The Complexity of Shapes: How the Circularity of Tumor Nodules Affects Prognosis in Colorectal Cancer.

The current stratification of tumor nodules in colorectal cancer (CRC) staging is subjective and leads to high interobserver variability. In this study, the objective assessment of the shape of lymph node metastases (LNMs), extranodal extension (ENE), and tumor deposits (TDs) was correlated with outcomes. A test cohort and a validation cohort were included from 2 different institutions. The test cohort consisted of 190 cases of stage III CRC. Slides with LNMs and TDs were annotated and processed using a segmentation algorithm to determine their shape. The complexity ratio was calculated for every shape and correlated with outcomes. A cohort of 160 stage III CRC cases was used to validate findings. TDs showed significantly more complex shapes than LNMs with ENE, which were more complex than LNMs without ENE (P < .001). In the test cohort, patients with the highest sum of complexity ratios had significantly lower disease-free survival (P < .01). When only the nodule with the highest complexity was considered, this effect was even stronger (P < .001). This maximum complexity ratio per patient was identified as an independent prognostic factor in the multivariate analysis (hazard ratio, 2.47; P < .05). The trends in the validation cohort confirmed the results. More complex nodules in stage III CRC were correlated with significantly worse disease-free survival, even if only based on the most complex nodule. These results suggest that more complex nodules reflect more invasive tumor biology. As most of the more complex nodules were diagnosed as TDs, we suggest providing a more prominent role for TDs in the nodal stage and include an objective complexity measure in their definition.

Pseudobudding: ruptured glands do not represent true tumor buds.

Tumor budding (TB) is a strong biomarker of poor prognosis in colorectal cancer and other solid cancers. TB is defined as isolated single cancer cells or clusters of up to four cancer cells at the invasive tumor front. In areas with a large inflammatory response at the invasive front, single cells and cell clusters surrounding fragmented glands are observed appearing like TB. Occurrence of these small groups is referred to as pseudobudding (PsB), which arises due to external influences such as inflammation and glandular disruption. Using a combination of orthogonal approaches, we show that there are clear biological differences between TB and PsB. TB is representative of active invasion by presenting features of epithelial-mesenchymal transition and exhibiting increased deposition of extracellular matrix within the surrounding tumor microenvironment (TME), whereas PsB represents a reactive response to heavy inflammation where increased levels of granulocytes within the surrounding TME are observed. Our study provides evidence that areas with a strong inflammatory reaction should be avoided in the routine diagnostic assessment of TB. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Transcriptomics and proteomics reveal distinct biology for lymph node metastases and tumour deposits in colorectal cancer.

Both lymph node metastases (LNMs) and tumour deposits (TDs) are included in colorectal cancer (CRC) staging, although knowledge regarding their biological background is lacking. This study aimed to compare the biology of these prognostic features, which is essential for a better understanding of their role in CRC spread. Spatially resolved transcriptomic analysis using digital spatial profiling was performed on TDs and LNMs from 10 CRC patients using 1,388 RNA targets, for the tumour cells and tumour microenvironment. Shotgun proteomics identified 5,578 proteins in 12 different patients. Differences in RNA and protein expression were analysed, and spatial deconvolution was performed. Image-based consensus molecular subtype (imCMS) analysis was performed on all TDs and LNMs included in the study. Transcriptome and proteome profiles identified distinct clusters for TDs and LNMs in both the tumour and tumour microenvironment segment, with upregulation of matrix remodelling, cell adhesion/motility, and epithelial-mesenchymal transition (EMT) in TDs (all p < 0.05). Spatial deconvolution showed a significantly increased abundance of fibroblasts, macrophages, and regulatory T-cells (p < 0.05) in TDs. Consistent with a higher fibroblast and EMT component, imCMS classified 62% of TDs as poor prognosis subtype CMS4 compared to 36% of LNMs (p < 0.05). Compared to LNMs, TDs have a more invasive state involving a distinct tumour microenvironment and upregulation of EMT, which are reflected in a more frequent histological classification of TDs as CMS4. These results emphasise the heterogeneity of locoregional spread and the fact that TDs should merit more attention both in future research and during staging. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Computer-Assisted Diagnosis of Lymph Node Metastases in Colorectal Cancers Using Transfer Learning With an Ensemble Model.

Screening of lymph node metastases in colorectal cancer (CRC) can be a cumbersome task, but it is amenable to artificial intelligence (AI)-assisted diagnostic solution. Here, we propose a deep learning-based workflow for the evaluation of CRC lymph node metastases from digitized hematoxylin and eosin-stained sections. A segmentation model was trained on 100 whole-slide images (WSIs). It achieved a Matthews correlation coefficient of 0.86 (±0.154) and an acceptable Hausdorff distance of 135.59 µm (±72.14 µm), indicating a high congruence with the ground truth. For metastasis detection, 2 models (Xception and Vision Transformer) were independently trained first on a patch-based breast cancer lymph node data set and were then fine-tuned using the CRC data set. After fine-tuning, the ensemble model showed significant improvements in the F1 score (0.797-0.949; P <.00001) and the area under the receiver operating characteristic curve (0.959-0.978; P <.00001). Four independent cohorts (3 internal and 1 external) of CRC lymph nodes were used for validation in cascading segmentation and metastasis detection models. Our approach showed excellent performance, with high sensitivity (0.995, 1.0) and specificity (0.967, 1.0) in 2 validation cohorts of adenocarcinoma cases (n = 3836 slides) when comparing slide-level labels with the ground truth (pathologist reports). Similarly, an acceptable performance was achieved in a validation cohort (n = 172 slides) with mucinous and signet-ring cell histology (sensitivity, 0.872; specificity, 0.936). The patch-based classification confidence was aggregated to overlay the potential metastatic regions within each lymph node slide for visualization. We also applied our method to a consecutive case series of lymph nodes obtained over the past 6 months at our institution (n = 217 slides). The overlays of prediction within lymph node regions matched 100% when compared with a microscope evaluation by an expert pathologist. Our results provide the basis for a computer-assisted diagnostic tool for easy and efficient lymph node screening in patients with CRC.

The Impact of Primary Tumor Location in Synchronous Metastatic Colorectal Cancer: Differences in Metastatic Sites and Survival.

PURPOSE: We explored differences in survival between primary tumor locations, hereby focusing on the role of metastatic sites in synchronous metastatic colorectal cancer (mCRC). METHODS: Data for patients diagnosed with synchronous mCRC between 1989 and 2014 were retrieved from the Netherlands Cancer registry. Relative survival and relative excess risks (RER) were analyzed by primary tumor location (right colon (RCC), left colon (LCC), and rectum). Metastatic sites were reported per primary tumor location. Survival was analyzed for metastatic sites combined and for single metastatic sites. RESULTS: In total, 36,297 patients were included in this study. Metastatic sites differed significantly between primary tumor locations, with liver-only metastases in 43%, 54%, and 52% of RCC, LCC, and rectal cancer patients respectively (p < 0.001). Peritoneal metastases were most prevalent in RCC patients (33%), and lung metastases were most prevalent in rectal cancer patients (28%). Regardless of the location of metastases, patients with RCC had a worse survival compared with LCC (RER 0.81, 95% CI 0.78-0.83) and rectal cancer (RER 0.73, 95% CI 0.71-0.76). The survival disadvantage for RCC remained present, even in cases with metastasectomy for liver-only disease (LCC: RER 0.66, 95% CI 0.57-0.76; rectal cancer: RER 0.84, 95% CI 0.66-1.06). CONCLUSIONS: This study showed significant differences in relative survival between primary tumor locations in synchronous mCRC, which can only be partially explained by distinct metastatic sites. Our findings support the concept that RCC, LCC and rectal cancer should be considered distinct entities in synchronous mCRC.

An overview of 25 years of incidence, treatment and outcome of colorectal cancer patients.

Regarding the continuous changes in the diagnostic process and treatment of colorectal cancer (CRC), it is important to evaluate long-term trends which are relevant in giving direction for further research and innovations in cancer patient care. The aim of this study was to analyze developments in incidence, treatment and survival for patients diagnosed with CRC in the Netherlands. For this population-based retrospective cohort study, all patients diagnosed with CRC between 1989 and 2014 in the Netherlands were identified using data of the nationwide population-based Netherlands Cancer Registry (n = 267,765), with follow-up until January 1, 2016. Analyses were performed for trends in incidence, mortality, stage distribution, treatment and relative survival measured from the time of diagnosis. The incidence of both colon and rectal cancer has risen. The use of postoperative chemotherapy for Stage III colon cancer increased (14-60%), as well as the use of preoperative (chemo)radiotherapy for rectal cancer (2-66%). The administration of systemic therapy and metastasectomy increased for Stage IV disease patients. The 5-year relative survival increased significantly from 53 to 62% for colon cancer and from 51 to 65% for rectal cancer. Ongoing advancements in treatment, and also improvement in other factors in the care of CRC patients-such as diagnostics, dedicated surgery and pre- and postoperative care-lead to a continuous improvement in the relative survival of CRC patients. The increasing incidence of CRC favors the implementation of the screening program, of which the effects should be monitored closely.

Incidence, treatment and relative survival of early-onset colorectal cancer in the Netherlands since 1989.

AIM: Previous studies showed that the incidence of early-onset colorectal cancer (EO-CRC, diagnosis <50 years) is rising in Western countries. Additionally, young patients present with more advanced disease. Integrated nationwide assessment of epidemiologically and clinically relevant trends would provide more insight into this specific group of patients with CRC. We aimed to provide an analysis of trends in age- and stage-specific incidence, characteristics, treatment and relative survival of patients with EO-CRC in the Netherlands and compare these with 50- to 59-year-old patients. METHODS: Data from 1989 to 2018 were retrieved from the Netherlands Cancer Registry. Non-standardised age-specific incidence rates were calculated, and trends were assessed using Joinpoint regression. Treatment and 5-year relative survival trends were provided and compared between EO-CRC and 50- to 59-year-old patients. RESULTS: The EO-CRC incidence annually increased with 0.7-2.1% over the last decades. CRC incidence for the 50- to 59-year-old population annually increased with 0.8-1.7% until 2006 and showed a major increase in incidence after the introduction of nationwide screening in 2014. Stage III and Stage IV CRC primarily increased across the studied age groups, while Stage I and Stage II CRC did not. Patients with EO-CRC received multimodal treatment more often than 50- to 59-year-old patients, but differences were minor. Relative survival increased over time and showed little differences between EO-CRC and 50- to 59-year-old patients. CONCLUDING STATEMENT: Only few epidemiological and clinical differences were found between EO-CRC and 50- to 59-year-old patients; hence, the urge for a specific approach of EO-CRC in screening and treatment guidelines might be tempered.

Interobserver variation in the classification of tumor deposits in rectal cancer-is the use of histopathological characteristics the way to go?

The focus on lymph node metastases (LNM) as the most important prognostic marker in colorectal cancer (CRC) has been challenged by the finding that other types of locoregional spread, including tumor deposits (TDs), extramural venous invasion (EMVI), and perineural invasion (PNI), also have significant impact. However, there are concerns about interobserver variation when differentiating between these features. Therefore, this study analyzed interobserver agreement between pathologists when assessing routine tumor nodules based on TNM 8. Electronic slides of 50 tumor nodules that were not treated with neoadjuvant therapy were reviewed by 8 gastrointestinal pathologists. They were asked to classify each nodule as TD, LNM, EMVI, or PNI, and to list which histological discriminatory features were present. There was overall agreement of 73.5% (κ 0.38, 95%-CI 0.33-0.43) if a nodal versus non-nodal classification was used, and 52.2% (κ 0.27, 95%-CI 0.23-0.31) if EMVI and PNI were classified separately. The interobserver agreement varied significantly between discriminatory features from κ 0.64 (95%-CI 0.58-0.70) for roundness to κ 0.26 (95%-CI 0.12-0.41) for a lone arteriole sign, and the presence of discriminatory features did not always correlate with the final classification. Since extranodal pathways of spread are prognostically relevant, classification of tumor nodules is important. There is currently no evidence for the prognostic relevance of the origin of TD, and although some histopathological characteristics showed good interobserver agreement, these are often non-specific. To optimize interobserver agreement, we recommend a binary classification of nodal versus extranodal tumor nodules which is based on prognostic evidence and yields good overall agreement.

Interaction Between Primary Tumor Resection, Primary Tumor Location, and Survival in Synchronous Metastatic Colorectal Cancer: A Population-Based Study.

OBJECTIVES: Location of the primary tumor has prognostic value and predicts the effect of certain therapeutics in synchronous metastatic colorectal cancer. We investigated whether the association between primary tumor resection (PTR) and overall survival (OS) also depends on tumor location. METHODS: Data on synchronous metastatic colorectal cancer patients from the Netherlands Cancer Registry (n=16,106) and Surveillance, Epidemiology, and End Results (SEER) registry (n=19,584) were extracted. Cox models using time-varying covariates were implemented. Median OS for right-sided colon cancer (RCC), left-sided colon cancer, and rectal cancer was calculated using inverse probability weighting and a landmark point of 6 months after diagnosis as reference. RESULTS: The association between PTR and OS was dependent on tumor location (P<0.05), with a higher median OS of upfront PTR versus upfront systemic therapy in Netherlands Cancer Registry (NCR) of 1.9 (95% confidence interval: 0.9-2.8), 4.3 (3.3-5.6), and 3.4 (0.6-7.6) months in RCC, left-sided colon cancer and rectal cancer, respectively. In SEER data, the difference was 6.0 (4.0-8.0), 8.0 (5.0-10.0), and 10.0 (7.0-13.0) months, respectively. Hazard plots indicate a higher hazard of death 2 to 3 months after PTR in RCC. CONCLUSION: Upfront PTR is associated with improved survival regardless of primary tumor location. Patients with RCC appear to have less benefit because of higher mortality during 2 to 3 months after PTR.

Characteristics of Early-Onset vs Late-Onset Colorectal Cancer: A Review.

Importance: The incidence of early-onset colorectal cancer (younger than 50 years) is rising globally, the reasons for which are unclear. It appears to represent a unique disease process with different clinical, pathological, and molecular characteristics compared with late-onset colorectal cancer. Data on oncological outcomes are limited, and sensitivity to conventional neoadjuvant and adjuvant therapy regimens appear to be unknown. The purpose of this review is to summarize the available literature on early-onset colorectal cancer. Observations: Within the next decade, it is estimated that 1 in 10 colon cancers and 1 in 4 rectal cancers will be diagnosed in adults younger than 50 years. Potential risk factors include a Westernized diet, obesity, antibiotic usage, and alterations in the gut microbiome. Although genetic predisposition plays a role, most cases are sporadic. The full spectrum of germline and somatic sequence variations implicated remains unknown. Younger patients typically present with descending colonic or rectal cancer, advanced disease stage, and unfavorable histopathological features. Despite being more likely to receive neoadjuvant and adjuvant therapy, patients with early-onset disease demonstrate comparable oncological outcomes with their older counterparts. Conclusions and Relevance: The clinicopathological features, underlying molecular profiles, and drivers of early-onset colorectal cancer differ from those of late-onset disease. Standardized, age-specific preventive, screening, diagnostic, and therapeutic strategies are required to optimize outcomes.

Gfi1b regulates the level of Wnt/ß-catenin signaling in hematopoietic stem cells and megakaryocytes.

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with ß-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/ß-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite ß-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/ß-catenin target genes. Consistently, numerous canonical Wnt/ß-catenin target genes, co-occupied by Gfi1b, ß-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/ß-catenin signaling pathway.

The gap in postoperative outcome between older and younger patients with stage I-III colorectal cancer has been bridged; results from the Netherlands cancer registry.

AIM OF THE STUDY: Previous studies have shown that older patients benefited less than younger patients from surgical treatment for colorectal cancer (CRC). However, CRC care has advanced over time, and it is time to assess whether the difference in postoperative mortality between older and younger CRC patients is still present. METHODS: Patients with primary stage I-III CRC diagnosed between 2005 and 2016 were selected from the Netherlands Cancer Registry (N = 111,778). Trends in postoperative mortality and 1-year postoperative relative survival (RS) were analysed, stratified according to age (<75 versus ≥75 years) and tumour location (colon versus rectum). One-year postoperative RS was analysed to correct for background mortality in the older population. RESULTS: Between 2005 and 2016, 30-day postoperative mortality showed a stronger decrease for older patients (from 10.0% to 4.0% for colon cancer [p < 0.001] and from 8.3% to 2.7% for rectal cancer [p < 0.001]) compared with younger patients (from 2.0% to 0.9% for colon cancer [p < 0.001] and from 1.4% to 0.7% for rectal cancer [p = 0.01]). Between 2005 and 2016, also 1-year RS increased more for older patients (from 84.8% to 94.6% for colon cancer and from 86.1% to 97.2% for rectal cancer) compared with younger patients (from 94.0% to 97.8% for colon cancer and from 96.3% to 98.8% for rectal cancer). CONCLUSION: Between 2005 and 2016, differences in postoperative mortality between older and younger CRC patients decreased. One-year postoperative RS was almost equal for older and younger patients in 2015-2016. This information is crucial for shared decision-making on surgical treatment.

Clinical lymph node staging in colorectal cancer; a flip of the coin?

BACKGROUND: This study aims to provide insight in the quality of current daily practice in clinical lymph node staging in colorectal cancer (CRC) in the Netherlands. METHODS: Data of the nationwide population-based Netherlands Cancer Registry between 2003 and 2014 were used to analyze lymph node staging for cM0 CRC patients. Accuracy of clinical lymph node staging was calculated for the period 2011-2014. Analyses were performed for patients without preoperative treatment or treated with short-course radiotherapy (SCRT) followed by resection. RESULTS: 100,211 patients were included for analysis. The proportion clinically positive lymph nodes increased significantly between 2003 and 2014 (6%-22% for colon cancer; 7%-53% for rectal cancer). The proportion histological positive lymph nodes remained stable (±35% colon, ±33% rectum). Data from 2011 to 2014 yielded a sensitivity, specificity, positive and negative predictive value of 41%, 84%, 59% and 71% for colon cancer, respectively (n = 21,629). This was 38%, 87%, 56%, 76% for rectal cancer without SCRT, (n = 2178) and 56%, 67%, 47% and 75% for rectal cancer with SCRT (n = 3401), respectively. CONCLUSION: Accuracy of clinical lymph node staging in colorectal cancer patients is about as accurate as flipping a coin. This may lead to overtreatment of rectal cancer patients. Acceptable specificity and NPV limit the risk of undertreatment.

Generation and characterization of human iPSC line MML-6838-Cl2 from mobilized peripheral blood derived megakaryoblasts.

Mobilized peripheral blood (MPB) CD34+ cells were cultured to CD41+/CD34+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from megakaryoblasts.