Jan Klos

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.

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.

Comparing subjective and digital image analysis HER2/neu expression scores with conventional and modified FISH scores in breast cancer

Background: HER2/neu expression and fluorescence in situ hybridisation (FISH) amplification have therapeutic significance. Aims: To compare subjective HER2/neu expression scores with digital image analysis (DIA) and conventional and modified FISH scores in breast cancer. Methods: Sixty HercepTest-immunostained breast carcinomas, prospectively scored as consensus 2+ and 3+ (DAKO protocol) by two observers, were analysed with DIA, and conventional (Vysis) and modified FISH scoring protocols. Results: With consensus scoring, 23 (38%) of the 60 cases were 2+ and 37 (62%) were 3+. Agreement with DIA scores was 100%. With conventional FISH scoring, 4 of the 3+ cases did not show amplification, but all of those negative cases had high HER2/neu copy numbers. With the modified FISH scoring protocol, all HercepTest immunohistochemical 3+ cases were amplified. Of the 2+ cases, 3 were amplified with the modified FISH protocol and 4 with the conventional FISH protocol. Conclusions: Modified FISH scores were better correlated with HercepTest 3+ consensus and DIA scores than were conventional FISH scores. HER2/neu DIA scoring is a cost-effective supplementary tool in surgical pathology.

Digital image analysis improves the quality of subjective HER-2 expression scoring in breast cancer.

AimTo compare HER-2 scoring reproducibility by subjective and digital image analysis (DIA) scores with each other and with fluorescence in situ hybridization (FISH) assessed HER-2 amplification. MethodsHerceptest-stained Tissue Micro Arrays of 219 breast carcinomas were scored (DAKO protocol) by 3 observers (both independent and as consensus), scored by DIA and both scores were compared with FISH amplification results. ResultsInterobserver subjective scores reproducibility was good (κ 0.82 to 0.86) but therapeutically important 3+/2+discrepancies occurred in 11% to 16% of all 3+ cases. Subjective scores and FISH results differed considerably. Consensus scores by 3 pathologists correlated better with FISH, reducing the number of both Immunohistochemical (IHC) negative/FISH positives and IHC 3+/FISH negatives. DIA scores were well reproducible and correlated better with FISH amplification than did subjective scores. ConclusionsDIA scores were comparable with consensus scores between 3 expert pathologists, were very well reproducible and performed better in classifying IHC 3+/FISH+ cases than did subjective scores.

Evaluation of 5 different labeled polymer immunohistochemical detection systems.

Immunohistochemical staining is important for diagnosis and therapeutic decision making but the results may vary when different detection systems are used. To analyze this, 5 different labeled polymer immunohistochemical detection systems, REAL EnVision, EnVision Flex, EnVision Flex+ (Dako, Glostrup, Denmark), NovoLink (Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK) and UltraVision ONE (Thermo Fisher Scientific, Fremont, CA) were tested using 12 different, widely used mouse and rabbit primary antibodies, detecting nuclear, cytoplasmic, and membrane antigens. Serial sections of multitissue blocks containing 4% formaldehyde fixed paraffin embedded material were selected for their weak, moderate, and strong staining for each antibody. Specificity and sensitivity were evaluated by subjective scoring and digital image analysis. At optimal primary antibody dilution, digital image analysis showed that EnVision Flex+ was the most sensitive system (P<0.005), with means of 8.3, 13.4, 20.2, and 41.8 gray scale values stronger staining than REAL EnVision, EnVision Flex, NovoLink, and UltraVision ONE, respectively. NovoLink was the second most sensitive system for mouse antibodies, but showed low sensitivity for rabbit antibodies. Due to low sensitivity, 2 cases with UltraVision ONE and 1 case with NovoLink stained false negatively. None of the detection systems showed any distinct false positivity, but UltraVision ONE and NovoLink consistently showed weak background staining both in negative controls and at optimal primary antibody dilution. We conclude that there are significant differences in sensitivity, specificity, costs, and total assay time in the immunohistochemical detection systems currently in use.

The prognostic value of molecular biomarkers in tissue removed by curettage from FIGO stage 1 and 2 endometrioid type endometrial cancer.

OBJECTIVE To analyze the prognostic value of molecular biomarkers in curettages of endometrioid endometrial cancer pathologic FIGO stages 1 and 2. STUDY DESIGN Population-based survival analysis in 258 patients of classical prognostic features and molecular biomarkers of cell cycle regulation, (anti)apoptosis, proliferation, squamous differentiation, and PTEN/Akt pathway. RESULTS With 74 months median follow-up (range, 1-209), 24 (9.3%) patients had metastases develop. Pathologic FIGO stage 2B (6% of all cases) and age > 68 years had independent multivariate prognostic value. Many molecular biomarkers were prognostic, particularly cell-cycle regulators p16, p21, p27, p53, p63, and the antiapoptosis marker survivin (which mostly stains mitoses). The strong prognostic value of a multivariate model with survivin, p21, and p53 overshadowed all other prognosticators in pathologic FIGO 1 and 2A. CONCLUSION In pathologic FIGO stage 1 and 2A endometrioid endometrial cancer curettages, combined biomarkers survivin, p21, and p53 expression patterns are prognostically stronger than classical feature combinations.

Prognostic comparison of the proliferation markers mitotic activity index, phosphohistone H3, Ki67, steroid receptors, HER2, high molecular weight cytokeratins and classical prognostic factors in T 1-2 N 0 M 0 breast cancer

The proliferation factors: mitotic activity index (MAI), phosphohistone H3 (PPH3) and Ki67 have strong prognostic value in early breast cancer but their independent value to each other and other prognostic factors has not been evaluated. In 237 T₁₋₂N₀M₀ breast cancers without systemic adjuvant treatment, formalized MAI assessment and strictly standardized, fully automated quantitative immunohistochemistry (IHC) for Ki67, PPH3, estrogen (ER) and progesterone receptor (PR), HER2, cytokeratins-5/6 and -14, and automated digital image analysis (DIA) for measuring PPH3 and Ki67 were performed. Section thickness was measured to further control IHC measurements. All features were measured in the periphery of tumors. The different proliferation assessments and other well-established clinicopathological and biomarker prognostic factors were compared. DIA-Ki67 added prognostically to PPH3. None of the other biomarkers or clinicopathological variables added prognostically to this PPH3/Ki67 combination. However, when PPH3 is replaced by MAI the prognostic value is nearly the same. In early operable node negative breast cancer without adjuvant systemic treatment, Ki67 with a threshold of 6.5% assessed by digital image analysis in the periphery of the tumor is prognostically strong. The combination of either PPH3/Ki67 or MAI/Ki67 overshadowed the prognostic value of all other features including Ki67 alone.

Reduced Number of CD8+ Cells in Tonsillar Germinal Centres in Children with the Periodic Fever, Aphthous Stomatitis, Pharyngitis and Cervical Adenitis Syndrome.

The syndrome of periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) is an autoinflammatory disorder of unknown aetiology. Tonsillectomy may cause a prompt resolution of the syndrome. The aim was to study the histologic and immunological aspects of the palatine tonsils in PFAPA, to help understand the pathophysiology of the syndrome. Tonsils from children with PFAPA (n = 11) and children with tonsillar hypertrophy (n = 16) were evaluated histologically after haematoxylin and eosin staining. The number of different cell types was identified immunohistochemically by cluster of differentiation (CD) markers: CD3 (T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD15 (neutrophils), CD20 (B cells), CD45 (all leucocytes), CD57 (NK cells) and CD163 (monocytes and macrophages). Tonsils from children with PFAPA showed reactive lymphoid hyperplasia dominated by well‐developed germinal centres with many tingible body macrophages. The histologic findings were unspecific, and a similar morphologic appearance was also found in the tonsils from controls. The number of CD8+ cells in germinal centres differed between children with PFAPA [median 9 cells (quartiles: 5, 15)] and controls [18 cells (12, 33) (P = 0.001)] and between children with PFAPA with (median 14 cells; 9, 16) and without (4 cells; 3, 8) aphthous stomatitis (P = 0.015). For the other cell types, no differences in germinal centres were found between children with PFAPA and controls. In conclusion, a lower number of CD8+ cells were found in germinal centres of tonsils in children with PFAPA compared to controls, which may be a feature linked to the aetiology of the syndrome.

Improved diagnostic segregation of mantle cell lymphoma by determination of cyclin D1/D3 expression ratio in formalin-fixed tissue.

Mantle cell lymphomas (MCLs) are associated with a characteristic t(11;14)(q13;q32) chromosomal translocation. This causes the CCND1 gene on chromosome 11 to be co-localized with the immunoglobulin heavy chain gene on chromosome 14, resulting in increased expression of cyclin D1. The cyclin D1/D3 expression ratio, as an approach to segregate MCLs from other small B-cell lymphomas, has not previously been evaluated in formalin-fixed, paraffin-embedded tissue. We found that mean cyclin D3 expression was lower in MCLs (P<0.05) than in chronic lymphocytic leukemias (CLLs), follicular lymphomas (FLs), marginal zone/mucosa-associated lymphoid tissue lymphomas (MALTs), multiple myelomas (MMs), and reactive lymph nodes. As expected, mean cyclin D1 expression was increased in MCL (P<0.05), but in several cases the expression of cyclin D1 did overlap with the level observed in CLLs, FLs, MALTs, MMs, and reactive lymph nodes. The cyclin D1/D3 expression ratio, however, did fully separate MCLs from FLs, CLLs, and reactive lymph nodes. The mean expression ratio was also significantly different between MCL and MALT (P<0.05), but 3 MCL cases had values overlapping those of some MALTs. The expression ratio was not significantly different between MCL and MM. In conclusion, the cyclin D1/D3 expression ratio gave an improved segregation of MCLs from CLLs, FLs, MALTs, and reactive lymph nodes, as compared with determination of cyclin D1 alone in formalin-fixed, paraffin-embedded tissue.

GATA3 differential expression in neuroblastoma and nephroblastoma

We have read with great interest the report by Wiles et al, published in Cancer Cytopathology, about the immunohistochemical expression of the GATA3 antibody in cytological material from neuroblastomas. They evaluated GATA3 immunohistochemical expression in neuroblastoma cytology and/or biopsy samples, and diffuse nuclear positivity was found in all 30 specimens studied. Each sample revealed either strong (n 5 26) or moderate nuclear staining (n 5 4) in more than 75% of neuroblastoma cells, regardless of the presence or lack of stromata, necrosis, or differentiation. GATA3 is a relatively recent marker useful for diagnosing tumors of various epithelial and nonepithelial origins such as mammary tumors, urothelial tumors, renal tumors, germ cell tumors, mesotheliomas, and paragangliomas. Moreover, it is usually negative in round cell sarcomas. In contrast, little is known about GATA3 expression in blastemal or other small round cell pediatric tumors. Nonaka et al reported the presence of diffuse GATA3 expression in the neuroblastoma family of tumors and a lack of expression in other small round cell tumors. The distinction between neuroblastomas and nephroblastomas may be delicate because the clinical and radiological presentation may be overlapping. Moreover, we have previously encountered important difficulties in the cytological differential diagnosis between poorly differentiated neuroblastomas without neuropils or differentiating cells from blastema-rich monophasic nephroblastomas. We initiated this study to evaluate GATA3’s immunohistochemical utility in the cytological differential diagnosis between neuroblastomas and nephroblastomas. Six cell blocks from the most recent consecutive ultrasound-guided fine-needle aspirates from neuroblastomas and 6 cell blocks from nephroblastomas were investigated. Paraffin-embedded sections were stained with the GATA3 antibody (clone L50-823; Biocare, Concord, California). Immunostaining was scored as negative, weakly positive, or strongly positive. The percentage of positive cells was also estimated. Nuclear staining was found in all 6 cases of neuroblastoma but in only 1 case of nephroblastoma. No cytoplasmic staining was observed. The intensity varied from weak (4 cases) to strong (2 cases; Figs. 1 and 2). The percentage of positive cells varied from 5% to 100%. Only 1 of the 6 cases of nephroblastoma showed weak nuclear positivity in 5% of tumor cells. The clinical data and the results of immunohistochemistry are presented in Table 1 On the basis of our results, we conclude that GATA3 may be a useful marker for differentiating neuroblastomas from nephroblastomas, especially when it is used with Phox2b and tyrosine-hydroxylase antibodies. Moreover, in some cases, GATA3 may be not specific, and positivity in a blastemal nephroblastoma may be a pitfall of a differential diagnosis with a poorly differentiated neuroblastoma. Figure 2. Weak GATA3 nuclear positivity in nephroblastoma. Figure 1. Strong GATA3 nuclear positivity in neuroblastoma.