Spatial effects of infiltrating T cells on neighbouring cancer cells and prognosis in stage III CRC patients.

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single-cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil (5FU)-based chemotherapy. Images underwent segmentation for tumour, stroma, and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell-T-cell interactions at single-cell level. In our discovery cohort (Memorial Sloan Kettering samples), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (Huntsville Clearview Cancer Center samples) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between the percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (discovery cohort: p = 0.07; validation cohort: p = 0.19). We next utilised our region-based nearest neighbour approach to determine the spatial relationships between cytotoxic T cells, helper T cells, and cancer cell clusters. In both cohorts, we found that shorter distance between cytotoxic T cells, T helper cells, and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (discovery cohort: p = 0.01; validation cohort: p = 0.003). © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Tumour deposits are independently associated with recurrence in colon cancer.

AIM: Tumour deposits are focal aggregates of cancer cells in pericolic fat and mesentery, distinct from vessels, nerves and lymphatics. Their presence upstages lymph node negative patients but is ignored in lymph node positive patients. We investigated the clinicopathological factors associated with tumour deposits and their impact on recurrence in lymph node positive and negative patients. METHOD: Clinicopathological variables were collected from the medical records of patients with Stage I-III colon cancer who underwent resection in 2017-2019. Pathology was reviewed by a gastrointestinal pathologist. Patients with rectal cancer, metastasis, and concurrent malignancy were excluded. RESULTS: Tumour deposits were noted in 69 (9%) of 770 patients. They were associated with the presence of lymph node metastasis, advanced T category, poorly differentiated tumours, microsatellite stable subtype and lymphovascular and perineural invasion (p < 0.05). The presence of tumour deposits (hazard ratio 2.48, 95% CI 1.49-4.10) and of lymph node metastasis (hazard ratio 3.04, 95% CI 1.72-5.37) were independently associated with decreased time to recurrence. There was a weak correlation (0.27) between the number of tumour deposits and the number of positive lymph nodes. CONCLUSION: Tumour deposits are associated with more advanced disease and high-risk pathological features. The presence of tumour deposits and lymph node metastasis were found to be independent risk factors for decreased time to recurrence. A patient with both lymph node metastasis and tumour deposits is more than twice as likely to have recurrence compared with a patient with only lymph node metastasis. Tumour deposits independently predict recurrence and should not be ignored in lymph node positive patients.

Extramural Venous Invasion and Tumor Deposit at Diffusion-weighted MRI in Patients after Neoadjuvant Treatment for Rectal Cancer.

Background Diffusion-weighted (DW) imaging is useful in detecting tumor in the primary tumor bed in locally advanced rectal cancer (LARC) after neoadjuvant therapy, but its value in detecting extramural venous invasion (EMVI) and tumor deposit is not well validated. Purpose To evaluate diagnostic accuracy and association with patient prognosis of viable EMVI and tumor deposit on DW images in patients with LARC after neoadjuvant therapy using whole-mount pathology specimens. Materials and Methods This retrospective study included patients who underwent neoadjuvant therapy and surgery from 2018 to 2021. Innovative five-point Likert scale was used by two radiologists to independently evaluate the likelihood of viable EMVI and tumor deposit on restaging DW MRI scans in four axial quadrants (12 to 3 o'clock, 3 to 6 o'clock, 6 to 9 o'clock, and 9 to 12 o'clock). Diagnostic accuracy was assessed at both the per-quadrant and per-patient level, with whole-mount pathology as the reference standard. Weighted κ values for interreader agreement and Cox regression models for disease-free survival and overall survival analyses were used. Results A total of 117 patients (mean age, 56 years ± 12 [SD]; 70 male, 47 female) were included. Pathologically proven viable EMVI and tumor deposit was detected in 29 of 117 patients (25%) and in 44 of 468 quadrants (9.4%). Per-quadrant analyses showed an area under the receiver operating characteristics curve of 0.75 (95% CI: 0.68, 0.83), with sensitivity and specificity of 55% and 96%, respectively. Good interreader agreement was observed between the radiologists (κ = 0.62). Per-patient analysis showed sensitivity and specificity of 62% and 93%, respectively. The presence of EMVI and tumor deposit on restaging DW MRI scans was associated with worse disease-free survival (hazard ratio [HR], 5.6; 95% CI: 2.4, 13.3) and overall survival (HR, 8.9; 95% CI: 1.6, 48.5). Conclusion DW imaging using the five-point Likert scale showed high specificity and moderate sensitivity in the detection of viable extramural venous invasion and tumor deposits in LARC after neoadjuvant therapy, and its presence on restaging DW MRI scans is associated with worse prognosis. Published under a CC BY 4.0 license. Supplemental material is available for this article. See also the editorial by Méndez and Ayuso in this issue.

Characteristics of Mismatch Repair-Deficient Colon Cancer in Relation to Mismatch Repair Protein Loss, Hypermethylation Silencing, and Constitutional and Biallelic Somatic Mismatch Repair Gene Pathogenic Variants.

BACKGROUND: Mismatch repair-deficient colon cancer is heterogeneous. Differentiating inherited constitutional variants from somatic genetic alterations and gene silencing is important for surveillance and genetic counseling. OBJECTIVE: This study aimed to determine the extent to which the underlying mechanism of loss of mismatch repair influences molecular and clinicopathologic features of microsatellite instability-high colon cancer. DESIGN: This is a retrospective analysis. SETTINGS: This study was conducted at a comprehensive cancer center. PATIENTS: Patients with microsatellite instability-high colon cancer of stage I, II, or III were included. INTERVENTION: Patients underwent a curative surgical resection. MAIN OUTCOME MEASURES: The main outcome measures were hypermethylation of the MLH1 promoter, biallelic inactivation, constitutional pathogenic variants, and loss of specific mismatch repair proteins. RESULTS: Of the 157 identified tumors with complete genetic analysis, 66% had hypermethylation of the MLH1 promoter, 18% had constitutional pathogenic variants, (Lynch syndrome), 11% had biallelic somatic mismatch repair gene pathogenic variants, and 6% had unexplained high microsatellite instability. The distribution of mismatch repair loss was as follows: MLH1 and PMS2 co-loss, 79% of the tumors; MSH2 and MSH6 co-loss, 10%; MSH6 alone, 3%; PMS2 alone, 2%; other combinations, 2%; no loss, 2%. Tumor mutational burden was lowest in MLH1- and PMS2-deficient tumors. MSH6-deficient tumors had the lowest levels of tumor-infiltrating lymphocytes, lowest MSI scores, and fewest frameshift deletions. Patients with MLH1 promoter hypermethylation were significantly more likely to be older and female and to have right-sided colon lesions than patients with biallelic inactivation. Mutation was the most prevalent second hit in tumors with biallelic inactivation and tumors of patients with Lynch syndrome. LIMITATIONS: This study was limited by potential selection or referral bias, missing data for some patients, and relatively small sizes of some subgroups. CONCLUSIONS: Clinical characteristics of mismatch repair-deficient colon cancer vary with the etiology of microsatellite instability, and its molecular characteristics vary with the affected mismatch repair protein. See Video Abstract at http://links.lww.com/DCR/B984 . CARACTERSTICAS DEL CNCER DE COLON CON DEFICIENCIA EN LA REPARACIN DE ERRORES DE EMPAREJAMIENTO EN RELACIN CON LA PRDIDA DE PROTENAS MMR, SILENCIAMIENTO DE LA HIPERMETILACIN Y LAS VARIANTES PATGENAS SOMTICAS DE GENES MMR CONSTITUCIONAL Y BIALLICO: ANTECEDENTES:El cáncer de colon deficiente en la reparación de errores de emparejamiento es heterogéneo. La diferenciación de las variantes constitucionales heredadas de las alteraciones genéticas somáticas y el silenciamiento de genes es importante para la vigilancia y el asesoramiento genético.OBJETIVO:Determinar hasta qué punto el mecanismo subyacente de pérdida de reparación de desajustes influye en las características moleculares y clinicopatológicas del cáncer de colon con alta inestabilidad de microsatélites.DISEÑO:Análisis retrospectivo.ESCENARIO:Centro integral de cáncer.PACIENTES:Pacientes con cáncer de colon con inestabilidad de microsatélites alta en estadio I, II, o III.INTERVENCIÓN:Resección quirúrgica con intención curativa.PRINCIPALES RESULTADOS Y MEDIDAS:Hipermetilación del promotor MLH1, inactivación bialélica, variante patógena constitucional y pérdida de proteínas específicas reparadoras de desajustes.RESULTADOS:De los 157 tumores identificados con un análisis genético completo, el 66 % tenía hipermetilación del promotor MLH1, el 18 % tenía una variante patogénica constitucional (síndrome de Lynch), el 11 % tenía variantes patogénicas somáticas bialélicas de algún gen MMR y el 6 % tenía una alta inestabilidad de microsatélites sin explicación. La distribución de la pérdida según la proteína de reparación del desajuste fue la siguiente: pérdida conjunta de MLH1 y PMS2, 79 % de los tumores; co-pérdida de MSH2 y MSH6, 10%; MSH6 solo, 3%; PMS2 solo, 2%; otras combinaciones, 2%; sin pérdida, 2%. La carga mutacional del tumor fue más baja en los tumores deficientes en MLH1 y PMS2. Los tumores con deficiencia de MSH6 tenían los niveles más bajos de linfocitos infiltrantes de tumores, las puntuaciones más bajas del sensor de IMS y la menor cantidad de deleciones por cambio de marco. Los pacientes con hipermetilación del promotor MLH1 tenían significativamente más probabilidades de ser mayores y mujeres y de tener lesiones en el colon derecho que los pacientes con inactivación bialélica. La mutación fue el segundo golpe más frecuente en tumores con inactivación bialélica y tumores de pacientes con síndrome de Lynch.LIMITACIONES:Sesgo potencial de selección o referencia, datos faltantes para algunos pacientes y tamaños relativamente pequeños de algunos subgrupos.CONCLUSIONES:Las características clínicas del cáncer de colon deficiente en reparación de desajustes varían con la etiología de la inestabilidad de microsatélites, y sus características moleculares varían con la proteína de reparación de desajustes afectada. Vea Resumen de video en http://links.lww.com/DCR/B984 . (Traducción-Dr. Felipe Bellolio ).

Three-Dimensional Vessel Segmentation in Whole-Tissue and Whole-Block Imaging Using a Deep Neural Network: Proof-of-Concept Study.

In the field of pathology, micro-computed tomography (micro-CT) has become an attractive imaging modality because it enables full analysis of the three-dimensional characteristics of a tissue sample or organ in a noninvasive manner. However, because of the complexity of the three-dimensional information, understanding would be improved by development of analytical methods and software such as those implemented for clinical CT. As the accurate identification of tissue components is critical for this purpose, we have developed a deep neural network (DNN) to analyze whole-tissue images (WTIs) and whole-block images (WBIs) of neoplastic cancer tissue using micro-CT. The aim of this study was to segment vessels from WTIs and WBIs in a volumetric segmentation method using DNN. To accelerate the segmentation process while retaining accuracy, a convolutional block in DNN was improved by introducing a residual inception block. Three colorectal tissue samples were collected and one WTI and 70 WBIs were acquired by a micro-CT scanner. The implemented segmentation method was then tested on the WTI and WBIs. As a proof-of-concept study, our method successfully segmented the vessels on all WTI and WBIs of the colorectal tissue sample. In addition, despite the large size of the images for analysis, all segmentation processes were completed in 10 minutes.

MRI at Restaging After Neoadjuvant Therapy for Rectal Cancer Overestimates Circumferential Resection Margin Proximity as Determined by Comparison With Whole-Mount Pathology.

BACKGROUND: Current guidelines recommend restaging with MRI after neoadjuvant therapy for rectal cancer, but the accuracy of restaging MRI in estimating circumferential margin involvement requires additional clarification. OBJECTIVE: The objective of this study was to measure the accuracy of circumferential resection margin assessment by MRI after neoadjuvant therapy and identify characteristics associated with accuracy. DESIGN: MRI data were retrospectively analyzed for concordance with the findings of whole-mount pathology analysis of the corresponding surgical specimens. Univariate and multivariate logistic regression analyses were performed to identify characteristics associated with accuracy. SETTING: This study was conducted at a comprehensive cancer center. PATIENTS: Included in the study were consecutive patients who underwent total mesorectal excision for rectal cancer between January 2018 and March 2020 after receiving neoadjuvant therapy and undergoing restaging with MRI. MAIN OUTCOME MEASURES: The primary outcome of this study included accuracy, sensitivity, specificity, and positive and negative predictive values for categorizing the circumferential resection margin as threatened; mean and paired mean differences were in proximity of the margin. RESULTS: Of the 94 patients included in the analysis, 39 (41%) had a threatened circumferential resection margin according to MRI at restaging, but only 17 (18%) had a threatened margin based on pathology. The accuracy of MRI in identifying a threatened margin was 63.8%, with margin proximity overestimated by 0.4 cm on average. In multivariate logistic regression, anterior location of the margin and tumor proximity to the anal verge were independently associated with reduced MRI accuracy. LIMITATIONS: A limitation was the retrospective design at a single institution. CONCLUSIONS: The knowledge that MRI-based restaging after neoadjuvant therapy overestimates circumferential margin proximity may render some surgical radicality unnecessary and thereby help avoid the associated morbidity. With the recognition that MRI-based assessment of margin proximity may not be reliable for anterior margin and for distal tumors, radiologists may want to use greater caution in interpreting images of tumors with these characteristics and to acknowledge the uncertainty in their reports. See Video Abstract at http://links.lww.com/DCR/B814. LA IRM EN LA REESTADIFICACIN LUEGO DE TERAPIA NEOADYUVANTE EN EL CNCER DE RECTO SOBRESTIMA LA PROXIMIDAD DEL MARGEN DE RESECCIN CIRCUNFERENCIAL SEGN LO DETERMINADO COMPARATIVAMENTE CON LA PIEZA DE ANATOMOPATOLOGA: ANTECEDENTES:Las pautas actuales recomiendan la re-estadificación por medio de la resonancia magnética luego de terapia neoadyuvante en los casos de cáncer de recto, pero la precisión de la reevaluación con la IRM para estimar el grado de implicación del margen circunferencial requiere aclaraciones adicionales.OBJETIVO:Medir el grado de exactitud en la evaluación del margen de resección circunferencial mediante resonancia magnética después de la terapia neoadyuvante e identificar las características asociadas con la precisión.DISEÑO:Se analizaron retrospectivamente los datos de resonancia magnética para determinar la concordancia entre los hallazgos del análisis de la pieza de anatamopatología y las muestras quirúrgicas correspondientes. Se realizó el análisis de regresión logística univariada y multivariada para identificar las características asociadas con la exactitud.AJUSTE:Centro oncológico integral.PACIENTES:Todos aquellos que se sometieron consecutivamente a una excisión total del mesorrecto por cáncer rectal entre Enero 2018 y Febrero 2020 luego de recibir terapia neoadyuvante y someterse a una re-estadificación por imágenes de resonancia magnética (IRM).PRINCIPALES MEDIDAS DE RESULTADO:La exactitud, la sensibilidad y especificidad; los valores predictivos positivos y negativos para categorizar el margen de resección circunferencial como amenazado; la diferencia media y las medias pareadas de proximidad a los margenes.RESULTADOS:De los 94 pacientes incluidos en el análisis, 39 (41%) tenían un margen de resección circunferencial amenazado según la resonancia magnética en la re-estadificación, pero solo 17 (18%) tenían un margen amenazado basado en la patología. La precisión de la resonancia magnética para identificar un margen amenazado fue del 63,8%, con la proximidad del margen sobreestimada en 0,4 cm en promedio. En la regresión logística multivariada, la ubicación anterior de los bordes de resección y la proximidad del tumor al margen anal se asociaron de forma independiente con la reducción en la precisión de la resonancia magnética.LIMITACIONES:Diseño retrospectivo en una institución única.CONCLUSIONES:El saber que la re-estadificación basada en la IRM, luego de terapia neoadyuvante sobreestima la proximidad de la lesión a los márgenes circunferenciales, hace innecesaria cierta radicalidad quirúrgica complementaria, lo que ayuda a evitar morbilidad asociada. Reconociendo que la evaluación de proximidad de los márgenes de resección basada en la resonancia magnética, no puede ser confiable en casos de márgenes anteriores y en casos de tumores distales. Los radiólogos recomiendan tener más precaución en la interpretación de imágenes de tumores con estas características y reconocen cierto desasosiego en sus informes. Consulte Video Resumen en http://links.lww.com/DCR/B814.

Colorectal carcinoma with double somatic mismatch repair gene inactivation: clinical and pathological characteristics and response to immune checkpoint blockade.

Double somatic mismatch-repair-gene mutation/alteration is a recently recognized molecular mechanism that underlies microsatellite instability-high in some colorectal carcinomas. It remains to be determined whether and how microsatellite instability-high tumors with this molecular defect differ from their counterparts caused by other mechanisms, specifically, Lynch syndrome-associated and MLH1-promoter hypermethylated. In this study, we evaluated the clinical and pathological characteristics of a series of 15 double somatic mutation/alteration-associated microsatellite instability-high colorectal carcinomas identified from our genetics service and 68 such cases reported in the literature. We observed that these cases presented at an age similar to MLH1-promoter hypermethylated (n = 20) and microsatellite-stable (n = 39) cases but older than Lynch syndrome-associated cases (n = 20, p < 0.05). While these tumors simulated other microsatellite instability-high tumors in their prevalent right-sided location, they appeared to differ in TNM stages at presentation (73% stage III/IV versus 25% stage III/IV in other microsatellite instability-high tumors, p = 0.04). Histologically, 40% of them had a dominant solid growth pattern. Inter-tumoral heterogeneity was a striking feature, spanning the spectrum from medullary type (with a tumor-infiltrating-lymphocyte/high-power-field count as high as 59) to conventional-type with only few tumor-infiltrating-lymphocytes (1/high-power-filed). As a group, these tumors seemed less likely to show robustly high lymphocytic infiltration than other microsatellite instability-high tumors (only 20% had ≥10 tumor-infiltrating-lymphocytes/high-power-filed, whereas this rate in Lynch syndrome-associated and MLH1-promoter hypermethylated tumors was 60% and 75%, respectively). Three double somatic mutation/alteration-associated tumors were treated with a PD1/PD-L1 checkpoint inhibitor. While all three had an elevated tumor-mutation-burden (>47 mut/megabase), only one had tumor-infiltrating-lymphocytes >10/high-power-field, yet all three exhibited measurable response. In summary, microsatellite instability-high colorectal carcinomas caused by double somatic mismatch-repair-gene mutation/alteration may have varied clinical and pathological characteristics, and some may have relatively low tumor-infiltrating-lymphocytes; response to immune checkpoint inhibitors can be achieved in this group even when the lymphocytic infiltration is not abundant.

Pendrin Expression in Preeclampsia: A Prospective Immunohistochemical Staining Study on Placental Bed Biopsies.

INTRODUCTION: To assess the pendrin expression density in placental bed biopsies from preeclampsia cases in comparison with healthy term controls. MATERIAL AND METHODS: A prospective case-control study with 106 placental bed biopsies obtained during cesarean deliveries. Pendrin expression was evaluated by immunohistochemical staining in different hypertensive disorders of pregnancy. RESULTS: Pendrin immunostaining frequency was higher in the hypertensive disorders group (p: 0.024), which was a result of the high frequency in the early-onset preeclampsia group. Uterine artery pulsatility indices were higher in pendrin positive patients than in the negatives in the case group. Gravidity was not found to affect the pendrin expression frequency in the placental bed. CONCLUSION: Placental ischemia seems to be an important determinant of pendrin expression in pregnant decidua. Increased pendrin density in early-onset preeclampsia could be a pathogenetic mechanism in or a part of the adaptational response to the development of the hypertension.

Deformable Mapping of Rectal Cancer Whole-Mount Histology with Restaging MRI at Voxel Scale: A Feasibility Study.

Purpose To develop a radiology-pathology coregistration method for 1:1 automated spatial mapping between preoperative rectal MRI and ex vivo rectal whole-mount histology (WMH). Materials and Methods This retrospective study included consecutive patients with rectal adenocarcinoma who underwent total neoadjuvant therapy followed by total mesorectal excision with preoperative rectal MRI and WMH from January 2019 to January 2022. A gastrointestinal pathologist and a radiologist established three corresponding levels for each patient at rectal MRI and WMH, subsequently delineating external and internal rectal wall contours and the tumor bed at each level and defining eight point-based landmarks. An advanced deformable image coregistration model based on the linearized iterative boundary reconstruction (LIBR) approach was compared with rigid point-based registration (PBR) and state-of-the-art deformable intensity-based multiscale spectral embedding registration (MSERg). Dice similarity coefficient (DSC), modified Hausdorff distance (MHD), and target registration error (TRE) across patients were calculated to assess the coregistration accuracy of each method. Results Eighteen patients (mean age, 54 years ± 13 [SD]; nine female) were included. LIBR demonstrated higher DSC versus PBR for external and internal rectal wall contours and tumor bed (external: 0.95 ± 0.03 vs 0.86 ± 0.04, respectively, P < .001; internal: 0.71 ± 0.21 vs 0.61 ± 0.21, P < .001; tumor bed: 0.61 ± 0.17 vs 0.52 ± 0.17, P = .001) and versus MSERg for internal rectal wall contours (0.71 ± 0.21 vs 0.63 ± 0.18, respectively; P < .001). LIBR demonstrated lower MHD versus PBR for external and internal rectal wall contours and tumor bed (external: 0.56 ± 0.25 vs 1.68 ± 0.56, respectively, P < .001; internal: 1.00 ± 0.35 vs 1.62 ± 0.59, P < .001; tumor bed: 2.45 ± 0.99 vs 2.69 ± 1.05, P = .03) and versus MSERg for internal rectal wall contours (1.00 ± 0.35 vs 1.62 ± 0.59, respectively; P < .001). LIBR demonstrated lower TRE (1.54 ± 0.39) versus PBR (2.35 ± 1.19, P = .003) and MSERg (2.36 ± 1.43, P = .03). Computation time per WMH slice for LIBR was 35.1 seconds ± 12.1. Conclusion This study demonstrates feasibility of accurate MRI-WMH coregistration using the advanced LIBR method. Keywords: MR Imaging, Abdomen/GI, Rectum, Oncology Supplemental material is available for this article. © RSNA, 2024.

Micro-Computed Tomography Whole-Block Imaging Reveals Origin and Path of Rectal Cancer Tumor Deposits: A Pilot Study.

In colorectal carcinoma (CRC), tumor deposits (TDs) are described as macroscopic/microscopic nests/nodules in the lymph drainage area discontinuous with the primary mass, without identifiable lymph node (LN) tissue, and not confined to vascular or perineural spaces. A TD is categorized as pN1C only when no bona fide LN metastasis exists. However, there has been an ongoing debate on whether TDs should be counted as LNs. The fact that the origin of TDs is not fully understood adds further uncertainty. This pilot study aims to evaluate whether whole-block imaging by micro-computed tomography (micro-CT WBI) that enables three-dimensional reconstruction of whole-mount (WM) blocks can serve as a tool to assess the origin and path of CRC TDs. We evaluated whole-slide imaging (WSI) and micro-CT WBI of 20 WM blocks from a rectal cancer resection that contained TDs. Each TD was tracked through the contiguous blocks to define their origin and path. Of eleven TDs identified on WSI, six were detected on WBI. Strikingly, six of six TDs trackable through the blocks on WBI revealed an origin from the main tumor. This pilot study provided evidence that micro-CT WBI can serve as an effective tool to evaluate the origin and path of CRC TDs.

Spatial Effects of Infiltrating T cells on Neighbouring Cancer Cells and Prognosis in Stage III CRC patients.

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks, as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil-based chemotherapy. Images underwent segmentation for tumour, stroma and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell - T cell interactions at single-cell level. In our discovery cohort (MSK), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (HV) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (Discovery cohort: p = 0.07, Validation cohort: p = 0.19). We next utilized our region-based nearest neighbourhood approach to determine the spatial relationships between cytotoxic T cells, helper T cells and cancer cell clusters. In the both cohorts, we found that lower distance between cytotoxic T cells, T helper cells and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (Discovery cohort: p = 0.01, Validation cohort: p = 0.003).

MSH6-proficient crypt foci in MSH6 constitutional mismatch repair deficiency: reversion of a frameshifted coding microsatellite to its wild-type sequence.

The discovery of "mismatch repair deficient (MMRd)-crypt foci" in non-neoplastic intestinal mucosa in Lynch syndrome (LS) has significantly enhanced our understanding of how tumors and tumor immunity form and evolve in LS. In this study, we report the frequent presence of "mismatch repair proficient (MMRp)-crypt foci" in both non-neoplastic and neoplastic intestinal mucosa in a patient with constitutional MMR deficiency (CMMRD), who carried a germline MSH6 pathogenic variant (c.3261dupC) in trans with an MSH6 likely pathogenic variant (c.3724_3726del) and whose tissues were otherwise deficient in MMR globally. The MMRp-crypts occurred at a rate of 1.1/100 crypts in non-neoplastic intestinal mucosa and were readily discernible in adenomas > 1 cm. Sequencing analysis revealed normalization of the MSH6c.3261dupC variant in MMRp-adenoma crypts, indicating reverse frameshifting of the exon 5 C8 microsatellite. Interestingly but not surprisingly, the MMRp-adenoma crypts remained microsatellite-instability-high (MSI-H), and shared oncogenic APC mutations with the background MMRd-adenoma. Contrasting with MSH6-CMMRD, no PMS2-CMMRD individuals (0/5) harbored MMRp-crypts. In conclusion, our study documents distinct MMRp-crypts in MSH6-CMMRD, a phenomenon in keeping with MSH6 being a frequent target of MSI-H due to its coding microsatellite and suggesting that MSH6-CMMRD can potentially serve as a unique model system to further our understanding of MSH6's role in MSI-H tumor formation and evolution. Our findings also bear diagnostic implications; when using MMR immunohistochemistry as an ancillary tool in detecting CMMRD, awareness of these MMRp crypts can help avoid diagnostic pitfalls.

Identification of a Subset of Stage I Colorectal Cancer Patients With High Recurrence Risk.

BACKGROUND: A challenge in early-stage colorectal cancer (CRC) is identifying biomarkers that predict an increased risk for recurrence. A potential clinically adaptable biomarker is focal adhesion kinase (FAK), a tyrosine kinase that promotes invasion and metastasis. METHODS: An initial, single-institution, 298-patient cohort with all stages of CRC and long-term follow-up was assessed for FAK with tissue microarrays using immunohistochemistry. FAK expression was scored and dichotomized into high and low. Subsequently, a validation cohort of 517 early-stage CRCs from a separate institution was evaluated. All statistical tests were 2-sided. RESULTS: FAK overexpression did not correlate with any known histologic feature and was an early event in CRC, increasing from normal colon to stage I, and stage I to II, but not different at higher stages. High FAK was associated with decreased 10-year recurrence-free survival (RFS) among stage I patients (70.2% for high FAK vs 94.1% for low, P = .02), but not among higher stages in the initial cohort. The same finding was seen in the validation cohort (73.1% for high FAK vs 93.1% for low, P = .004). Multivariable survival analysis for stage I patients showed only two statistically significant factors predicting RFS: FAK (hazard ratio = 5.27, 95% confidence interval = 1.81 to 15.33, P = .002) and perineural invasion (hazard ratio = 7.38, 95% confidence interval = 1.01 to 53.96, P = .049). FAK was the only statistically significant factor in multivariable analysis across RFS, overall, and disease-specific survivals. CONCLUSIONS: High FAK expression identified a subset of stage I CRC patients with high incidence of recurrence and reduced survival, suggesting that FAK has important prognostic value. These patients would immediately benefit from more rigorous surveillance protocols for recurrent disease.

Pathological Evaluation of Rectal Cancer Specimens Using Micro-Computed Tomography.

Whole-block imaging (WBI) using micro-computed tomography (micro-CT) allows the nondestructive reconstruction of a three-dimensional view of tissues, implying that WBI may be used for accurate pathological evaluation of patients with rectal cancer. HOWEVER, the clinical impact of this approach is unclear. We aimed to clarify the efficacy of WBI in the whole-mount specimens of locally advanced rectal cancer. A total of 237 whole-mount formalin-fixed paraffin-embedded blocks from 13 patients with rectal cancer who underwent surgical treatment were enrolled and scanned with micro-CT to generate three-dimensional images. WBI was evaluated following the conventional pathological review of the corresponding whole-slide imaging (WSI). WBI identified all tumor sites detected using WSI. Furthermore, WBI revealed one additional tumor site, which was not detected using WSI. Tumor resection margin was significantly closer to the soft-tissue edge when measured using WBI (7.7 mm vs. 6.6 mm, p < 0.01). Seventy-six percent of tumor deposits on WSI were changed according to the evidence of tumor interaction with the surrounding tissues confirmed using WBI. Furthermore, WBI revealed 25 additional lymph nodes, six of which were metastatic. The combination of conventional hematoxylin and eosin-stained imaging and WBI may contribute to an accurate pathological assessment.

Intratumoral T-cell repertoires in DNA mismatch repair-proficient and -deficient colon tumors containing high or low numbers of tumor-infiltrating lymphocytes.

Colon tumors with deficient DNA mismatch repair (dMMR) are generally infiltrated by T cells more densely than tumors with proficient mismatch repair (pMMR). However, high numbers of tumor-infiltrating lymphocytes (TILs) are found in select pMMR tumors, and low numbers of TILs are seen in select dMMR tumors. In this study, we compared T-cell repertoires in 20 pMMR and 27 dMMR colon tumors with high and low TIL counts. We found that T cells in dMMR tumors are more clonal and their repertoire is less rich compared with T cells in pMMR tumors. In the dMMR group, T cells in TIL-high tumors were more clonal and their repertoire was less rich compared with T cells in TIL-low tumors, but in the pMMR group, T-cell diversity in TIL-high tumors was comparable to T-cell diversity in TIL-low tumors. These findings suggest that T cells clonally expand in dMMR tumors, possibly in response to MMR deficiency-induced tumor neoantigens.

Acellular mucin in lymph nodes isolated from treatment-naïve colorectal cancer resections: a clinicopathologic analysis of 16 cases.

Lymph nodes with acellular mucin harvested from treated colorectal cancers (CRC) are staged as pN0. However, there is variability among pathologists while reporting the pN stage when acellular mucin is found within nodes of untreated CRCs. While the UICC guidelines suggest staging them as pN1, the AJCC and CAP do not offer any recommendations. In order to characterize their clinicopathologic features and outcome, we compared 16 untreated CRCs (study group; mean age: 68 years) harboring nodes with acellular mucin with 34 pN0 and 25 pN1 untreated CRC controls. All tumors were unifocal; 12 (75%) were right-sided lesions. Most cases (75%) showed one node with acellular mucin (range: 1-3). MMR-deficient tumors were significantly more common in the study group (83%) compared to pN0 (33%; p = 0.006) and pN1 controls (8%; p < 0.001). The overall survival of study group patients was closer to pN0 compared to pN1 controls; however, this difference was not statistically significant. In conclusion, untreated CRC that harbor acellular mucin within lymph nodes commonly present as right-sided, MMR-deficient tumors in older women that show a non-mucinous phenotype. While the limited number of cases precludes us from making any formal recommendations about staging, we suggest that the finding of acellular mucin in a node should prompt evaluation of deeper levels (with or without cytokeratin immunohistochemistry) and submission of all pericolonic fat for additional lymph node harvest. Whether acellular mucin in nodes of untreated CRCs is related to the indolent biology of the disease, a robust local immune response or MMR deficiency requires further investigation.

An immunocompetent rectal cancer model to study radiation therapy.

We describe a mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation in an immunocompetent setting. This approach uses simple mechanical disruption to allow engraftment, avoiding the use of dextran sulfate sodium. The resulting RC tumors invaded from the mucosal surface and metastasized to distant organs. Histologically, the tumors closely resemble human RC and mirror remodeling of the tumor microenvironment in response to radiation. This murine RC model thus recapitulates key aspects of human RC pathogenesis and presents an accessible approach for more physiologically accurate, preclinical efficacy studies.

Tumor-Infiltrating Lymphocytes, Tumor Mutational Burden, and Genetic Alterations in Microsatellite Unstable, Microsatellite Stable, or Mutant POLE/POLD1 Colon Cancer.

To characterize the relationship between tumor-infiltrating lymphocytes (TIL), tumor mutational burden (TMB), and genetic alterations in microsatellite stable (MSS), microsatellite instability (MSI), or mutant POLE/POLD1 colon cancer. MATERIALS AND METHODS: Four hundred ninety-nine resected stage I-III colon tumors treated between 2014 and 2019 were assessed for TIL; somatic mutations, copy number alterations, and structural changes in > 400 oncogenes; and MSI status. RESULTS: Of the 499 tumors analyzed, 313 were MSS, 175 were MSI, and 11 had POLE/POLD1 pathogenic mutations. Both the percentage of tumors with a high level of TIL (≥ 4 lymphocytes per high-power field) and the median TMB differed significantly between the three phenotypes: MSS, 4.5% and 6 mutations/Mb; MSI, 68% and 54 mutations/Mb; POLE/POLD1, 82% and 158 mutations/Mb (P < .05). Within each phenotype, TMB did not vary significantly with TIL level. Among MSI tumors, the median number of frameshift indels was significantly higher in tumors with high levels of TIL (20 v 17; P = .018). In the MSS group, significantly higher proportions of tumors with high levels of TIL had mutations in the transforming growth factor-ß (36% v 12%; P = .01), RAS (86% v 54%; P = .02), and Hippo (7% v 1%; P = .046) pathways; in contrast, TP53 alterations were associated with low levels of TIL (74% v 43%; P = .01). CONCLUSION: The association between TIL, TMB, and genetic alterations varies significantly between MSI, MSS, and mutant POLE/POLD1 colon tumors. These differences may help explain tumoral immunity and lead to predictors of response to immunotherapy.

Discordant DNA mismatch repair protein status between synchronous or metachronous gastrointestinal carcinomas: frequency, patterns, and molecular etiologies.

The widespread use of tumor DNA mismatch repair (MMR) protein immunohistochemistry in gastrointestinal tract (GIT) carcinomas has unveiled cases where the MMR protein status differs between synchronous/metachronous tumors from the same patients. This study aims at examining the frequency, patterns and molecular etiologies of such inter-tumoral MMR discordances. We analyzed a cohort of 2159 colorectal cancer (CRC) patients collected over a 5-year period and found that 1.3% of the patients (27/2159) had ≥ 2 primary CRCs, and 25.9% of the patients with ≥ 2 primary CRCs (7/27) exhibited inter-tumoral MMR discordance. We then combined the seven MMR-discordant CRC patients with three additional MMR-discordant GIT carcinoma patients and evaluated their discordant patterns and associated molecular abnormalities. The 10 patients consisted of 3 patients with Lynch syndrome (LS), 1 with polymerase proofreading-associated polyposis (PAPP), 1 with familial adenomatous polyposis (FAP), and 5 deemed to have no cancer disposing hereditary syndromes. Their MMR discordances were associated with the following etiologies: (1) PMS2-LS manifesting PMS2-deficient cancer at an old age when a co-incidental sporadic MMR-proficient cancer also occurred; (2) microsatellite instability-driven secondary somatic MSH6-inactivation occurring in only one-and not all-PMS2-LS associated MMR-deficient carcinomas; (3) "compound LS" with germline mutations in two MMR genes manifesting different tumors with deficiencies in different MMR proteins; (4) PAPP or FAP syndrome-associated MMR-proficient cancer co-occurring metachronously with a somatic MMR-deficient cancer; and (5) non-syndromic patients with sporadic MMR-proficient cancers co-occurring synchronously/metachronously with sporadic MMR-deficient cancers. Our study thus suggests that inter-tumoral MMR discordance is not uncommon among patients with multiple primary GIT carcinomas (25.9% in patients with ≥ 2 CRCs), and may be associated with widely varied molecular etiologies. Awareness of these patterns is essential in ensuring the most effective strategies in both LS detection and treatment decision-making. When selecting patients for immunotherapy, MMR testing should be performed on the tumor or tumors that are being treated.