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Name of the study

Uniform Noting for International Application of the Tumor-Stroma Ratio as an Easy Diagnostic Tool: Protocol for a Multicenter Prospective Cohort Study

Researchers and contact information

Lead investigator:
Dr. W.E. (Wilma) Mesker

Other principal investigators
Prof. dr. R.A.E.M. (Rob) Tollenaar
Prof. dr. J.H.J.M. (Han) van Krieken

Coordinating investigators
Drs. M. (Meaghan) Polack
T: + 31 71 526 5130

Summary study

Colon cancer treatment is highly dependent on stage at diagnosis. The current TNM (Tumor-NodeMetastasis) staging used for the selection of patients for adjuvant chemotherapy however, needs improvement. The research group of dr. Mesker and Prof. dr. Tollenaar developed a novel, easily applicable, practice changing method to better select colon cancer patients for adjuvant chemotherapy: the tumor-stroma ratio (TSR), proven in various cancer types to be an independent prognosticator of survival. The UNITED study consists of an international multicenter prospective validation trial of the TSR, to help distinguish in clinical practice within stage II-III colon cancer patients who will likely benefit of adjuvant (chemo)therapy and those who will not. Better shared decision making based on this, will result in reduced over- and undertreatment and reduced costs.

Primary objective

To implement the TSR in current routine pathology diagnosis, in addition to the TNM classification. First, pathologists will be trained in scoring the TSR in combination with a quality assessment program. The interobserver variation will be determined, and a certificate is awarded. After certification, a prospective study including patients will be conducted, validating the TSR as an independent prognosticator.

Study design
1. An E-learning module, with a quality assessment program in the framework of the European Society of Pathology EQA program. This module will be used to assess the reliability and reproducibility of the scoring of the TSR by national and international pathologists (as well as residents pathology/pathologist trainees), using scanned routine Hematoxylin and Eosin (H&E) stained tumor tissue sections. 

2. A prospective study of colon cancer stage II-III patients within this multicenter setting. This study will be used to determine whether the TSR can be validated as an independent prognosticator.

Study population
1. For the reproducibility and E-learning study, already scanned H&E stained slides of stage II-III colon cancer patients were selected; 40 cases for a training set and 40 different cases for the test set. All cases were available from the pathology archive of the Leiden University Medical Center (LUMC).
2. For the prospective cohort study, a minimum of 1172 patients in participating international centers will be included, undergoing or who have undergone complete curative treatment (R0 resection). Final inclusion criteria are a histologically proven p-stage II (T3-4, N0, M0) or p-stage III (any T, N1-2, M0) colon carcinoma. All patients will be ≥18 years of age and need a signed informed consent form. Exclusion criteria consist of rectal tumors, multiple synchronous malignant colon tumors, or if a patient had received neoadjuvant treatment. If patients are deceased within three months after surgery, or have had a malignancy within 10 years prior to the current colon carcinoma (except for basal cell carcinoma or cervical carcinoma in situ) or any colon carcinoma in their history, exclusion followed too. Through a collaboration with the Dutch Prospective Colorectal cohort (PLCRC), it is possible to include patients prospectively since the start of the UNITED study in February 2018.

Primary endpoint
The main endpoints of this study are:
1. Inclusion of well-trained (international) pathologists, who passed the E-learning module with low inter-observer variation.
2. Inclusion of the minimal number of patients with a minimum follow up period of three years or until death, to determine whether differences in overall survival between patients with a stromahigh tumor versus patients with a stroma-low tumor within each stage exist. Thus, patients can be more adequately selected for adjuvant (chemo)therapy.

Secondary endpoint
Disease free survival between stroma-high and stroma-low tumors, and differences in response to current treatment regimens between patients with a stroma-high tumor versus patients with a stroma-low tumor.

Sample size
For the prospective cohort, a sample size calculation was performed:

p-Stage II patients
Selection of patients for adjuvant therapy: with a Hazard ratio of 2.0, adjusted for TNM, and a known percentage of stroma-high patients in p-stage II of 20-25%, 114 recurrence events with 90% power are necessary. In order to achieve 114 recurrence events based on an event rate of 0.0575 per year (leading to a 5 years probability of 75% and 3-years recurrence probability of 84.2%), this leads to 722 patients.

p-Stage III patients
Selection of patients for adjuvant therapy: with a Hazard ratio of 2.0, adjusted for TNM, and a known percentage of stroma-high patients in stage III of 30-35%, 97 recurrence events with 90% power are necessary. In order to achieve 97 recurrence events based on an event rate of 0.081 per year (leading to a 5 years probability of 66.7% and 3-years recurrence probability of 78.4%), this leads to 450 patients.

In order to have 1172 evaluable p-stage II/II, approximately 1500 (+25%) patients will be registered, since patients could still be excluded.



1. Training of pathologists
An E-learning module was developed in the framework of the ESP. Before starting this module, an introduction film was shown for instructions on how to score the TSR. Participating pathologists (or pathology residents/trainees) then scored a training set of 40 p-stage II-III colon cancer slides, from patients of the LUMC. Their scores were compared to the scores of two experienced observers of the LUMC, used as reference to calculate a Cohen’s kappa for interobserver agreement. In case of ĸ ≥ 0.7, the participant continued with the test set of 40 slides of stage II-III colon cancer patients. If the test set was finished with ĸ ≥ 0.7 as well, the E-learning was passed, and certification followed (Figure 1). The pathologists were unaware of any clinical information of the patients of which theslides were used, or any of their previous scoring.

2. Histopathological scoring of the TSR
H&E stained sections of the primary colon adenocarcinoma of the included patients, used in routine pathology to determine the T-status (i.e. the most invasive part), are selected and analyzed using conventional microscopy. Areas with the largest amount of stroma are selected using the x2.5 or the x5 objective. An area where both tumor and stromal tissue are present within this vision site is selected using the x10 objective. Tumor cells have to be present at all borders of the selected image field (north-east-south-west) (Figure 2). Vessels, erythrocytes, necrosis, fatty or muscle tissue are to be avoided as much aspossible. The stroma percentage is scored in increments of 10%, and then categorized in two groups: stroma-high, defined as >50% stroma, and stroma-low, with ≤50% stroma. This was as determined a priori to have maximum discriminative power, by Mesker et al (2007). Even if there is only one image-field with a stroma-high score, this image-field is decisive to categorize the patient as stroma-high. The scoring protocol is also available in a film and on a website (










Participating centers

University Cancer Center Leiden-The Hague, the Netherlands
Radboud University Medical Center, Nijmegen, the Netherlands

International centers

Napoleão Laureano Hospital, João Pessoa, Brazil

Charles University Medical Faculty Hospital, Prague, Czech Republic
Vejle Sygehus, Vejle, Denmark
Hopital Saint-Antoine, Paris, France
Landspitali, Reykjavik, Iceland
University of Ss. Cyril and Methodius, Skopje, North-Macedonia
Hospital Cuf Descobertas, Lisbon, Portugal
Hospital Garcia de Orta, Almada, Portugal
Faculty of Medicine University of Belgrade, Belgrade, Serbia
Institut za onkologiju Vojvodine, Sremska Kamenica, Serbia Medicinski fakultet Novi Sad, Novi Sad, Serbia
Hospital Clinical Barcelona, Spain
Vall d’Hebron Institute of Oncology, Barcelona, Spain

United Kingdom centers:
NHS Greater Glasgow and Clyde, Glasgow, Scotland
North Tees and Hartlepool NHS foundation trust, Hartlepool
Royal free London NHS foundation trust, London

Prospective Dutch CRC cohort (PLCRC) centers:
Deventer Ziekenhuis, Deventer
LabPON, Twente
Meander Medical Center, Amersfoort
PATHAN Laboratories, Rotterdam region
Reinier de Graaf Gasthuis, Delft
Spaarne Gasthuis, Hoofddorp en Haarlem
Symbiant Laboratories, Alkmaar region
Universitair Medisch Centrum Utrecht, Utrecht