The risk of lymph node metastasis in patients with T2 colon cancer.

AIM: It is often safe to treat pT1 tumours with local resection due to the low risk of lymph node metastasis. The risk of lymph node metastasis in pT2 colon cancer is less well investigated. The recommendation for patients diagnosed with T2 colon cancer is a segmental resection including regional lymph nodes. The aim of this work was to determine the risk of lymph node metastasis in pT2 colon cancer and identify the possible associated clinical and pathological risk factors for lymph node metastasis. METHOD: PubMed and Embase were systematically searched for studies describing patients with T2 colon cancer and lymph node status after histopathological assessment. Lymph node metastasis and the effect of histological and clinical factors were included. RESULTS: Overall, 5489 studies were screened, and 10 studies consisting of a total of 91 460 patients were included in the review. The overall risk of lymph node metastasis was 19.3% (95% confidence interval 19.0%-19.5%). A meta-analysis was not possible as very few studies described the clinical and pathological risk factors for lymph node metastasis. CONCLUSION: The risk of lymph node metastasis in patients with pT2 colon cancers is higher than for pT1. The studies included patients operated on from 1985 to 2015 with variations in surgical procedure, pathological handling, and definition of lymph node metastasis. Further studies reporting risk factors for lymph node metastasis in pT2 colon cancer are warranted as more data are needed to determine if local resection for a subgroup of patients could be an alternative treatment modality.

The topographical distribution of lymph node metastases in colon cancer resections.

In accordance with international guidelines all lymph nodes in colon cancer specimens must be examined to obtain accurate staging. This study aimed to determine the topographical location of lymph node metastases and evaluate if a more limited sampling approach could be an alternative. Partial colectomies received at the Department of Pathology, Zealand University Hospital during a six-month period were included. At the macroscopic examination, each specimen was divided into three different segments: a segment containing the index tumor and the tumor-feeding artery, an oral and an anal segment. The number of lymph nodes and lymph node metastases were registered separately for each segment. Resections from 93 patients were included. Of 2466 lymph nodes, 1839 (74.6 %) were located in the tumor segment, 308 (12.5 %) in the oral, and 319 (12.9 %) in the anal segment, respectively. In 133 (5,4 %) lymph nodes a metastasis was present. Of these 129 (97.0 %) were located in the tumor segment, one (0.8 %) in the oral segment, and three (2.3 %) in the anal segment. No patients had metastasis in the oral or anal segments without metastases also being present in the tumor segment leading to consideration of the need for lymph node harvest of the complete specimen upon initial examination. As such, the segment containing the index tumor and tumor-feeding artery could be regarded as a sentinel segment indicating a potential need for lymph node dissection in the oral and anal segments.

An exploration of immunohistochemistry-based prognostic markers in patients undergoing curative resections for colon cancer.

BACKGROUND: The immune system recognizes and destroys cancer cells. However, cancer cells develop mechanisms to avoid detection by expressing cell surface proteins. Specific tumour cell surface proteins (e.g. HLA-G, PD-L1, CDX2) either alone or in combination with the relative presence of immune cells (CD3 and CD8 positive T-cells) in the tumour tissue may describe the cancer cells' ability to escape eradication by the immune system. The aim was to investigate the prognostic value of immunohistochemical markers in patients with colon cancer. METHODS: We conducted a retrospective study including patients diagnosed with pT3 and pT4 colon cancers. Immunohistochemical staining with HLA-G, PD-L1, CDX2, CD3, and CD8 was performed on tissue samples with representation of the invasive margin. PD-L1 expression in tumour cells and immune cells was reported conjointly. The expression of CD3 and CD8 was reported as a merged score based on the expression of both markers in the invasive margin and the tumour centre. Subsequently, a combined marker score was established based on all of the markers. Each marker added one point to the score when unfavourable immunohistochemical features was present, and the score was categorized as low, intermediate or high depending on the number of unfavourable stains. Hazard ratios for recurrence, disease-free survival and mortality were calculated. RESULTS: We included 188 patients undergoing colon cancer resections in 2011-2012. The median follow-up was 41.7 months, during which 41 (21.8%) patients had recurrence and 74 (39.4%) died. In multivariable regression analysis positive HLA-G expression (HR = 3.37, 95%CI [1.64-6.93]) was associated with higher recurrence rates, while a preserved CDX2 expression (HR = 0.23, 95%CI [0.06-0.85]) was associated with a lower risk of recurrence. An intermediate or high combined marker score was associated with increased recurrence rates (HR = 20.53, 95%CI [2.68-157.32] and HR = 7.56, 95%CI [1.06-54.16], respectively). Neither high expression of PD-L1 nor high CD3-CD8 score was significantly associated with recurrence rates. Patients with a high CD3-CD8 score had a significantly longer DFS and OS. CONCLUSIONS: In tumour cells, expression of HLA-G and loss of CDX2 expression were associated with cancer recurrence. In addition, a combination of certain tumour tissue biomarkers was associated with colorectal cancer recurrence.

Colonic Stent as Bridge to Surgery for Malignant Obstruction Induces Gene Expressional Changes Associated with a More Aggressive Tumor Phenotype.

BACKGROUND: Colonic stent is recommended as a bridge to elective surgery for malignant obstruction to improve short-term clinical outcomes for patients with colorectal cancer. However, since the oncological outcomes remain controversial, this study aimed to investigate the impact of self-expandable metallic stent (SEMS) on the tumor microenvironment. METHODS: Patients treated with colonic stent as a bridge to surgery from 2010 to 2015 were identified from hospital records. Tumor biopsies and resected tumor samples of the eligible patients were retrieved retrospectively. Gene expression analysis was performed using the NanoString nCounter PanCancer IO 360 gene expression panel. RESULTS: Of the 164 patients identified, this study included 21 who underwent colonic stent placement as a bridge to elective surgery. Gene expression analysis revealed 82 differentially expressed genes between pre- and post-intervention specimens, of which 72 were upregulated and 10 downregulated. Among the significantly upregulated genes, 46 are known to have protumor functions, of which 26 are specifically known to induce tumorigenic mechanisms such as proliferation, migration, invasion, angiogenesis, and inflammation. In addition, ten differentially expressed genes were identified that are known to promote antitumor functions. CONCLUSION: SEMS induces gene expressional changes in the tumor microenvironment that are associated with tumor progression in colorectal cancer and may potentiate a more aggressive phenotype. Future studies are warranted to establish optimal timing of surgery after SEMS insertion in patients with obstructive colorectal cancer.

Distribution of histopathological features along the colon in microscopic colitis.

PURPOSE: The diagnosis microscopic colitis (MC) consisting of collagenous colitis (CC) and lymphocytic colitis (LC) relies on histological assessment of mucosal biopsies from the colon. The optimal biopsy strategy for reliable diagnosis of MC is controversial. The aim of this study was to evaluate the distribution of histopathological features of MC throughout the colon. METHODS: Mucosal biopsies from multiple colonic segments of patients with MC who participated in one of the three prospective European multicenter trials were analyzed. Histological slides were stained with hematoxylin-and-eosin, a connective tissue stain, and CD3 in selected cases. RESULTS: In total, 255 patients were included, 199 and 56 patients with CC and LC, respectively. Both groups exhibited a gradient with more pronounced inflammation in the lamina propria in the proximal colon compared with the distal colon. Similarly, the thickness of the subepithelial collagenous band in CC showed a gradient with higher values in the proximal colon. The mean number of intraepithelial lymphocytes was > 20 in all colonic segments in patients within both subgroups. Biopsies from 86 to 94% of individual segments were diagnostic, rectum excluded. Biopsies from non-diagnostic segments often showed features of another subgroup of MC. CONCLUSION: Conclusively, although the severity of the histological changes in MC differed in the colonic mucosa, the minimum criteria required for the diagnosis were present in the random biopsies from the majority of segments. Thus, our findings show MC to be a pancolitis, rectum excluded, questioning previously proclaimed patchiness throughout the colon.

Quantifying intraepithelial lymphocytes and subepithelial collagen band in microscopic colitis, extracting insights into the interrelationship of lymphocytic and collagenous colitis.

Microscopic colitis (MC) is the umbrella term for the conditions termed lymphocytic colitis (LC) and collagenous colitis (CC). LC with thickening of the subepithelial collagen band or CC with increased number of intraepithelial T- lymphocytes (IELs) is often seen in MC and may lead to difficulties in correct histological classification. We investigated the extent of overlapping features of CC and LC in 60 cases of MC by measuring the exact thickness of the subepithelial collagen band in Van Gieson stained slides and quantifying number of IELs in CD3 stained slides by digital image analysis. A thickened collagen band was observed in nine out of 29 cases with LC (31%) and an increased number of IELs in all 23 cases of CC (100%). There was no correlation between the thickness of the collagen band and number of IELs. Due to the increased number of IELs in all cases of CC we consider the lymphocytic inflammatory infiltration of the mucosa to be the essential histopathological feature of MC. However, although LC and CC are related due to the lymphocytic inflammation, the non-linear correlation of number of IELs and thickness of the collagenous band indicate differences in their pathogenesis.

Isolated tumor cells in the regional lymph nodes in patients with squamous cell carcinoma of the esophagus are rarely observed but often represent part of a true metastasis.

The most common malignancy of the esophagus is squamous cell carcinoma (SCC) and regional lymph node metastases are an important prognostic factor. Isolated tumor cells (ITCs) are defined as single tumor cells or small clusters of tumor cells not exceeding 0.2 mm. The prognostic role of ITCs is not clear. This study aimed to determine the prevalence of ITCs in regional lymph nodes in patients with esophageal SCC and to investigate how frequently ITCs represent part of a true metastasis. Surgical specimens from 100 patients with SCC of the esophagus were included. All original H&E stained slides containing lymph nodes were reviewed by two gastrointestinal pathologists. In lymph nodes containing ITCs, additional levels were cut and stained with a H&E- and a cytokeratin stain. Areas of tumor cells that measured >0.2 mm on the deeper sections were classified as metastases. A total of 2460 lymph nodes were examined. ITCs were detected in 10 lymph nodes (0.4%) from nine patients (9%). Deeper sections revealed metastases in five out of the 10 lymph nodes (50%). ITCs in regional lymph nodes of patients with SCC of the esophagus is a rare finding compared with patients with adenocarcinoma of the esophagogastric junction. However, deeper sections often revealed metastases. Therefore, in patients with SCC of the esophagus, we recommend additional sectioning and immunohistochemical examination of lymph nodes when ITCs are detected on the first slide.

An investigation of European pathologists' approach to diagnose microscopic colitis.

Microscopic colitis (MC) comprising lymphocytic colitis (LC), collagenous colitis (CC) and the incomplete forms of microscopic colitis (MCi) are frequent causes of chronic watery diarrhea. The diagnosis is based on specific histological features in colonic biopsies. Especially regarding MCi, the histological features may be subtle. The PRO-MC collaboration was established in 2016 with the aims to systematically describe the disease course and to validate the diagnostic criteria of MC. In the present study, we analysed pathologists' initial approach to diagnose MC. Five pathologists with expertise in gastro-intestinal pathology reviewed the first 10 cases enrolled in the PRO-MC registry in six of the participating centres. Despite considerable differences in strategies in biopsy sampling, in choice of stains and in minimum number of biopsies and segments required for diagnosing MC, inter-observer agreement between the participating centres and expert pathologists as well as among the latter was substantial. Disagreed cases most often related to difficulties in distinguishing between MC subgroups. We recommend that pathologists as well as clinicians reach consensus in their diagnostic approach to MC, which is a prerequisite to compare MC cohorts internationally and to facilitate clinical MC trials and follow-up studies.

Digitally assessed lymphocyte infiltration in rectal cancer biopsies is associated with pathological response to neoadjuvant therapy.

A frequently used treatment strategy in locally advanced rectal cancer (RC) is neoadjuvant therapy followed by surgery. Patients treated with neoadjuvant therapy achieve varying pathological response, and currently, predicting the degree of response is challenging. This study examined the association between digitally assessed histopathological features in the diagnostic biopsies and pathological response to neoadjuvant therapy, aiming to find potential predictive biomarkers. 50 patients with RC treated with neoadjuvant chemotherapy and/or radiotherapy followed by surgery were included. Deep learning-based digital algorithms were used to assess the epithelium tumor area percentage (ETP) based on H&E-stained slides, and to quantify the density of CD3+ and CD8+ lymphocytes, as well as the CD8+/CD3+ lymphocyte percentage, based on immunohistochemically stained slides, from the diagnostic tumor biopsies. Pathological response was assessed according to the Mandard method. A good pathological response was defined as tumor regression grade (TRG) 1-2, and a complete pathological response was defined as Mandard TRG 1. Associations between the ETP and lymphocyte densities in the diagnostic biopsies and the pathological response were examined. The density of CD8+ lymphocytes, and the CD8+/CD3+ lymphocyte percentage, were associated with both good and complete response to neoadjuvant therapy, while the density of CD3+ lymphocytes was associated with complete response. The ETP did not correlate with response to neoadjuvant therapy. It is well-known that infiltration of lymphocytes in colorectal cancer is a prognostic biomarker. However, assessment of CD8+ and CD3+ lymphocytes in the diagnostic tumor biopsies of patients with RC may also be useful in predicting response to neoadjuvant therapy.

Number of intraepithelial lymphocytes and presence of a subepithelial band in normal colonic mucosa differs according to stainings and evaluation method.

Chronic watery diarrhea is a frequent symptom. In approximately 10% of the patients, a diagnosis of microscopic colitis (MC) is established. The diagnosis relies on specific, but sometimes subtle, histopathological findings. As the histology of normal intestinal mucosa vary, discriminating subtle features of MC from normal tissue can be challenging and therefore auxiliary stainings are increasingly used. The aim of this study was to determine the variance in number of intraepithelial lymphocytes (IELs) and presence of a subepithelial band in normal ileum and colonic mucosa, according to different stains and digital assessment. Sixty-one patients without diarrhea referred to screening colonoscopy due to a positive feacal blood test and presenting with endoscopically normal mucosa were included. Basic histological features, number of IELs, and thickness of a subepithelial band was manually evaluated and a deep learning-based algorithm was developed to digitally determine the number of IELs in each of the two compartments; surface epithelium and cryptal epithelium, and the density of lymphocytes in the lamina propria compartment. The number of IELs was significantly higher on CD3-stained slides compared with slides stained with Hematoxylin-and-Eosin (HE) (p<0.001), and even higher numbers were reached using digital analysis. No significant difference between right and left colon in IELs or density of CD3-positive lymphocytes in lamina propria was found. No subepithelial band was present in HE-stained slides while a thin band was visualized on special stains. Conclusively, in this cohort of prospectively collected ileum and colonic biopsies from asymptomatic patients, the range of IELs and detection of a subepithelial collagenous band varied depending on the stain and method used for assessment. As assessment of biopsies from patients with diarrhea constitute a considerable workload in the pathology departments digital image analysis is highly desired. Knowledge provided by the present study highlight important differences that should be considered before introducing this method in the clinic.

Detection of collision metastases.

Cancer is a cause of high morbidity and mortality. It is not uncommon for a patient to have more than one primary tumour. This review summarises the knowledge of collision tumours which are defined as two adjacent neoplasms in the same organ, while a collision metastasis is the rare occurrence of two different primary cancers metastasising to the same anatomical site. Identification of collision metastasis is a diagnostic challenge and relies on histopathological examination. As it might have profound impact on prognosis and treatment decisions, it is important to create awareness among both pathologists and clinicians of this phenomenon.

Endoscopic calcium electroporation for colorectal cancer: a phase I study.

Background and study aims Colorectal cancer is one of the most common malignancies, with approximately 20 % of patients having metastatic disease. Local symptoms from the tumor remain a common issue and affect quality of life. Electroporation is a method to permeabilize cell membranes with high-voltage pulses, allowing increased passage of otherwise poorly permeating substances such as calcium. The aim of this study was to determine the safety of calcium electroporation for advanced colorectal cancer. Patients and methods Six patients with inoperable rectal and sigmoid colon cancer were included, all presenting with local symptoms. Patients were offered endoscopic calcium electroporation and were followed up with endoscopy and computed tomography/magnetic resonance scans. Biopsies and blood samples were collected at baseline and at follow-up, 4, 8, and 12 weeks after treatment. Biopsies were examined for histological changes and immunohistochemically with CD3/CD8 and PD-L1. In addition, blood samples were examined for circulating cell-free DNA (cfDNA). Results A total of 10 procedures were performed and no serious adverse events occurred. Prior to inclusion, patients reported local symptoms, such as bleeding (N = 3), pain (N = 2), and stenosis (N = 5). Five of six patients reported symptom relief. In one patient, also receiving systemic chemotherapy, clinical complete response of primary tumor was seen. Immunohistochemistry found no significant changes in CD3 /CD8 levels or cfDNA levels after treatment. Conclusions This first study of calcium electroporation for colorectal tumors shows that calcium electroporation is a safe and feasible treatment modality for colorectal cancer. It can be performed as an outpatient treatment and may potentially be of great value for fragile patients with limited treatment options.

Neoadjuvant intratumoral influenza vaccine treatment in patients with proficient mismatch repair colorectal cancer leads to increased tumor infiltration of CD8+ T cells and upregulation of PD-L1: a phase 1/2 clinical trial.

BACKGROUND: In colorectal cancer, the effects of immune checkpoint inhibitors are mostly limited to patients with deficient mismatch repair tumors, characterized by a high grade infiltration of CD8+T cells. Interventions aimed at increasing intratumoral CD8+T-cell infiltration in proficient mismatch repair tumors are lacking. METHODS: We conducted a proof of concept phase 1/2 clinical trial, where patients with non-metastasizing sigmoid or rectal cancer, scheduled for curative intended surgery, were treated with an endoscopic intratumorally administered neoadjuvant influenza vaccine. Blood and tumor samples were collected before the injection and at the time of surgery. The primary outcome was safety of the intervention. Evaluation of pathological tumor regression grade, immunohistochemistry, flow cytometry of blood, tissue bulk transcriptional analyses, and spatial protein profiling of tumor regions were all secondary outcomes. RESULTS: A total of 10 patients were included in the trial. Median patient age was 70 years (range 54-78), with 30% women. All patients had proficient mismatch repair Union of International Cancer Control stage I-III tumors. No endoscopic safety events occurred, with all patients undergoing curative surgery as scheduled (median 9 days after intervention). Increased CD8+T-cell tumor infiltration was evident after vaccination (median 73 vs 315 cells/mm2, p<0.05), along with significant downregulation of messenger RNA gene expression related to neutrophils and upregulation of transcripts encoding cytotoxic functions. Spatial protein analysis showed significant local upregulation of programmed death-ligand 1 (PD-L1) (adjusted p value<0.05) and downregulation of FOXP3 (adjusted p value<0.05). CONCLUSIONS: Neoadjuvant intratumoral influenza vaccine treatment in this cohort was demonstrated to be safe and feasible, and to induce CD8+T-cell infiltration and upregulation of PD-L1 proficient mismatch repair sigmoid and rectal tumors. Definitive conclusions regarding safety and efficacy can only be made in larger cohorts. TRIAL REGISTRATION NUMBER: NCT04591379.

Deeper sections reveal residual tumor cells in rectal cancer specimens diagnosed with pathological complete response following neoadjuvant treatment.

Guidelines and requirements for diagnosing pathological complete response (pCR) in rectal adenocarcinoma following neoadjuvant treatment vary, and there is currently no consensus on the appropriate number of sections to examine per formalin-fixed, paraffin-embedded (FFPE) tissue block. The consequences of systematic use of deeper sections on the diagnostic accuracy and prognosis for patients classified as ypT0 rectal cancer were investigated. In this retrospective study, 23 out of 155 patients who underwent neoadjuvant therapy and surgical resection between 2015 and 2020 were diagnosed with ypT0 rectal cancer. Three additional deeper sections were cut from each FFPE block from the primary tumor site and reviewed for presence of residual tumor cells. Additional sections revealed residual viable tumor cells in seven patients (30.4%), reducing the rate of pCR in the cohort from 14.8 to 10.3%. Of the seven patients, three patients later had local recurrence or distant metastasis during the follow-up period, compared with one patient with no residual tumor cells in deeper sections (p = 0.07). A nonsignificant reduction in disease-free survival (p = 0.08) was observed in the patients with residual tumor. Systematic use of deeper sections in evaluation of tumor regression in rectal cancer reveals the presence of residual tumor cells in a subset of patients originally diagnosed with pCR based on a single section per FFPE block. Although the results are not statistically significant, it cannot be excluded that accurately distinguishing complete from near-complete response may be clinically relevant for prognostic prediction.

Development of a method for digital assessment of tumor regression grade in patients with rectal cancer following neoadjuvant therapy.

Neoadjuvant chemo-radiotherapy (nCRT) followed by surgical resection is the standard treatment strategy in patients with locally advanced rectal cancer (RC). The pathological effect of nCRT is assessed by determining the tumor regression grade (TRG) of the resected tumor. Various methods exist for assessing TRG and all are performed manually by the pathologist with an accompanying risk of interobserver variation. Automated digital image analysis could be a more objective and reproducible approach to evaluate TRG. This study aimed at developing a digital method to assess TRG in RC following nCRT, and correlate the results to the currently used Mandard method. A deep learning-based semi-automatic Epithelium-Tumor area Percentage (ETP) algorithm enabling quantification of tumor regression by determining the percentage of residual tumor epithelium out of the total tumor area was developed. The ETP was quantified in 50 cases treated with nCRT and 25 cases with no prior nCRT served as controls. Median ETP was 39.25% in untreated compared with 6.64% in patients who received nCRT (P < .001). The ETP of the resected tumors treated with nCRT increased along with increasing Mandard grade (P < .001). As new treatment strategies in RC are emerging, performing an accurate and reproducible evaluation of TRG is important in the assessment of treatment response and prognosis. TRG is often used as an outcome point in clinical trials. The ETP algorithm is capable of performing a precise and objective value of tumor regression.

Histological disease activity in patients with microscopic colitis is not related to clinical disease activity or long-term prognosis.

BACKGROUND: Microscopic colitis (MC) is a common cause of chronic watery diarrhea. Biopsies with characteristic histological features are crucial for establishing the diagnosis. The two main subtypes are collagenous colitis (CC) and lymphocytic colitis (LC) but incomplete forms exist. The disease course remains unpredictable varying from spontaneous remission to a relapsing course. AIM: To identify possible histological predictors of course of disease. METHODS: Sixty patients from the European prospective MC registry (PRO-MC Collaboration) were included. Digitised histological slides stained with CD3 and Van Gieson were available for all patients. Total cell density and proportion of CD3 positive lymphocytes in lamina propria and surface epithelium were estimated by automated image analysis, and measurement of the subepithelial collagenous band was performed. Histopathological features were correlated to the number of daily stools and daily watery stools at time of endoscopy and at baseline as well as the clinical disease course (quiescent, achieved remission after treatment, relapsing or chronic active) at 1-year follow-up. RESULTS: Neither total cell density in lamina propria, proportion of CD3 positive lymphocytes in lamina propria or surface epithelium, or thickness of collagenous band showed significant correlation to the number of daily stools or daily watery stools at any point of time. None of the assessed histological parameters at initial diagnosis were able to predict clinical disease course at 1-year follow-up. CONCLUSIONS: Our data indicate that the evaluated histological parameters were neither markers of disease activity at the time of diagnosis nor predictors of disease course.

ERCC1 expression in advanced colorectal cancer and matched liver metastases.

BACKGROUND: Platinum-based chemotherapy is part of the standard treatment for patients with colorectal cancer. ERCC1 is a potential predictive biomarker for platinum-based chemotherapy. The aim of this study was to examine interobserver agreement on ERCC1 protein expression in primary colorectal cancer as well as corresponding liver metastasis. Furthermore, comparison of ERCC1-expression in primary tumor and the corresponding liver metastasis was performed. METHODS: Forty patients with primary colorectal cancers and corresponding liver metastases were included. One slide was stained with the anti-ERCC1 antibody, 4F9 clone (DAKO) and evaluated by two gastrointestinal pathology consultants and a pathology registrar separately. Interobserver agreement was evaluated for primary tumors and liver metastases using kappa (κ) statistics. Discordant scorings were reviewed, and consensus was obtained. The expression in primary tumor was compared with the corresponding liver metastases. RESULTS: For the primary tumors agreement was found in 85% of the tumors corresponding to an unweighted kappa value of 0,79 (95% CI 0,64-0,94). For the liver metastases agreement was found in 76% corresponding to an unweighted kappa value of 0,64 (95% CI 0,49-0,79). When comparing primary tumors to the corresponding metastases, no concordance in ERCC1-expression was observed. CONCLUSION: Interobserver agreement of ERCC1 expression was good for both primary tumors and liver metastases, which is crucial for a potential predictive biomarker. As no concordance between primary tumor and liver metastases was found it seems to be of high importance to use tissue from actual tumor burden for evaluation of ERCC1 expression. Further studies and correlation to clinical outcome are warranted.

Is revision of cutoff values needed when using CD3 immunohistochemical staining in histopathologic diagnosis of lymphocytic colitis?

Lymphocytic colitis (LC) and LC incomplete (LCi) are common causes of chronic watery diarrhea. The diagnosis relies on clinical findings and histopathologic evaluation. The diagnostic criteria of LC are based on hematoxylin and eosin (HE) staining. However, supplementary immunohistochemical staining for highlighting the lymphocytes in borderline cases is now widely used. This change in diagnostics could lead to incorrectly diagnosing patients with LC and LCi if the present histologic criteria are used. The number of intraepithelial lymphocytes (IELs) was estimated and categorized in intervals based on HE- versus CD3-stained slides from patients with an HE diagnosis of normal colonic mucosa (n = 19), mucosa with nonspecific reactive changes (n = 24), LCi (n = 24), and LC (n = 40). The number of IELs was compared with clinical symptoms. Overall, the number of IELs was higher with CD3 stain compared with HE stain in 73% of cases, unchanged in 26% of cases, and lower in 1 case. The number of IELs detected was higher using the CD3 stain in 53%, 79%, 79%, and 75% of cases included as normal colonic mucosa, nonspecific reactive changes, LCi, and LC, respectively. Based on CD3 stain, 58% of the cases with nonspecific reactive changes fulfilled the HE criteria for LCi, and 79% of the cases with LCi fulfilled the HE criteria for LC. Automated image analysis of CD3-stained slides resulted in even higher numbers of IELs in all 4 diagnostic groups. Conclusively, our data support considering increased cutoff values for LCi and LC when assessed in CD3-stained specimens.

Isolated tumor cells in regional lymph nodes in patients with adenocarcinoma of the esophagogastric junction might represent part of true metastases.

Regional lymph node metastases in patients with carcinoma of the esophagogastric junction (EGJ) are an important prognostic factor. According to the tumor, node, and metastasis classification, isolated tumor cells (ITCs) are single tumor cells or small clusters of tumor cells not exceeding 0.2 mm. Tumor clusters >0.2 mm are classified as metastases. The significance of lymph nodes with ITCs is unclear, although not contributing to the pN category. The aim of this study was to determine the prevalence of regional lymph nodes with ITCs on the primary hematoxylin and eosin-stained slide and to examine how often deeper sections reveal a true metastasis. The study included surgical specimens of 126 patients with adenocarcinoma of the EGJ. Lymph nodes with ITCs were identified. Additional sections were cut and stained with hematoxylin and eosin and with cytokeratin. All slides were evaluated for the presence of tumor cells, and it was determined whether the criteria for a metastasis were met on the additional sections. ITCs were detected in 59 (1.7%) of 3454 lymph nodes and in 41 (32.5%) of 126 patients. In 29 (49.2%) lymph nodes with ITCs on the primary slide, further sections resulted in a changed status from ITCs to a metastasis. In 7 (17.1%) of 41 patients, the pN category was changed. In patients with adenocarcinoma of the EGJ, the presence of ITCs in regional lymph nodes is a common observation. ITCs often represent part of a real metastasis. To obtain a pN category as accurate as possible, we strongly recommend thorough examination of regional lymph nodes with additional sections when ITCs are observed.

Establishment of digital cutoff values for intraepithelial lymphocytes in biopsies from colonic mucosa with lymphocytic colitis.

BACKGROUND/INTRODUCTION: Lymphocytic colitis (LC) and the incomplete form (LCi) are common causes of chronic watery diarrhea. Endoscopy is often inconspicuous, and the diagnosis relies on histopathological assessment of colonic biopsies. Digital Image Analysis (DIA) eliminates interobserver variation. The aim of this study was to establish digital cutoff values for LC and LCi on CD3 stained slides. MATERIAL AND METHODS: One hundred and six patients with a hematoxylin and eosin (HE) diagnosis of normal colonic mucosa (N = 19), non-specific reactive changes (N = 24), LCi (N = 23) and LC (N = 40) were eligible for analysis. The number of intraepithelial lymphocytes (IELs) reached by DIA in the total surface epithelium and in hot spots of the biopsies was compared with the diagnostic category assigned by the pathologists based on HE stained slides. The digitalized slides were analyzed for number of IELs using Visiopharm Quantitative Digital Pathology software. All digitalized slides were examined manually to identify differences in the approach to the evaluation of the biopsies by the pathologists and DIA. RESULTS: The median IEL counts and interquartile range in the total surface epithelium were 3.6 (3.2-4.3), 4.4 (3.4-5.3), 19.8 (16.6-30.0) and 41.3 (37.0-47.8) in normal colon mucosa, mucosa with non-specific reactive changes, LCi and LC, respectively. Discrimination between normal mucosa and non-specific reactive changes was not possible. Digital cutoff values with the best separation between non-LC, LCi and LC were > 13 IELs/100 epithelial cells for LCi and > 36 IELs/100 epithelial cells for LC. These cutoff values resulted in an agreement between the pathologist's and DIA that was very good with a kappa value of 0.90. CONCLUSION: Despite differences among the approach of DIA and the pathologist's assessment of IELs in colonic mucosa DIA is able discriminate between the HE based diagnoses of the three subgroups non-LC, LCi and LC with high accuracy.