). Patients with vitamin D deficiency and insufficiency had shorter progression-free survival compared to those with sufficiency (p = 0.014), whereas no correlation was found with disease-specific survival. CONCLUSIONS: Vitamin D deficiency is highly prevalent among GEP-NETs and could be associated with high tumor grade and disease progression. Therefore, the monitoring of 25(OH)D levels is relevant in these patients and vitamin D supplementation should be considered in the management of GEP-NET patients with vitamin D deficiency or insufficiency.

The Tumor Dynamism Is the Dark Matter of the NGS Galaxy: How to Understand It?

Since its discovery, there has been a great enthusiasm around NGS (next generation sequencing) technology due to extensive (from restricted gene panels to entire genomes) and rapid (few hours) DNA sequencing [...].

Prospective Evaluation of Radiotherapy-Induced Immunologic and Genetic Effects in Colorectal Cancer Oligo-Metastatic Patients with Lung-Limited Disease: The PRELUDE-1 Study.

BACKGROUND: in recent years, the management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). The administration of SRT has been demonstrated, particularly in oligo-metastatic (om) CRC, to be a safe and effective option. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions ("abscopal effect") through stimulation of anti-tumor immune effects ("radiation-induced immunity"). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. AIMS: to assess the genetic evolution on tumor cancer cells induced by SRT in lung-limited omCRC. Secondary objectives included descriptions of the abscopal effect, responses' duration, toxicity, and progression-free survival. A translational research will be performed to evaluate tumor genetic evolution (through liquid biopsies and Next Generation Sequencing), HLA class I repertoire, peripheral immune cells, and cytokine dynamics. METHODS: PRELUDE-1 is a prospective translational study. SRT will be administered only to the largest nodule (with a maximum diameter ≤ 25 mm) in omCRC with two or three radiologically evident lesions. The sample size is based on the innovative hypothesis that radiation-induced immunity could induce regression of tumor clones bearing KRAS oncogene mutations. According to the binomial test, considering the frequency of KRAS mutations and assuming a probability of mutant KRAS→wild type KRAS of p0 = 0.0077, with α = 0.05 and 1-ß = 0.60, the final sample size is 25 patients.

Genetic regressive trajectories in colorectal cancer: A new hallmark of oligo-metastatic disease?

Colorectal cancer (CRC) originates as consequence of multiple genetic alterations. Some of the involved genes have been extensively studied (APC, TP53, KRAS, SMAD4, PIK3CA, MMR genes) in highly heterogeneous and poly-metastatic cohorts. However, about 10% of metastatic CRC patients presents with an indolent oligo-metastatic disease differently from other patients with poly-metastatic and aggressive clinical course. Which are the genetic dynamics underlying the differences between oligo- and poly-metastatic CRC? The understanding of the genetic trajectories (primary→metastatic) of CRC, in patients selected to represent homogenous clinical models, is crucial to make genotype/phenotype correlations and to identify the molecular events pushing the disease towards an increasing malignant phenotype. This information is crucial to plan innovative therapeutic strategies aimed to reverse or inhibit these phenomena. In the present study, we review the genetic evolution of CRC with the intent to give a developmental perspective on the border line between oligo- and poly-metastatic diseases.

KRAS Mutational Regression Is Associated With Oligo-Metastatic Status and Good Prognosis in Metastatic Colorectal Cancer.

BACKGROUND: We previously reported that loss of KRAS mutations ("regressive" mutational trajectories) from primary tumors to metastases associated with the oligo-metastatic status in colorectal cancer (CRC). The present study was undertaken in order to analyze the mutational trajectories of KRAS in a well-characterized cohort of CRC patients who developed poly- or oligo-metastatic disease. MATERIAL AND METHODS: Patients were treated and followed-up according to European Society of Medical Oncology guidelines. Primary CRC FFPE tissue and metastatic circulating-free DNA were extracted using the QIAamp DNA specific kits (Qiagen, Hilden, Germany). Samples were sequenced with the Oncomine Solid Tumour DNA kit (Thermo Fisher Scientific, Waltham, MA, USA). Plasma collection for liquid biopsy was done from 1 to 14 days before starting first-line chemotherapy. Analysis of the prognostic power of KRAS evolutionary trajectories was done with uni- and multivariate analyses. RESULTS: One-hundred-fourteen patients were enrolled. Sixty-three patients presented with mutated KRAS (mutKRAS) and 51 with wild-type KRAS (wtKRAS). KRAS mutational concordance was high (70.1%).Two divergent subsets were identified: mutKRAS in primary tumors and wtKRAS in metastatic ones (regressive: mutKRAS → wtKRAS in 8.8% of patients), and vice versa (progressive: wtKRAS → mutKRAS in 21.1% of patients). An association between KRAS regressive trajectory and the oligo-metastatic status (P <0.0001) was found. At multivariate analysis, regressive and progressive mutational trajectories emerged as independent prognostic factors for survival, with Hazard Ratios of 0.22 (CI 95%: 0.08-0.61; median survival: not reached) and 2.70 (CI 95%: 1.11-6.56, median survival: 12.1 months), respectively. CONCLUSIONS: Our data provide evidence that the evolutionary trajectories of KRAS can have a strong clinical prognostic role and that they can be involved in discriminating between poly-metastatic aggressive vs oligo-metastatic indolent CRC.

Evolution of Mutational Landscape and Tumor Immune-Microenvironment in Liver Oligo-Metastatic Colorectal Cancer.

Genetic dynamics underlying cancer progression are largely unknown and several genes involved in highly prevalent illnesses (e.g., hypertension, obesity, and diabetes) strongly concur to cancer phenotype heterogeneity. To study genotype-phenotype relationships contributing to the mutational evolution of colorectal cancer (CRC) with a focus on liver metastases, we performed genome profiling on tumor tissues of CRC patients with liver metastatic disease and no co-morbidities. We studied 523 cancer-related genes and tumor-immune microenvironment characteristics in primary and matched metastatic tissues. We observed a loss of KRAS and SMAD4 alterations and a high granzyme-B+ T-cell infiltration when the disease did not progress. Conversely, gain in KRAS, PIK3CA and SMAD4 alterations and scarce granzyme-B+ T-cells infiltration were observed when the tumor evolved towards a poly-metastatic spread. These findings provide novel insights into the identification of tumor oligo-metastatic status, indicating that some genes are on a boundary line between these two clinical settings (oligo- vs. poly-metastatic CRC). We speculate that the identification of these genes and modification of their evolution could be a new approach for anti-cancer therapeutic strategies.

Genetic trajectory and immune microenvironment of lung-specific oligometastatic colorectal cancer.

Genetics and immunologic dynamics pushing the evolution of colorectal cancer (CRC) from the primary tumor to the metastases are largely unknown; cancer heterogeneity makes challenging both therapy and mechanistic studies. We selected patients developing CRC with lung-limited metastatic disease as only illness during their life in order to find any relevant genotype-phenotype relationship. Analysis of 523 cancer-relevant genes and of immune cells infiltration in primary and metastatic tissues revealed atypical genomic trajectories (TMB decrease, KRAS and SMAD4 regressive mutations), specific genetic events (ERBB2 point mutations) and scarce T-cell infiltration. These insights provide novel information in oligometastatic CRC biology and new perspectives for cancer monitoring and anti-cancer therapeutic strategies.

Use of adjuvants for immunotherapy.

Cancer vaccines are designed to stimulate the body's immune system to kill tumor cells. To improve their immunogenicity, vaccine antigens must be combined with adjuvants which are able to stimulate the innate immunity and potentiate the adaptive immune response. In the last years a new generation of adjuvants mimicking the natural microbial ligands have been developed. In particular, several TLR ligands have been extensively explored as vaccine adjuvants and many preclinical and clinical studies have been conducted. However, the road to approval of such adjuvants for clinical use is still to go.

Epigenome-wide association study in hepatocellular carcinoma: Identification of stochastic epigenetic mutations through an innovative statistical approach.

Hepatocellular carcinoma (HCC) results from accumulation of both genetic and epigenetic alterations. We investigated the genome-wide DNA methylation profile in 69 pairs of HCC and adjacent non-cancerous liver tissues using the Infinium HumanMethylation 450K BeadChip array. An innovative analytical approach has been adopted to identify Stochastic Epigenetic Mutations (SEMs) in HCC.HCC and peritumoral tissues showed a different epigenetic profile, mainly characterized by loss of DNA methylation in HCC. Total number of SEMs was significantly higher in HCC tumor (median: 77,370) than in peritumoral (median: 5,656) tissues and correlated with tumor grade. A significant positive association emerged between SEMs measured in peritumoral tissue and hepatitis B and/or C virus infection status. A restricted number of SEMs resulted to be shared by more than 90% of HCC tumor samples and never present in peritumoral tissue. This analysis allowed the identification of four epigenetically regulated candidate genes (AJAP1, ADARB2, PTPRN2, SDK1), potentially involved in the pathogenesis of HCC.In conclusion, HCC showed a methylation profile completely deregulated and very far from adjacent non-cancerous liver tissues. The SEM analysis provided valuable clues for further investigations in understanding the process of tumorigenesis in HCC.

Immunological effects of a novel RNA-based adjuvant in liver cancer patients.

Evaluation of biological effects of adjuvants on immune cells has been assessed in a limited number of studies. Moreover, no data are available on samples derived from cancer patients who may have a severe immune impairment. The effects of a novel RNA-based adjuvant (RNAdjuvant® developed by CureVac) were assessed in an ex vivo setting on PBMCs obtained from 8 healthy volunteers and 17 HCC patients, using a multiparametric approach to analyze network dynamics of early immune responses. Evaluation of CD80, CD86 and HLA-DR expression, cytokine production as well as gene expression was performed. Moreover, the downstream effect on CD4+ T cell phenotyping was evaluated. Treatment with RNAdjuvant® showed comparable effects on PBMCs of both HCC and healthy subjects. In particular, CD80, CD86 and HLA-DR expression was found up-regulated in circulating dendritic cells, which promoted a CD4+ T cell differentiation toward an effector phenotype. A mixed Th1/Th2 cytokine pattern was induced, although a more predominant production of TNFα and IFNγ was observed in HCC patients versus healthy controls. The cytokine profile was further confirmed by gene transcriptional analysis, which showed up-regulation of several genes involved in innate and adaptive immune-related pathways. The present study is the first demonstration that HCC patients and healthy subjects are equally responsive to an adjuvant. This may suggest that the same vaccine formulation including the RNAdjuvant® might have similar potency in healthy subjects and cancer patients.

Animal models of medullary thyroid cancer: state of the art and view to the future.

Medullary thyroid carcinoma is a neuroendocrine tumour originating from parafollicular C cells accounting for 5-10% of thyroid cancers. Increased understanding of disease-specific molecular targets of therapy has led to the regulatory approval of two drugs (vandetanib and cabozantinib) for the treatment of medullary thyroid carcinoma. These drugs increase progression-free survival; however, they are often poorly tolerated and most treatment responses are transient. Animal models are indispensable tools for investigating the pathogenesis, mechanisms for tumour invasion and metastasis and new therapeutic approaches for cancer. Unfortunately, only few models are available for medullary thyroid carcinoma. This review provides an overview of the state of the art of animal models in medullary thyroid carcinoma and highlights future developments in this field, with the aim of addressing salient features and clinical relevance.

CXCR4/CXCL12/CXCR7 axis is functional in neuroendocrine tumors and signals on mTOR.

OBJECTIVE: To evaluate the possible crosstalk between C-X-C chemokine receptor 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12)/C-X-C chemokine receptor 7 (CXCR7) axis with the mammalian target of rapamycin (mTOR) pathway in neuroendocrine tumors (NETs). METHODS: Sixty-one human NETs were included into the study. CXCR4/CXCL12/CXCR7 axis and mTOR pathway were assessed by qRT-PCR and immunohistochemistry (IHC). The effect of mTOR inhibitor, RAD001, was evaluated on CXCR4 pathway through proliferation and p-Erk and p-AKT induction. RESULTS: CXCR4/CXCL12/CXCR7 axis and p-mTOR were found to be active and correlated with grading, Ki67 index and tumor stage. mTOR pathway activation significantly correlated with poor prognosis. In human NET cells, CXCL12 induced mTOR signalling while AMD3100 (CXCR4-antagonist) impaired it. The mTOR-antagonist, RAD001, impaired the CXCL12-dependent induction of CXCR4 downstream effectors. Combination of AMD3100 and RAD001 potentiate cell growth inhibition. CONCLUSIONS: CXCR4/CXCL12/CXCR7 axis is active in NETs and signals on mTOR. CXCR4 might be considered a prognostic factor in NETs. Combined treatment with AMD3100 and RAD001 may provide clinical benefits in NET patients with drug-resistant.