Lymph Node Molecular Analysis with OSNA Enables the Identification of pT1 CRC Patients at Risk of Recurrence: A Multicentre Study.

Early-stage colorectal carcinoma (CRC)-pT1-is a therapeutic challenge and presents some histological features related to lymph node metastasis (LNM). A significant proportion of pT1 CRCs are treated surgically, resulting in a non-negligible surgical-associated mortality rate of 1.5-2%. Among these cases, approximately 6-16% exhibit LNM, but the impact on survival is unclear. Therefore, there is an unmet need to establish an objective and reliable lymph node (LN) staging method to optimise the therapeutic management of pT1 CRC patients and to avoid overtreating or undertreating them. In this multicentre study, 89 patients with pT1 CRC were included. All histological features associated with LNM were evaluated. LNs were assessed using two methods, One-Step Nucleic Acid Amplification (OSNA) and the conventional FFPE plus haematoxylin and eosin (H&E) staining. OSNA is an RT-PCR-based method for amplifying CK19 mRNA. Our aim was to assess the performance of OSNA and H&E in evaluating LNs to identify patients at risk of recurrence and to optimise their clinical management. We observed an 80.9% concordance in LN assessment using the two methods. In 9% of cases, LNs were found to be positive using H&E, and in 24.7% of cases, LNs were found to be positive using OSNA. The OSNA results are provided as the total tumour load (TTL), defined as the total tumour burden present in all the LNs of a surgical specimen. In CRC, a TTL ≥ 6000 CK19 m-RNA copies/µL is associated with poor prognosis. Three patients had TTL > 6000 copies/µL, which was associated with higher tumour budding. The discrepancies observed between the OSNA and H&E results were mostly attributed to tumour allocation bias. We concluded that LN assessment with OSNA enables the identification of pT1 CRC patients at some risk of recurrence and helps to optimise their clinical management.

Tumour Cell Seeding to Lymph Nodes from In Situ Colorectal Cancer.

Lymph node (LN) metastasis is an important prognostic factor in colorectal cancer (CRC). We aimed to demonstrate the presence of lymphatic vessels (LV) in the mucosa of in-situ (pTis) CRC, and of detectable tumour burden in regional LNs. This is an observational retrospective study of 39 surgically resected in situ CRCs. The number of LVs was evaluated in both pTis and normal mucosa using D2-40 immunostains. All LNs were assessed with both H&E and the One Step Nucleic Acid Amplification (OSNA) assay, and the results were correlated with clinicopathological features. D2-40 immunohistochemisty revealed LVs in the lamina propria of all pTis CRC (100%), being absent in normal mucosa. A median of 16 LNs were freshly dissected per patient, and all cases were pN0 with H&E. Molecular LN analysis with OSNA revealed the presence of low amounts of tumour burden in 11/39 (28%) cases (range 400 to 4270 CK19 mRNA copies/µL), which had no clinical consequences. This study demonstrates the presence of LVs in the lamina propria in 100% of pTis CRC, as well as the presence of low amounts of tumour burden in regional LNs, only detected by molecular methods. Given the prognostic value of LN tumour burden, its molecular quantification may help a patient's clinical management.

Cytology Smears: An Enhanced Alternative Method for Colorectal Cancer pN Stage-A Multicentre Study.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p < 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and H&E. Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients.

Microwave-Based Colonoscopy: Preclinical Evaluation in an Ex Vivo Human Colon Model.

INTRODUCTION: Microwave imaging can obtain 360° anatomical and functional images of the colon representing the existing contrast in dielectric properties between different tissues. Microwaves are safe (nonionizing) and have the potential of reducing the visualization problems of conventional colonoscopy. This study assessed the efficacy of a microwave-based colonoscopy device to detect neoplastic lesions in an ex vivo human colon model. METHODS: Fresh surgically excised colorectal specimens containing cancer or polyps were fixed to a 3D positioning system, and the accessory device was introduced horizontally inside the ex vivo colon lumen and moved along it simulating a real colonoscopy exploration. Measurements of the colon were taken every 4 mm with the microwave-based colonoscopy device and processed with a microwave imaging algorithm. RESULTS: 14 ex vivo human colorectal specimens with carcinomas (n = 11) or adenomas with high grade dysplasia (n = 3) were examined with a microwave-based device. Using a detection threshold of 2.79 for the dielectric property contrast, all lesions were detected without false positives or false negatives. CONCLUSIONS: This study demonstrates the use of a microwave-based device to be used as an accessory of a standard colonoscope to detect neoplastic lesions in surgically excised colorectal specimens.

Copy-number intratumor heterogeneity increases the risk of relapse in chemotherapy-naive stage II colon cancer.

Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy. Here, we investigated the prognostic value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined targeted sequencing, SNP arrays, fluorescence in situ hybridization, and immunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the clonality of copy-number alterations (CNAs) and mutations, CD8+ lymphocyte infiltration, and their association with time to recurrence. Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients displayed a greater proportion of CNAs compared with non-recurrent (mean 31.3% versus 23%, respectively; p = 0.014). Furthermore, patients with elevated tumor CNA load exhibited a higher risk of recurrence compared with those with low levels [p = 0.038; hazard ratio (HR) 2.46], which was confirmed in an independent cohort (p = 0.004; HR 3.82). Candidate chromosome-specific aberrations frequently observed in recurrent cases included gain of the chromosome arm 13q (p = 0.02; HR 2.67) and loss of heterozygosity at 17q22-q24.3 (p = 0.05; HR 2.69). CNA load positively correlated with intratumor heterogeneity (R = 0.52; p < 0.0001). Consistently, incremental subclonal CNAs were associated with an elevated risk of relapse (p = 0.028; HR 2.20), which we did not observe for subclonal single-nucleotide variants and small insertions and deletions. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared with clinicopathological markers (p = 0.047). In conclusion, tumor aneuploidy and copy-number intratumor heterogeneity were predictive of poor outcome and improved discriminative performance in early-stage colon cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ex vivo Fusion Confocal Microscopy of Colorectal Polyps: A Fast Turnaround Time of Pathological Diagnosis.

BACKGROUND: Colorectal cancer screening programs have accomplished a mortality reduction from the disease but have created bottlenecks in endoscopy units and pathology departments. We aimed to explore the feasibility of ex vivo fusion confocal microscopy (FuCM) to improve the histopathology diagnostic efficiency and reduce laboratory workload. METHODS: Consecutive fresh polyps removed at colonoscopy were scanned using ex vivo FuCM, then went through histopathologic workout and hematoxylin and eosin (H&E) diagnosis. Two pathologists blinded to H&E diagnosis made a diagnosis based on FuCM scanned images. RESULTS: Thirty-six fresh polyps from 22 patients were diagnosed with FuCM and H&E. Diagnostic agreement between H&E and FuCM was 97.2% (kappa = 0.96) for pathologist #1 and 91.7% (kappa = 0.87) for pathologist #2. Diagnostic performance concordance between FuCM and H&E to discern adenomatous from nonadenomatous polyps was 100% (kappa = 1) for pathologist #1 and 97.2% (kappa = 0.94) for pathologist #2. Global interobserver agreement was 94.44% (kappa = 0.91) and kappa = 0.94 to distinguish adenomatous from nonadenomatous polyps. CONCLUSIONS: Ex vivo FuCM shows an excellent correlation with standard H&E for the diagnosis of colorectal polyps. The clinical direct benefit for patients, pathologists, and endoscopists allows adapting personalized surveillance protocols after colonoscopy and a workload decrease in pathology departments.

Lymph Node Tumor Burden Correlates With Tumor Budding and Poorly Differentiated Clusters: A New Prognostic Factor in Colorectal Carcinoma?

INTRODUCTION: Molecular lymph node (LN) staging in early colorectal cancer (CRC) has demonstrated to be more precise than conventional histopathology pN staging. Tumor budding (TB) and poorly differentiated clusters (PDCs) are associated with LN metastases, recurrences, and lower survival in CRC. We evaluated the correlation between the total tumor load (TTL) in LNs from CRC surgical specimens with patient outcome, TB, and PDC. METHODS: In this retrospective multicentre study, 5,931 LNs from 342 stage I-III CRC were analyzed by both hematoxylin and eosin and molecular detection of tumor cytokeratin 19 mRNA by one-step nucleic acid amplification. TB and PDC were evaluated by hematoxylin and eosin and cytokeratin 19 immunohistochemistry. RESULTS: One-step nucleic acid was positive in 38.3% patients (n = 131). Tumor Budding was low in 45% cases, intermediate in 25%, and high in 30%. Poorly Differentiated Clusters were low-grade G1 in 53%, G2 in 32%, and G3 in 15%. TB and PDC correlated with TTL, high-grade, lymphovascular and perineural invasion, pT, pN and stage (P < 0.001). TB, PDC, and TTL ≥ 6,000 copies/µL were associated with worse overall survival (P = 0.002, P = 0.013, and P = 0.046) and disease-free survival (P < 0.001). DISCUSSION: The implementation of more sensitive molecular methods to assess LN status is a promising alternative approach to pN staging, which could be integrated to other factors to help risk stratification and management of patients with early-stage CRC. This study demonstrates the correlation of the amount of LN tumor burden with TB and PDCs. TTL is related to the outcome and could be used as a new prognostic factor in CRC (see Visual Abstract, Supplementary Digital Content 2, http://links.lww.com/CTG/A512).