Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression.

mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.

Synthetic Knockout Protein Standard for Evaluating Interference in Tandem Mass Tag-Based Proteomics.

Isobaric labeling is widely used for unbiased, proteome-wide studies, and it provides several advantages, such as fewer missing values among samples and higher quantitative precision. However, ion interference may lead to compressed or distorted observed ratios due to the coelution and coanalysis of peptides. Here, we introduced a synthetic KnockOut standard (sKO) for evaluating interference in tandem mass tags-based proteomics. sKO is made by mixing TMTpro-labeled tryptic peptides derived from four nonhuman proteins and a whole human proteome as background at different proportions. We showcased the utility of the sKO standard by exploring ion interference at different peptide concentrations (up to a 30-fold change in abundance) and using a variety of mass spectrometer data acquisition strategies. We also demonstrated that the sKO standard could provide valuable information for the rational design of acquisition strategies to achieve optimal data quality and discussed its potential applications for high-throughput proteomics workflows development.

Genome-wide methylation profiling identifies a novel gene signature for patients with synchronous colorectal cancer.

BACKGROUND: There are no robust tools for the diagnosis of synchronous colorectal cancer (SyCRC). Herein, we developed the first methylation signature to identify and characterise patients with SyCRC. METHODS: For biomarker discovery, we analysed the genome-wide methylation profiles of 16 SyCRC and 18 solitary colorectal cancer (SoCRC) specimens. We thereafter established a methylation signature risk-scoring model to identify SyCRC in an independent cohort of 38 SyCRC and 42 SoCRC patients. In addition, we evaluated the prognostic value of the identified methylation profile. RESULTS: We identified six differentially methylated CpG probes/sites that distinguished SyCRC from SoCRC. In the validation cohort, we developed a methylation panel that identified patients with SyCRC from not only larger tumour (AUC = 0.91) but also the paired remaining tumour (AUC = 0.93). Moreover, high risk scores of our panel were associated with the development of metachronous CRC among patients with SyCRC (AUC = 0.87) and emerged as an independent predictor for relapse-free survival (hazard ratio = 2.72; 95% CI = 1.12-6.61). Furthermore, the risk stratification model which combined with clinical risk factors was a diagnostic predictor of recurrence (AUC = 0.90). CONCLUSIONS: Our novel six-gene methylation panel robustly identifies patients with SyCRC, which has the clinical potential to improve the diagnosis and management of patients with CRC.

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.

Sample multiplexing for targeted pathway proteomics in aging mice.

Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (∼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community.

Telomere Length as a New Risk Marker of Early-Onset Colorectal Cancer.

Early-onset colorectal cancer (EOCRC; age younger than 50 years) incidence has been steadily increasing in recent decades worldwide. The need for new biomarkers for EOCRC prevention strategies is undeniable. In this study, we aimed to explore whether an aging factor, such as telomere length (TL), could be a useful tool in EOCRC screening. The absolute leukocyte TL from 87 microsatellite stable EOCRC patients and 109 healthy controls (HC) with the same range of age, was quantified by Real Time Quantitative PCR (RT-qPCR). Then, leukocyte whole-exome sequencing (WES) was performed to study the status of the genes involved in TL maintenance (hTERT, TERC, DKC1, TERF1, TERF2, TERF2IP, TINF2, ACD, and POT1) in 70 sporadic EOCRC cases from the original cohort. We observed that TL was significantly shorter in EOCRC patients than in healthy individuals (EOCRC mean: 122 kb vs. HC mean: 296 kb; p < 0.001), suggesting that telomeric shortening could be associated with EOCRC susceptibility. In addition, we found a significant association between several SNPs of hTERT (rs79662648), POT1 (rs76436625, rs10263573, rs3815221, rs7794637, rs7784168, rs4383910, and rs7782354), TERF2 (rs251796 and rs344152214), and TERF2IP (rs7205764) genes and the risk of developing EOCRC. We consider that the measurement of germline TL and the status analysis of telomere maintenance related genes polymorphisms at early ages could be non-invasive methods that could facilitate the early identification of individuals at risk of developing EOCRC.

Assessing interference in isobaric tag-based sample multiplexing using an 18-plex interference standard.

Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO viewing tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that are acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.

Familial component of early-onset colorectal cancer: opportunity for prevention.

BACKGROUND: Individuals with a non-syndromic family history of colorectal cancer are known to have an increased risk. There is an opportunity to prevent early-onset colorectal cancer (age less than 50 years) (EOCRC) in this population. The aim was to explore the proportion of EOCRC that is preventable due to family history of colorectal cancer. METHODS: This was a retrospective multicentre European study of patients with non-hereditary EOCRC. The impact of the European Society of Gastrointestinal Endoscopy (ESGE), U.S. Multi-Society Task Force (USMSTF), and National Comprehensive Cancer Network (NCCN) guidelines on prevention and early diagnosis was compared. Colorectal cancer was defined as potentially preventable if surveillance colonoscopy would have been performed at least 5 years before the age of diagnosis of colorectal cancer, and diagnosed early if colonoscopy was undertaken between 1 and 4 years before the diagnosis. RESULTS: Some 903 patients with EOCRC were included. Criteria for familial colorectal cancer risk in ESGE, USMSTF, and NCCN guidelines were met in 6.3, 9.4, and 30.4 per cent of patients respectively. Based on ESGE, USMSTF, and NCCN guidelines, colorectal cancer could potentially have been prevented in 41, 55, and 30.3 per cent of patients, and diagnosed earlier in 11, 14, and 21.1 per cent respectively. In ESGE guidelines, if surveillance had started 10 years before the youngest relative, there would be a significant increase in prevention (41 versus 55 per cent; P = 0.010). CONCLUSION: ESGE, USMSTF, and NCCN criteria for familial colorectal cancer were met in 6.3, 9.4, and 30.4 per cent of patients with EOCRC respectively. In these patients, early detection and/or prevention could be achieved in 52, 70, and 51.4 per cent respectively. Early and accurate identification of familial colorectal cancer risk and increase in the uptake of early colonoscopy are key to decreasing familial EOCRC.

Deciphering the complex interplay between pancreatic cancer, diabetes mellitus subtypes and obesity/BMI through causal inference and mediation analyses.

OBJECTIVES: To characterise the association between type 2 diabetes mellitus (T2DM) subtypes (new-onset T2DM (NODM) or long-standing T2DM (LSDM)) and pancreatic cancer (PC) risk, to explore the direction of causation through Mendelian randomisation (MR) analysis and to assess the mediation role of body mass index (BMI). DESIGN: Information about T2DM and related factors was collected from 2018 PC cases and 1540 controls from the PanGenEU (European Study into Digestive Illnesses and Genetics) study. A subset of PC cases and controls had glycated haemoglobin, C-peptide and genotype data. Multivariate logistic regression models were applied to derive ORs and 95% CIs. T2DM and PC-related single nucleotide polymorphism (SNP) were used as instrumental variables (IVs) in bidirectional MR analysis to test for two-way causal associations between PC, NODM and LSDM. Indirect and direct effects of the BMI-T2DM-PC association were further explored using mediation analysis. RESULTS: T2DM was associated with an increased PC risk when compared with non-T2DM (OR=2.50; 95% CI: 2.05 to 3.05), the risk being greater for NODM (OR=6.39; 95% CI: 4.18 to 9.78) and insulin users (OR=3.69; 95% CI: 2.80 to 4.86). The causal association between T2DM (57-SNP IV) and PC was not statistically significant (ORLSDM=1.08, 95% CI: 0.86 to 1.29, ORNODM=1.06, 95% CI: 0.95 to 1.17). In contrast, there was a causal association between PC (40-SNP IV) and NODM (OR=2.85; 95% CI: 2.04 to 3.98), although genetic pleiotropy was present (MR-Egger: p value=0.03). Potential mediating effects of BMI (125-SNPs as IV), particularly in terms of weight loss, were evidenced on the NODM-PC association (indirect effect for BMI in previous years=0.55). CONCLUSION: Findings of this study do not support a causal effect of LSDM on PC, but suggest that PC causes NODM. The interplay between obesity, PC and T2DM is complex.

Iron Deficiency and Recovery in Yeast: A Quantitative Proteomics Approach.

Iron is an essential element for life, as it is critical for oxygen transport, cellular respiration, DNA synthesis, and metabolism. Disruptions in iron metabolism have been associated with several complex diseases like diabetes, cancer, infection susceptibility, neurodegeneration, and others; however, the molecular mechanisms linking iron metabolism with these diseases are not fully understood. A commonly used model to study iron deficiency (ID) is yeast, Saccharomyces cerevisiae. Here, we used quantitative (phospho)proteomics to explore the early (4 and 6 h) and late (12 h) response to ID. We showed that metabolic pathways like the Krebs cycle, amino acid, and ergosterol biosynthesis were affected by ID. In addition, during the late response, several proteins related to the ubiquitin-proteasome system and autophagy were upregulated. We also explored the proteomic changes during a recovery period after 12 h of ID. Several proteins recovered their steady-state levels, but some others, such as cytochromes, did not recover during the time tested. Additionally, we showed that autophagy is active during ID, and some of the degraded proteins during ID can be rescued using KO strains for several key autophagy genes. Our results highlight the complex proteome changes occurring during ID and recovery. This study constitutes a valuable data set for researchers interested in iron biology, offering a temporal proteomic data set for ID, as well as a compendium the proteomic changes associated with episodes of iron recovery.

Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative Proteomics.

Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).

Benchmarking the Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics.

The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a ∼2× throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.

Physical activity practice and sports preferences in a group of Spanish schoolchildren depending on sex and parental care: a gender perspective.

BACKGROUND: Physical activity plays an important role in the maintenance of health, and it is especially important during childhood. However, the lack of information about differences in physical activity practice and sports preferences of children considering gender differences can result in non-effective policies that enhance inequalities between sexes. The aim of this study is to identify the sports preferences of Spanish schoolchildren and their physical activity practice behaviors depending on their sex and their parental care, analyzing the possible differences from a gender perspective. METHOD: Three hundred sixty-four Spanish schoolchildren (179 girls, 185 boys) participated in this cross-sectional study. A daily physical activity questionnaire was used to evaluate physical activity level (PAL), moderate-to-vigorous physical activity (MVPA) and sports preferences and a socio-health questionnaire were used to collect data about parental care. Statistical analysis was performed using SPSS and applying Student's T-test for normal variables, Mann-Whitney U-test for non-parametrical variables, and chi-square (χ2) test for categorical variables. Subsequently, odds ratios were used to analyze associations between the physical activity practice of the children and parental care. RESULTS: PAL and time spent in MVPA was significantly lower for girls compared to boys (1.44 ± 0.07 vs. 1.46 ± 0.07, p < 0.001 and 0.74 ± 0.40 h/day vs. 0.90 ± 0.45 h/day; p < 0.001, respectively). Dancing, rhythmic gymnastics, skating, and water sports were practiced more by girls, while football, wrestling sports, handball, and racket sports were practiced more by boys (p < 0.05). Children cared for by their fathers had higher odds for physical activity practice (OR = 1.995 (1.202-3.310), p = 0.008). CONCLUSION: Physical activity among girls was less frequent and less intense. Girls opted for individual sports with artistic connotations, while boys often practiced more team contact sports. Furthermore, children are more physically actives when their father is in charge of them.

TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.

Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses.

Web-Based Search Tool for Visualizing Instrument Performance Using the Triple Knockout (TKO) Proteome Standard.

Multiplexing strategies are at the forefront of mass-spectrometry-based proteomics, with SPS-MS3 methods becoming increasingly commonplace. A known caveat of isobaric multiplexing is interference resulting from coisolated and cofragmented ions that do not originate from the selected precursor of interest. The triple knockout (TKO) standard was designed to benchmark data collection strategies to minimize interference. However, a limitation to its widespread use has been the lack of an automated analysis platform. We present a TKO Visualization Tool (TVT). The TVT viewer allows for automated, web-based, database searching of the TKO standard, returning traditional figures of merit, such as peptide and protein counts, scan-specific ion accumulation times, as well as the TKO-specific metric, the IFI (interference-free index). Moreover, the TVT viewer allows for plotting of two TKO standards to assess protocol optimizations, compare instruments, or measure degradation of instrument performance over time. We showcase the TVT viewer by probing the selection of (1) stationary phase resin, (2) MS2 isolation window width, and (3) number of synchronous precursor selection (SPS) ions for SPS-MS3 analysis. Using the TVT viewer will allow the proteomics community to search and compare TKO results to optimize user-specific data collection workflows.

Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows.

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

Influence of Thiazole-Modified Carbon Nitride Nanosheets with Feasible Electronic Properties on Inverted Perovskite Solar Cells.

Effective, solution-processable designs of interfacial electron-transporting layers (ETLs) or hole-blocking layers are promising tools in modern electronic devices, e.g., to improve the performance, cost, and stability of perovskite-based solar cells. Herein, we introduce a facile synthetic route of thiazole-modified carbon nitride with 1.5 nm thick nanosheets which can be processed to a homogeneous, metal-free ETL for inverted perovskite solar cells. We show that thiazole-modified carbon nitride enables electronic interface enhancement via suppression of charge recombination, achieving 1.09 V in Voc and a rise to 20.17 mA/cm2 in Jsc. Hence, this report presents the successful implementation of a carbon-nitride-based structure to boost charge extraction from the perovskite absorber toward the electron transport layer in p-i-n devices.

Redefining synchronous colorectal cancers based on tumor clonality.

To analyze the possible clonal origin of a part of Synchronous colorectal cancer (SCRC), we studied 104 paired-SCRCs from 52 consecutive patients without hereditary forms of CRC. We used a Single-Nucleotide Polymorphism array to characterize the genomic profiles, and subsequently used a statistical application to define them according to clonality within the same individual. We categorized the ensuing groups according to colonic location to identify differential phenotypes. The SCRC Monoclonal group (M) (19 cases) was divided into Monosegmental (MM) and Pancolonic (MP) groups. The SCRC Polyclonal group (P) (33 cases) was also divided into Monosegmental (PM) and Pancolonic (PP), the first exhibiting preference for left colon. The MM group showed a high rate of mucinous tumors, the lowest mean-number of tumors and associated-polyps, and the worst prognosis. The MP group included the largest mean-number of associated-polyps, best prognosis and familial cancer component. The PM group seemed to be a "frontier" group. Finally, the PP group also exhibited a mucin component, the highest mean-number of tumors (4.6) compared with the mean-number of polyps (7.7), poor prognosis and sporadic cases. Most relevant differential genomic regions within M groups were gains on 1q24 and 8q24, and deletions on 1p21 and 1p23 for MM, while within P were the gains on 7q36 and deletions on 1p36 for PM. The statistical application employed seems to define clonality more accurately in SCRC -more likely to be polyclonal in origin-, and together with the tumor locations, helped us to configure a classification with prognostic and clinical value.