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Background: Cell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients., Methods: Our study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5., Results: NKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome., Conclusions: We demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.

Background: Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers., Materials and Methods: A small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using (99m)Technetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 μM., Results: The Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1-3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 μM. No adverse reactions were observed., Conclusions: We describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

Background: Sentinel lymph node (SLN) mapping in colorectal cancer may have prognostic and therapeutic significance; however, currently available techniques are not optimal. We hypothesized that the combination of invisible near-infrared (NIR) fluorescent light and ex vivo injection could solve remaining problems of SLN mapping in colorectal cancer., Methods: The FLARE imaging system was used for real-time identification of SLNs after injection of the NIR lymphatic tracer HSA800 in the colon and rectum of (n = 4) pigs. A total of 32 SLN mappings were performed in vivo and ex vivo after oncologic resection using an identical injection technique. Guided by these results, SLN mappings were performed in ex vivo tissue specimens of 24 consecutive colorectal cancer patients undergoing resection., Results: Lymph flow could be followed in real-time from the injection site to the SLN using NIR fluorescence. In pigs, the SLN was identified in 32 of 32 (100%) of SLN mappings under both in vivo and ex vivo conditions. Clinically, SLNs were identified in all patients (n = 24) using the ex vivo strategy within 5 min after injection of fluorescent tracer. Also, 9 patients showed lymph node involvement (N1 disease). In 1 patient, a 3-mm mesenteric metastasis was found adjacent to a tumor-negative SLN., Conclusions: The current pilot study shows proof of principle that ex vivo NIR fluorescence-guided SLN mapping can provide high-sensitivity, rapid, and accurate identification of SLNs in colon and rectum. This creates an experimental platform to test optimized, non-FDA-approved NIR fluorescent lymphatic tracers in a clinical setting.

In breast cancer, the prognostic impact of COX2 expression varies widely between studies. We examined the prognostic value of COX2 expression in a large cohort of breast cancer patients treated with primary surgery between 1985 and 1994 and explained the variable results of COX2 expression found in the literature. A tissue microarray was constructed of available tumour material, and ER, PgR, HER2, Ki67 and COX2 were examined by immunohistochemistry. Median follow-up was 19 years. Fifty-five percent (n = 369/677) of patients received no systemic treatment. COX2 was scored using a weighted histoscore. Analysis of COX2 expression in two groups based on the median (148; below vs. above) showed an increased hazard ratio (HR) of 1.35 (95% CI 1.05-1.75, P = 0.021) for disease-free survival (DFS) and of 1.39 (95% CI 1.03-1.82, P = 0.016) for overall survival (OS). However, COX2 did not remain independent in multivariate analysis. In patients with hormone receptor positive tumours, COX2 expression had a negative influence on outcome (low vs. high: DFS: HR 1.37, 95% CI 1.07-1.76, P = 0.013). This effect disappeared when endocrine therapy was administered (low vs. high: DFS: HR 0.93, 95% CI 0.51-1.70, P = 0.811) while it remained statistically significant when endocrine therapy was omitted (low vs. high: DFS: HR 1.48, 95% CI 1.12-1.94, P = 0.005). Our results show that COX2 plays a role in hormonal pathways. Our results can explain the results found in previously published studies.

Stroma tissue surrounding cancer cells plays an important role in tumor development and behavior. In colorectal cancer, it has been found that the amount of stroma within the primary tumor is of prognostic value. We therefore have evaluated the prognostic value of this tumor-stroma ratio for breast cancer. A cohort of 574 early breast cancer patients, primarily treated with surgery between 1985 and 1994 was analyzed for the tumor-stroma ratio. The percentage of stroma was visually estimated on Haematoxylin-Eosin (H&E) stained histological sections. Patients with more than 50% intra-tumor stroma were quantified as stroma rich and patients with less than 50% as stroma poor. For the total group of patients, stroma-rich tumors had a shorter relapse-free period (RFP) (P = 0.001) and overall survival (OS) (P = 0.025) compared to stroma-poor tumors. Tumor-stroma ratio was an independent prognostic parameter for the total group of patients (P < 0.001) and also in stratified analysis based on systemic treatment. Importantly, in the triple-negative cancer subpopulation, patients with stroma-rich tumors had a 2.92 times higher risk of relapse (P = 0.006) compared to those with stroma-poor tumors, independently of other clinico-pathological parameters. Five-year RFP-rates for triple-negative cancer patients with stroma-rich compared to stroma-poor tumors were 56 and 81%, respectively. Tumor-stroma ratio has proven to be an independent prognostic factor for RFP in breast cancer patients and especially in the triple-negative cancer subpopulation. Tumor-stroma ratio could be easily implemented in routine daily pathology diagnostics, as it is simple to determine, reproducible, and performed in quick time.

Background: Tumour stroma ratio (TSR) in histological sections of resected oesophageal adenocarcinomas proved to be a prognostic factor for patients' survival. The objectives of this study were to assess inter- and intraobserver agreement for TSR scoring on biopsy material and to validate these biopsy results with the results derived from surgical specimens., Methods: Biopsies and surgical specimens of 91 patients with oesophageal adenocarcinoma were available. TSR was determined on the original haematoxylin-eosin (H&E) tissue sections from primary tumour biopsies. To assess interobserver variation, TSR was scored by three pathologists as 0-25%, 25-50%, 50-75% or 75-100%. A second scoring was done to examine intraobserver variation. The definitive TSR biopsy score was compared with the corresponding resection specimen score. Kappa statistics were applied to evaluate agreement., Results: Biopsies of 10 (11%) patients were rejected because of poor quality. For 81 TSR biopsy scores, interobserver correlations ranged between 0.239 and 0.486 (P < 0.001 for all). By classifying scores into two groups (<50% and ≥ 50%), interobserver correlations ranged between 0.372 and 0.886 (P < 0.001 for all). Intraobserver agreement was substantial to near-perfect (κ = 0.780-0.848; P < 0.001 for all). Definitive TSR biopsy score showed moderate correlation with TSR scores on surgical specimens (κ = 0.506), but it was an independent prognostic factor for survival., Conclusion: Reproducibility of tumour stroma ratio scoring on oesophageal adenocarcinoma biopsies was good. The ease of TSR scoring on H&E sections together with its correlation with patients' survival may have clinical relevance in this era of neoadjuvant therapy., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the prognostic value of the tumour stroma ratio (TSR) in resected adenocarcinoma of the oesophagus., Background: In the literature, a refinement of oesophageal cancer staging has been proposed. Recently, TSR has been identified as a histological characteristic of the tumour itself that proved to be a strong predictor for survival in colorectal cancer., Methods: In our cancer registry database, we identified 93 consecutive patients who underwent resection for oesophageal adenocarcinoma between 1990 and 2004 in two hospitals in our region. Using a predefined histopathological protocol, TSR was determined on the original haematoxylin-eosin (H&E) tissue sections of oesophagectomy specimens by two independent investigators., Results: With a cut-off value of 50% tumour/stroma, patients were classified as TSR high (n=60) or TSR low (n=33). There were no significant differences in patient, tumour and treatment characteristics between the two groups, except for M status (M1a) and radicality of resection. The (disease-free) survival in the TSR high group was significantly better than in the TSR low group. By multivariate analysis, TSR was identified as a highly significant prognostic factor for overall survival (HR 2.0; P=0.010), independent of depth of tumour invasion, nodal status, lymph node ratio, extracapsular involvement, TNM stage, histological grade and radicality of resection., Conclusion: TSR is a new and practicable prognostic tumour characteristic for oesophageal adenocarcinoma that can discriminate patients with a poor outcome from those with a better outcome., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Purpose: We hypothesized that T-cell immune interaction affects tumor development and thus clinical outcome. Therefore, we examined the clinical impact of human leukocyte antigen (HLA) class I tumor cell expression and regulatory T-cell (Treg) infiltration in breast cancer., Experimental Design: Our study population (N = 677) is consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed, paraffin-embedded tumor tissue was immunohistochemically stained using HCA2, HC10, and Foxp3 monoclonal antibodies., Results: HLA class I expression was evaluated by combining results from HCA2 and HC10 antibodies and classified into three groups: loss, downregulation, and expression. Remarkably, only in patients who received chemotherapy, both presence of Treg (P = 0.013) and higher HLA class I expression levels (P = 0.002) resulted in less relapses, independently of other variables. Treg and HLA class I were not of influence on clinical outcome in patients who did not receive chemotherapy., Conclusions: We showed that HLA class I and Treg affect prognosis exclusively in chemotherapy-treated patients and are therefore one of the few predictive factors for chemotherapy response in early breast cancer patients. Chemotherapy may selectively eliminate Treg, thus enabling CTLs to kill tumor cells that have retained HLA class I expression. As a consequence, HLA class I and Treg can predict response to chemotherapy with high discriminative power. These markers could be applied in response prediction to chemotherapy in breast cancer patients.

PURPOSE Studies on quality of life (QOL) among women with endometrial cancer have shown that patients who undergo pelvic radiotherapy report lower role functioning and more diarrhea and fatigue. In the Post Operative Radiation Therapy in Endometrial Cancer (PORTEC) trial, patients with endometrial carcinoma were randomly assigned to receive external-beam radiotherapy (EBRT) or vaginal brachytherapy (VBT). QOL was evaluated by using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire C30 and subscales from the prostate cancer module, PR-25, and the ovarian cancer module, OV-28. PATIENTS AND METHODS PORTEC-2 accrued 427 patients between 2002 and 2006, of whom 214 were randomly assigned to EBRT, and 213 were randomly assigned to VBT. Three-hundred forty-eight patients (81%) were evaluable for QOL. QOL outcomes were analyzed at a median follow-up of 2 years. Results At baseline after surgery, patient functioning was at the lowest level, and it increased during and after radiotherapy to reach a plateau after 12 months. Patients in the VBT group reported better social functioning (P < .002) and lower symptom scores for diarrhea, fecal leakage, the need to stay close to the toilet, and limitation in daily activities because of bowel symptoms (P < .001). At baseline, 15% of patients were sexually active; this increased significantly to 39% during the first year (P < .001). Sexual functioning and symptoms did not differ between the treatment groups. CONCLUSION Patients who received EBRT reported significantly higher levels of diarrhea and bowel symptoms. This resulted in a higher need to remain close to a toilet and, as a consequence, more limitation of daily activities because of bowel symptoms and decreased social functioning. Vaginal brachytherapy provides a better QOL, and should be the preferred treatment from a QOL perspective.

Atypical mycobacterial skin infections are difficult to diagnose owing to their aspecific histopathologic presentations and to the presence of few bacteria. Therefore, these infections are often not recognized. Molecular detection of mycobacterial DNA has proven to be useful in clinical samples. The aim of this study was to investigate the incidence of mycobacterial involvement in skin biopsies showing granulomatous inflammation, using real-time polymerase chain reaction (PCR). Real-time PCR specific for the genus Mycobacterium and the species Mycobacterium avium and Mycobacterium haemophilum was performed on formalin-fixed/paraffin-embedded biopsies from patients with granulomatous inflammation of the skin, from the period 1984 to 2004. A control group was assembled from patients with proven basal cell carcinoma. Amplicons of all positive reactions were sequenced to confirm or identify the mycobacterial species. Of 30 patients, 13 (43%) were found to be positive for mycobacterial infection, of whom only 5 patients had been previously diagnosed with a mycobacterial disease. M. haemophilum was identified as the most common species (n=7). The other identified species were Mycobacterium malmoense, Mycobacterium gordonae, and Mycobacterium marinum. The results show that real-time PCR is useful in detecting mycobacterial infections in undiagnosed formalin-fixed/paraffin-embedded skin samples and that the application of molecular approaches would improve the diagnoses of mycobacterial skin infections.

Purpose: Women carrying a CHEK2*1100delC germline mutation have an increased risk of developing breast cancer. This study aims to determine the proportion of CHEK2*1100delC carriers in a premenopausal breast cancer population, unselected for family history of breast cancer, and to investigate tumor characteristics and disease outcome with sufficient follow-up., Patients and Methods: We identified a retrospective cohort of 1,479 patients, who received surgery for invasive breast cancer between 1970 and 1994. All patients were diagnosed before age 50. Paraffin-embedded tissue blocks were collected for DNA isolation (normal tissue), subsequent CHEK2*1100delC analysis, and tumor revision. Median follow-up was 10.1 years., Results: We detected a CHEK2*1100delC germline mutation in 54 patients (3.7%). Tumor characteristics of CHEK2*1100delC carriers did not differ significantly from those of noncarriers. CHEK2*1100delC carriers had a two-fold increased risk (hazard ratio [HR], 2.1; 95% CI, 1.0 to 4.3; P = .049) of developing a second breast cancer and they had worse recurrence-free survival (HR, 1.7; 95% CI, 1.2 to 2.4; P = .006) and worse breast cancer-specific survival (HR, 1.4; 95% CI, 1.0 to 2.1; P = .072) compared with noncarriers. The poorer disease outcome of CHEK2*1100delC carriers could not be explained by the increased risk of second breast cancer., Conclusion: Our study, which is representative for the premenopausal breast cancer population, reveals approximately 4% CHEK2*1100delC carriers have an increased risk of second breast cancer and a worse long-term recurrence-free survival rate. Their identification at time of diagnosis and prolonged intensive follow-up should be considered to optimize clinical management.

Purpose: In 2000, the results of the multicenter Post Operative Radiation Therapy in Endometrial Carcinoma (PORTEC) trial were published. This trial included 714 Stage I endometrial carcinoma patients randomly assigned to postoperative pelvic radiotherapy (RT) or no further treatment, excluding those with Stage IC, Grade 3, or Stage IB, Grade 1 lesions. Radiotherapy significantly decreased the risk of locoregional recurrence (4% vs. 14%), without affecting overall survival. In this report the long-term outcome and results with central pathology review are presented., Methods and Materials: The slides of 569 patients (80%) could be obtained for pathology review. Median follow-up for patients alive was 97 months. Analysis was done according to the intention-to-treat principle. The primary study endpoints were locoregional recurrence and death., Results: Ten-year locoregional relapse rates were 5% (RT) and 14% (controls; p < 0.0001), and 10-year overall survival was 66% and 73%, respectively (p = 0.09). Endometrial cancer related death rates were 11% (RT) and 9% (controls; p = 0.47). Pathology review showed a substantial shift from Grade 2 to Grade 1, but no significant difference for Grade 3. When cases diagnosed at review as Grade 1 with superficial myometrial invasion were excluded from the analysis, the results remained essentially the same, with 10-year locoregional recurrence rates of 5% (RT) and 17% (controls; p < 0.0001)., Conclusions: In view of the significant locoregional control benefit, radiotherapy remains indicated in Stage I endometrial carcinoma patients with high-risk features for locoregional relapse.

A patient with tricuspid and pulmonary regurgitation due to carcinoid syndrome successfully underwent double bioprosthetic valve replacement. This technique avoids anticoagulation treatment in a patient with hepatic dysfunction and facilitates future hepatic de-arterialization as a treatment option in carcinoid disease. Advances in treatment of carcinoid syndrome may have reduced the risk of early bioprosthetic degeneration.

Promoter hypermethylation is known to result in transcriptional downregulation of many genes including the CDH1 gene. In this study we set out to determine CDH1 promoter methylation in breast tumors with decreased or absent E-cadherin protein expression and without CDH1 gene mutations by methylation-specific PCR (MSP). Interestingly, some tumor samples with normal E-cadherin expression yielded a methylation-specific PCR product. We hypothesized that other cells than tumor cells contribute to these products. Since in normal breast tissue no CDH1 promoter methylation is detected, infiltrating leukocytes, often present in tumors, might account for these methylation-specific fragments. Indeed, a methylation-specific fragment is found in all twelve leukocyte samples tested. Furthermore, activated T-cells also yielded a methylation-specific fragment. Sequencing of these fragments reveals two distinct methylation profiles. Leukocytes have only partial methylation of some CpGs, while the tumor-associated methylation profile shows complete methylation of most CpGs. Therefore, to assess whether CDH1 methylation is tumor associated, sequencing of MSP products is a prerequisite. Here we show that out of six lobular tumors lacking E-cadherin protein expression, three have tumor-associated CDH1 promoter methylation while in three other tumors no methylation is detected.

Background: Alterations in synthesis and breakdown of extracellular matrix components play a role in acute rejection after orthotopic liver transplantation (OLT). Matrix metalloproteinases (MMPs) are capable of degrading basement membranes and are involved in the process of tissue remodelling in inflammation and liver fibrosis., Methods: We examined MMP-2 and MMP-9 in serum of 33 patients before and during 1 year after OLT, in 60 controls as well as in some specimens of cirrhotic liver and control liver tissue., Results: Serum MMP-2 levels before OLT were significantly higher compared with controls and decreased approximately 50% after OLT. Also, the MMP-2 content of cirrhotic liver specimens was significantly higher compared with normal liver. MMP-9 in serum and liver tissue of patients were similar to controls, but serum levels showed a peak at 1 week after OLT. At this time-point, total and active/inhibitor-complexed MMP-9 was significantly higher in patients with rejection (n=13) compared with those without rejection (n=20). The relative amount of MMP-9 in the active/inhibitor-complexed form did not differ between each group over time. Immunohistochemical staining at 1 week after OLT showed increased numbers of MMP-9-positive inflammatory cells in the portal triads of patients with rejection., Conclusions: Patients with acute allograft rejection have elevated serum levels of MMP-9 1 week after OLT, which was most likely derived from inflammatory cells. An increased MMP-2 serum level and liver tissue content was found in patients with cirrhosis, which decreased after OLT. These observations indicate active involvement of MMP-2 and -9 in end-stage liver disease and OLT.

Invasive ductal carcinoma is by far the largest histological subtype of breast cancer, but clinical behavior can differ greatly. Reliable morphological markers are, therefore, of invaluable help to distinguish between patients with good and poor prognosis. Histological patterns stained with periodic acid-Schiff (PAS) were previously shown to be of prognostic significance in cutaneous and uveal melanoma. In this study, we examined the presence of different PAS-positive (PAS+) structures in 54 women with infiltrating ductal adenocarcinoma of the breast and at least one axillary lymph node metastasis but no distant metastases who were followed for at least 11 years. We found that the complexity of the thin PAS+ patterns in lymph node metastases is associated with a shorter period of disease free survival (DFS) as well as of total survival (Kaplan-Meier curves). Furthermore, the presence of PAS+ networks - the most complex thin PAS+ pattern - in lymph node metastases is one of the two independent factors associated with the occurrence of a distant metastasis (multivariate Cox model). Moreover, the presence of PAS+ networks in positive lymph nodes is the feature most strongly associated with DFS. In conclusion, the presence of PAS+ networks in lymph node metastases is a new, reliable and convenient indicator for prognosis of breast cancer patients.

Purpose: Endometrial carcinoma is the most common malignancy of the female genital tract. Recognized prognostic factors include FIGO stage, histologic grade, depth of myometrial invasion, and age. Although determination of these factors may seem clear and reproducible, the histologic grade has recently been the subject of debate. A retrospective analysis of long-term outcome and predictive factors in endometrial carcinoma was conducted, focusing on the prognostic value of tumor grade., Materials and Methods: The study included 253 patients with endometrial carcinoma Stages I to III, who were treated between 1984 and 1993. The histologic slides were reviewed and the prognostic value of stage, age, myometrial invasion (depth and pattern), tumor grade, and histologic subtype was analyzed. The end point was cancer-specific death; the median follow-up time was 11.7 years., Results: The actuarial 5- and 10-year cancer-specific survival rates (CSS) were 85% and 82%, respectively. Five-year vaginal and/or pelvic recurrence and distant relapse rates were 7% and 15%. In multivariate analysis, stage, pattern of myometrial invasion, tumor grade, and age were independent prognostic factors. At pathology review, a shift from Grade 2 to Grade 1 was seen in 112 of the original 144 Grade 2 (78%). There was no difference in CSS between Grade 1 and Grade 2 (94 vs. 90% for original grade and 92 vs. 95% for grade after review), whereas Grade 3 was found to be a significant adverse prognostic factor (p < 0.001)., Conclusions: The independent prognostic factors for patients with endometrial cancer were stage, pattern of myometrial invasion, tumor grade, and age. Systematic grading led to a considerable shift from Grade 2 to Grade 1. However, there was no difference in prognostic significance between Grade 1 and 2, whereas Grade 3 was a major adverse prognostic factor. A two-tiered grading system, instead of the currently used three-tiered system seems preferable, because it has a better correlation with clinical outcome and is expected to have less interobserver variability.