Emiel A.M. Janssen

Microsatellite instability in colorectal cancer

Microsatellite instability (MSI) causes hereditary non‐polyposis colorectal cancer (HNPCC), and occurs in about 15 per cent of sporadic colorectal cancers. Although the basic mechanisms are not clear, there is increased understanding of the clinicopathological consequences of MSI.

Prospective Multicenter Validation of the Independent Prognostic Value of the Mitotic Activity Index in Lymph Node–Negative Breast Cancer Patients Younger Than 55 Years

PURPOSE To validate the independent strong prognostic value of mitotic activity index (MAI) in lymph node (LN) -negative invasive breast cancer patients younger than 55 years in a nationwide multicenter prospective study. PATIENTS AND METHODS Analysis of routinely assessed MAI and other prognosticators in 516 patients (median follow-up, 118 months; range, 8 to 185 months), without systemic adjuvant therapy or previous malignancies. RESULTS Distant metastases occurred in 127 patients (24.6%); 90 (17.4%) died as a result of metastases. MAI (< 10, > or = 10) showed strong association with recurrence (hazard ratio [HR], 3.12; 95% CI, 2.17 to 4.50; P < or = .0001) and mortality (HR, 4.42; 95% CI, 2.79 to 7.01; P < .0001). The absolute difference in 10-year Kaplan-Meier estimates of time to distant recurrence as well as survival was 22% between MAI less than 10 versus > or = 10. This effect was independent of age, estrogen receptor (ER) status, and tumor diameter (which were significant prognosticators). In multivariate analysis with regard to patient age, tumor diameter, grade, ER status, and the St Gallen criterion, MAI proved to be an independent and the strongest prognosticator. Tubular formation (TF) and nuclear atypia (NA), as constituents of (expert revised) grade, had no (for TF) or limited (for NA, P = .048) additional prognostic value to the MAI. In the group with MAI less than 10, MAI less than 3 versus more than 3 had additional value but the classical threshold of 0 to 5 v 6 to 10 did not. With this additional subdivision of MAI as less than 3, 3 to 9, and more than 9, NA lost its additive prognostic value. CONCLUSION The MAI is the strongest, most widely available, easily assessable, inexpensive, well-reproducible prognosticator and is well suited to routinely differentiate between high- and low-risk LN-negative breast cancer patients younger than 55 years.

Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis.

Trypsin is involved in colorectal carcinogenesis and promotes proliferation, invasion, and metastasis. Although a well‐known pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers, most importantly of the colorectum. Moreover, colorectal cancers with trypsin expression have a poor prognosis and shorter disease‐free survival. Biological understanding of how trypsin causes cancer progression is emerging. It seems to act both directly and indirectly through a ‘proteinase–antiproteinase‐system’, and by activation of other proteinase cascades. Invasion of the basal membrane by cancer cells may be promoted directly by trypsin digestion of type I collagen. Trypsin activates, and is co‐expressed with matrix metalloproteinases (MMPs), which are known to facilitate invasion and metastasis. MMP‐2, MMP‐7, and MMP‐9 are co‐expressed together with trypsin and seem to be of particular importance in proliferation, progression, and invasion. MMPs may play a role in both conversion from adenoma to carcinoma, and in the initiation of invasion and metastasis. Co‐segregation of trypsin and MMPs within the tumour environment is important for the activation of MMPs, and may explain the deleterious effect of trypsin on prognosis in colorectal cancer. Trypsin and proteinase‐activated receptor 2 (PAR‐2) act together in an autocrine loop that promotes proliferation, invasion, and metastasis through various mechanisms, of which prostaglandin synthesis is important. Stimulated by trypsin, both MMP and PAR‐2 may activate the mitogenic MAPK–ERK pathway through activation of the epidermal growth factor receptor. Experimental trypsin inhibition is feasible but not very effective, and trypsin as a target for clinical therapy is unlikely to be successful owing to its universal distribution. However, as the pathways of trypsin and co‐activated protein cascades emerge, biological understanding of colorectal carcinogenesis will be further illuminated and may pave the way for prognosticators, predictors, and novel targets of therapy. Copyright

Phosphohistone H3 expression has much stronger prognostic value than classical prognosticators in invasive lymph node-negative breast cancer patients less than 55 years of age

The proliferation factor mitotic activity index is the strongest prognostic factor in early breast cancer, but it may lack reproducibility. We analyzed the prognostic value of phosphohistone H3, a marker of cells in late G2 and M phase, measuring highly standardized immunohistochemical nuclear phosphohistone H3 expression by subjective counts and digital image analysis. Expression was compared with classical clinico-pathologic prognostic variables and the mitotic activity index in 119 node-negative invasive breast cancers in patients less than 55 years old treated with adjuvant systemic chemotherapy with long-term follow-up (median 168 months). Nineteen patients (16%) developed distant metastases and 16 (13%) died. Strong phosphohistone H3 expression occurred preferentially in the peripheral growing front; counts were highly reproducible between observers (R=0.92) and highly consistent with digital image analysis (R=0.96). Phosphohistone H3 correlated (P<0.05) with tumor diameter, estrogen receptor, carcinoma grade, and mitotic activity index. Phosphohistone H3 values were systematically (80%) higher than the mitotic activity index. Receiver-operating curve analysis objectively showed that phosphohistone H3 <13 (n=53; 45% of all cases) vs phosphohistone H3≥13 (n=66; 55% of all cases) was the strongest prognostic threshold, with 20-year recurrence-free survival of distant metastases of 96 and 58%, respectively (P=0.0002, HR=9.6). Mitotic activity index was the second strongest prognostic variable (P=0.003, HR=3.9). In multivariate analysis, phosphohistone H3 <13 vs≥13 exceeded the prognostic value of the mitotic activity index. None of the other classical prognostic factors examined offered prognostic value additional to phosphohistone H3. Phosphohistone H3 is by far the strongest prognostic variable in early invasive node-negative breast cancer patients less than 55 years old with long-term follow-up.

Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer

Gudlaugsson E, Skaland I, Janssen E A M, Smaaland R, Shao Z, Malpica A, Voorhorst F & Baak J P A 
(2012) Histopathology
Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer

Quantitative molecular parameters to identify low-risk and high-risk early CIN lesions: role of markers of proliferative activity and differentiation and Rb availability.

Summary:In early cervical intraepithelial neoplasia (CIN), the Ki67 stratification index 90th percentile (Si90) is a strong predictor of progression. This study was designed to further investigate the mechanisms leading to elevated Ki67 levels in lesions that progress and to try to improve the prognostic accuracy of Ki67-Si90. We studied 90 CIN lesions in which consensus existed regarding the grade between two experienced gynecologic pathologists. All CINs were p16-positive and showed Ki67 cell clusters above the lower third of the epithelium (both features diagnostic for CIN). Ki67 parameters, cell cycle regulators (Rb, p53, Cyclin A, E and D, p16, p21, p27, and telomerase), and cellular differentiation products (involucrin, CK13, CK14) were compared in the basal zone as well as the deeper and upper halves of the epithelium. Fifteen CIN cases (17%) progressed to a higher CIN grade, including 2 of 25 CIN1 (8%) and 13 of 65 CIN2 (20%) (these proportions of progressing CINs are similar to those in a large meta-analysis). Ki67 quantitation effectively predicted CIN progression as 0 of 40 “Ki67 low-risk” and 15 of 50 (30%) “Ki67 high-risk” lesions progressed. CIN progressors showed decreased Rb, CK13, CK14, and involucrin, but increased p21 and p27 expression. Ki67-Si90 and Rb in the deeper half of the epithelium (RbDeep) were the strongest multivariate independent predictors of progression. Ki67-Si90>0.57 was unfavorable, but only if it coexisted with RbDeep <45% (progression risk = 47%). All early CINs with combined Si90>0.57+RbDeep>45% or any Ki67-Si90 value below 0.57 were nonprogressors. In the high-risk progression subgroup (Ki67-Si90>0.57+RbDeep<45%), all cases with combined CK14<50% and CK13<80% (both in the basal cell layer) (4% of all lesions) progressed. We hypothesize that onco-HPV E7 expression reduces Rb, causing increased and upward proliferation (Ki67-Si90>0.57). Increased RbDeep can reduce proliferation, including its upward spread. Combined quantitation of Ki67, Rb, CK13, and CK14 gives accurate information about the progression risk of early CIN lesions.

Evaluation of MIB-1-positive cell clusters as a diagnostic marker for cervical intraepithelial neoplasia

The objects of the study were to evaluate MIB-1-positive cell clusters (MIB-C) for distinguishing normal, reactive, and cervical intraepithelial neoplasia (CIN) biopsies and to determine possible pitfalls. Seventy-seven consecutive cervical specimens routinely diagnosed (Dx_orig) as CIN 1 or 2, or no-CIN, were revised independently by two expert gynecopathologists. MIB-1 staining and oncogenic human papillomavirus (HPV) assessment (by polymerase chain reaction) were performed. Independent diagnoses (plus oncogenic HPV status, in case of disagreement between the experts) were used to obtain a final diagnosis (Dx_final) and compared with MIB-C. Four of the 27 (15%) Dx_final = normal were HPV positive. Agreement between the gynecopathologists was 72 of 77 (94%). There were 30 (39%) discrepancies between Dx_orig and Dx_final (23 = 30% downgrades and 7 = 9% upgrades). All 23 downgrades were HPV negative and all seven upgrades were HPV positive. Overall agreement between Dx_orig and MIB-C was 73%, and with Dx_final 99%. Sensitivity, specificity, and positive and negative predictive values of MIB-C were very high without false negatives. Tangential cutting of MIB-1-positive parabasal cells and inflammatory cells can erroneously be overdiagnosed as a MIB-C. One single false positive of the 48 non-CIN cases (an immature squamous metaplasia) showed a special, easily recognizable MIB-1 pattern, different from CIN because the MIB-1 staining in the nuclei is not diffuse (as in CIN) but clumped. Moreover, positive nuclei are somewhat less densely packed than in CIN. When tangentially cut parabasal cells and inflammatory cells are carefully excluded, MIB-C is a strong diagnostic adjunct in distinguishing CIN from normal or benign cervical squamoepithelial lesions.

In lymph node-negative invasive breast carcinomas, specific chromosomal aberrations are strongly associated with high mitotic activity and predict outcome more accurately than grade, tumour diameter, and oestrogen receptor

The objectives of this study were to analyse whether specific chromosomal gains and losses in lymph‐node negative breast cancer correlate with other features and to evaluate their prognostic value. Seventy‐six lymph node‐negative breast carcinomas (median follow‐up 46 months; range 9–105 months) were used. Histological grade, tumour type, maximal tumour diameter, oestrogen/progesterone receptor (ER/PR), mitotic activity index (MAI), and mean nuclear area (MNA) were assessed. Whole genome DNA analysis was performed by comparative genomic hybridization (CGH). Chromosomal aberrations were compared with classical and other prognostic features. Kaplan–Meier curves and multivariate survival analysis (Cox model) were used to assess the prognostic value of the CGH and other data. Fifteen (21.4%) out of 70 patients (six cases were lost to follow‐up) developed locoregional (n = 3) or distant metastases (n = 12). The following criteria were prognostic for (any) recurrence (in decreasing significance): 3q gain, simultaneous gain at 1q and 8q, MAI < versus ≥ 10, MNA < versus ≥ 63 μm. Loss of 1p occurred significantly more often in the large group of ductal breast carcinomas with a MAI ≥ 10 (n = 38) than in cancers with a MAI < 10. Moreover, 8/15 (53%) patients with recurrences had a gain at 3q, as opposed to three (5.5%) of the 55 recurrence‐free patients. This association was even stronger in ductal carcinomas (hazard ratio = 10.9, p < 0.0001). Cox regression revealed that the 3q gain was the strongest prognostic factor; other features did not have additional prognostic value. In conclusion, loss of 1p is associated with a high MAI. A gain of 3q is a stronger predictor of recurrence than grade, MAI, and other features in invasive breast cancers. Copyright

Biologic profiling of lymph node negative breast cancers by means of microRNA expression.

Breast cancer is a heterogeneous disease. Different subgroups can be recognized on the basis of the steroid receptors, HER-2, cytokeratin expression and proliferation patterns. As a result of mRNA-profiling studies, five major groups can be recognized, of which the triple-negative and basal-like tumors have the worst prognosis. Many of these tumors have a high proliferation that has the strongest prognostic value in node negative breast cancer. In the current study we analyzed the microRNA pattern in 103 lymph node negative breast cancers and compared these profiles with different biological characteristics and clinicopathological features. Unsupervised hierarchical cluster analysis divides the patients into four main groups, of which the basal-like/triple-negative group is the most prominent (11% of all cases), the luminal A cancers containing the Her2 negative and estrogen receptor/progesterone receptor-positive tumors is the largest group (57%), and the group of luminal B (32%) is more heterogeneous and contains the Her2 positive/estrogen receptor-negative patients as well. The highest overall classification values by analysis of variance followed by cross validation (leave one sample out and reselect genes) were found for cytokeratin 5 and 6, triple-negative and estrogen receptor, with 97, 90 and 90% accuracy, respectively. MiR-106b gene is prominent in all of these signatures and correlates strongest with high proliferation. Other interesting observations are the presence of several microRNAs (miR532-5p, miR-500, miR362-5p, and miR502-3p) located at Xp11.23 in cancers with a triple-negative signature, and the upregulation of several miR-17 cluster members in estrogen receptor-negative tumors. The current study shows that estrogen receptor negativity and cytokeratin 5 and 6 expression are important, and specific biological processes in lymph node negative breast cancer, as microRNA signatures are strongest in these subgroups.

Low- and high-risk CIN 1 and 2 lesions: prospective predictive value of grade, HPV, and Ki-67 immuno-quantitative variables

The aim of this study was to evaluate in small cervical biopsies (non‐cone, non‐large loop excision of the transformation zone, LLETZ) the prognostic value of both routinely assessed and reviewed cervical intraepithelial neoplasia (CIN) grades 1 and 2, oncogenic human papillomavirus (onco‐HPV) DNA (HPV status) and Ki‐67 immuno‐quantitative features for the prediction of progression. In biopsies from 44 CIN patients (the learning set), subjective CIN grade, onco‐HPV by PCR, and Ki‐67 immuno‐quantitative features were assessed. We followed development of the lesions by colposcopy and cytology, but the final endpoint was the histological grade (again in small biopsies). The outcome was defined as progression (histological (CIN 1 to (CIN 2 or 3)) or CIN 2 to CIN 3) or not (all other cases). Single and multivariate (Cox regression) and survival analyses were applied. The resulting predictive combination of quantitative features was then applied to a new test set of 35 consecutive CIN 2 (small) biopsies followed by large (cone or LLETZ) biopsies. In the learning set, mean follow‐up of non‐progression cases was 18.8 months (range 4.7–35.9), and of progression cases 13.1 months (range 6.4–32.9) (p = 0.18). Five cases progressed (11%). Of the 16 CIN 1 and 28 CIN 2 lesions, 31 cases (70%) were onco‐HPV positive (5 of the CIN 1 and 26 of the CIN 2). The age of women with progression or not did not differ (p = 0.68). All 5 progression cases were CIN 2 (on review, one of these was reclassified as CIN 1), and positive for onco‐HPV. Cox regression analysis showed that the percentage of Ki‐67‐positive cells located in the middle third layer of the epithelium (MIDTHIRD) and the 90th percentile of the stratification index (SI90) was the best combination to predict progression (log rank = 5.1, p = 0.02). Furthermore, sensitivity (100%), specificity (56%), positive predictive value (23%), negative predictive value (100%), and overall percentage correctly classified cases (61%) of this Ki‐67 combination were higher than that of subjective CIN grade or HPV status, either single or combined (both for routine and review CIN grades). Adding CIN grade or HPV status did not improve the Ki‐67 prognostic results. Application of the prognostic Ki‐67 combination to the test set of 35 small biopsies followed by large (cone or LLETZ biopsies) gave comparable results. Analyses on homogeneous subgroups (CIN 2 only, onco‐HPV+ only, or CIN2/onco‐HPV+ only) gave similar results. In conclusion, Ki‐67 immuno‐quantitation of small biopsies showing CIN 1 or CIN 2 has strong independent prognostic value for progression. Copyright