Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index

AIMS : Counting mitotic figures is considered to be a reliable prognosticator, but evaluation of Ki67 immunohistochemistry has become more popular in evaluating proliferation. Our previous studies suggested an occasional discrepancy between mitotic figures and Ki67 fraction. The aim of this study was to investigate this more closely and also to study the associations between bcl-2 and p53 expression and proliferation. METHODS AND RESULTS: Two hundred and sixty-five infiltrating breast carcinomas were immunostained for Ki67, p53 and bcl-2. The standardized mitotic index (SMI) was determined. Four proliferation groups were based on Ki67 positivity fraction and SMI at optimal cut-off points. Cox's multivariate model was used to test the power of the prognosticators. SMI and nodal status were the most powerful individual prognosticators. Ki67 was an independent prognosticator if nodal status, tumour size, age and histological grade were included in the analysis but not if analysed with SMI. The group with low SMI and low Ki67 fraction had the best prognosis. Groups with high SMI had the poorest prognosis. The group with low SMI and high Ki67 fraction had a favourable prognosis. Bcl-2 negativity and p53 positivity correlated with proliferation. CONCLUSIONS: We have found a 'wrong positive' Ki67 group with favourable prognosis. SMI cannot be replaced by Ki67 because of the danger of misclassification of some patients.     

PTTG基因蛋白的表达与垂体腺瘤侵袭性和增殖能力的关系

目的探讨人垂体腺瘤中垂体肿瘤转化基因(PTTG)蛋白的表达与肿瘤侵袭性和增殖程度的关系。方法采用免疫组织化学染色方法检测手术切除石蜡包埋的63例垂体腺瘤(侵袭组45例,非侵袭组18例)组织中PTTG蛋白的表达,染色增殖细胞核抗原(PCNA),同时计数组织内微血管数量(MVD)。结果 PTTG在侵袭性垂体腺瘤中的表达水平显著高于非侵袭性垂体腺瘤。侵袭性垂体腺瘤中PCNA标记指数和微血管密度也显著高于非侵袭性垂体腺瘤。相关分析显示PTTG表达与垂体腺瘤内PCNA标记指数和微血管密度呈正相关(P<0.05)。结论 PTTG在垂体腺瘤形成过程中起重要作用,并与垂体腺瘤的侵袭性和增殖程度密切相关。     

Tumor apparent diffusion coefficient as an imaging biomarker to predict tumor aggressiveness in patients with estrogen‐receptor‐positive breast cancer

The purpose of this retrospective study was to evaluate whether tumor apparent diffusion coefficient (ADC) was correlated with pathologic biomarkers such as tumor cellularity, Ki67, tumor‐infiltrating lymphocytes (TILs), and peritumoral lymphocytic infiltrate (PLI) in patients with estrogen receptor (ER)‐positive breast cancer. The study was approved by the institutional review board and informed consent was waived. From July 2014 to December 2014, we reviewed 140 ER‐positive tumors in 138 consecutive patients (range, 28–77 years; mean, 52 years) who underwent preoperative breast MRI and definitive surgery. All patients underwent diffusion‐weighted imaging with a 3T scanner. Two radiologists drew the region of interest of the entire tumor and obtained the mean and pixel‐based histogram of ADC. On pathology, two pathologists reviewed tumor cellularity, Ki67, TILs, and PLI. Multiple linear regression analysis was used to determine pathologic variables independently associated with ADC. Tumors with high tumor cellularity and high Ki67 had significantly lower ADCs than those with low tumor cellularity and low Ki67 (P < 0.05 for all). Tumors without PLI had significantly higher standard deviation than those with PLI (0.23 ± 0.08 versus 0.18 ± 0.05; P < 0.001). Median ADC was negatively correlated with tumor cellularity (r = ?0.441), and Ki67 (r = ?0.382). The standard deviation of ADC was also negatively correlated with the degree of PLI (r = ?0.319). On multivariate linear regression analysis, tumor cellularity and Ki67 were independently associated with tumor ADC. Tumor ADC would be an MRI biomarker for the prediction of tumor aggressiveness indicators such as Ki67, tumor cellularity, and PLI in ER‐positive breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.     

Elevated Ki‐67 labeling index in ‘synchronous liver metastases’ of well differentiated enteropancreatic neuroendocrine tumor

There is no consensus as to whether or not metastatic nodules in the liver should be biopsied for tumor grading in cases of neuroendocrine tumors with ‘synchronous liver metastasis’. In this study, we compared the Ki‐67 labeling index between the primary tumor and synchronous liver metastasis in 30 patients, who had received simultaneous resections. Examined tumors were of the small bowel (n = 18) or pancreas (n = 12), and G1 or G2 in primary histologic grade. In 20 patients (67%), the Ki‐67 index was similar between the primary tumor and liver metastasis, but 10 (33%) showed an elevation of 3.4–14.4% in the liver, which increased the tumor grade in 4 cases. The Ki‐67 elevation in the liver was more common in G2 than G1 neoplasms (P = 0.002). The size, but not number, of liver metastases was significantly larger in patients with an elevated Ki‐67 index (P = 0.006). Using 40 mm as a provisional cutoff for the greatest diameter of liver metastases, the positive predictive value of this discriminator for elevated Ki‐67 was 56%, and the negative predictive value was 93%. In conclusion, synchronous liver metastases can yield a higher Ki‐67 labeling index than primary neuroendocrine tumours, particularly when the secondary is greater than 40 mm.     

Phosphohistone‐H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage,PHH3, Ki‐67 or mitotic indices

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone‐H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV‐positive or ‐negative MCC patients, and assessed its prognostic value in comparison to Ki‐67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV‐positive and 9 MCPyV‐negative MCC patients. PHH3‐positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003–0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23–20.51) were observed as significantly independent prognostic factors for OS. PHH3‐positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93–26.55). PHH3‐positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV‐infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices.     

Overexpression of pituitary tumor transforming gene (PTTG) is associated with tumor progression and poor prognosis in patients with esophageal squamous cell carcinoma

Pituitary tumor transforming gene (PTTG) is a newly identified proto-oncogene that has been shown to be aberrantly overexpressed in a subset of human cancers. The aim of the present study was to examine PTTG expression in patients with esophageal squamous cell cancer (ESCC) and explore its clinical significance. PTTG protein expression was analyzed in 108 archived, paraffin-embedded primary ESCC specimens by immunohistochemistry and correlated with clinicopathological parameters and patients’ outcome. Overexpression of PTTG was observed in 38.0% (41/108) of primary ESCC tissues and significantly correlated with differentiation, TNM stage, lymph node metastasis, and depth of invasion (P < 0.05). Kaplan–Meier curves showed that ESCC patients with tumors expressing high levels of PTTG had substantially shorter overall survival compared with patients expressing low levels of PTTG (P = 0.022, log-rank test). Cox multivariate regression analysis revealed that overexpression of PTTG was an independent prognostic factor in overall survival for ESCC patients (hazard ratio was 2.35, P = 0.009). Overall, our data suggest that overexpression of PTTG may contribute to the malignant progression of ESCC and serve as a novel prognostic indicator for patients with ESCC.     

The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure.

The expression of the nuclear protein Ki-67 (pKi-67) is strictly correlated with cell proliferation. Because of this, anti-Ki-67 antibodies can be used as operational markers to estimate the growth fraction of human neoplasia in situ. For a variety of tumours, the assessment of pKi-67 expression has repeatedly been proven to be of prognostic value for survival and tumour recurrence, but no cellular function has yet been ascribed to the Ki-67 protein. This study shows that a C-terminal domain of pKi-67 (Kon21) is able to bind to all three members of the mammalian heterochromatin protein 1 (HP1) family in vitro and in vivo. This interaction can be manipulated in living cells, as evidenced by ectopic expression of GFP-tagged HP1 proteins in HeLa cells, which results in a dramatic relocalization of endogenous pKi-67. Taken together, the data presented in this study suggest a role for pKi-67 in the control of higher-order chromatin structure.     

Validity and reproducibility of Ki‐67 assessment in gastrointestinal stromal tumors and leiomyosarcomas

With the aim of standardizing Ki‐67 immunohistochemistry, we assessed interobserver and interlaboratory variability of the Ki‐67 labeling index and Ki‐67 score among eight general pathologists for 24 gastrointestinal stromal tumors (GISTs) and 12 leiomyosarcomas, which were predominantly of the gastrointestinal (GI) tract, mesentery and retroperitoneum, based on a review of a tissue microarrays subjected to immunohistochemistry with antibodies for Ki‐67. For Ki‐67 immunostaining of mesenchymal tumors of the GI tract, including GISTs, differences were seen in the scores given by regional hospitals. Conversely, for two categories of the Ki‐67 labeling index, namely <10% and ≥10%, concordance of the Ki‐67 score between microscopic observation and image analysis, and between the observers, was good, but it was not good for the other four categories of the index for <5%, 5–9%, 10–29%, and ≥30%. The concordance of the Ki‐67 scores between the observers in two categories was higher using the Ki‐67 pre‐stained tissue microarrays (TMAs) within each participating institute than that using the Ki‐67 stained TMAs between the participating institutes. The reproducibility of a 10% cut‐off value for the Ki‐67 labeling index to predict the prognosis of GISTs was relatively high, but there is an urgent need to standardize the staining technique.     

Well‐differentiated pancreatic neuroendocrine tumours (PanNETs) and poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs): concepts,issues and a practical diagnostic approach to high‐grade (G3) cases

With increasing accessibility and advancements in abdominal imaging modalities, the incidence of pancreatic neuroendocrine neoplasms has increased steadily during the past few decades. By definition, neuroendocrine neoplasms of the pancreas show neuroendocrine differentiation, but they represent a broad and heterogeneous group of neoplasms with diverse clinical and pathological characteristics. The majority of pancreatic neuroendocrine neoplasms can be classified as well‐differentiated pancreatic neuroendocrine tumours (PanNETs) or poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs). While PanNETs and PanNECs are distinct entities with respect to clinical presentation, outcome and therapeutic approach, they may exhibit overlapping histopathological features. Moreover, the frequent modifications in nomenclature and prognostic grading systems over the years of not only pancreatic neuroendocrine neoplasms, but neuroendocrine neoplasms from other organ sites, has created confusion for both pathologists and clinicians as to the appropriate use of terminology and grading when evaluating these neoplasms. This review examines the current concepts and issues of nomenclature and grading of PanNETs and PanNECs. In addition, considering the morphological overlap between high‐grade (G3) PanNETs and PanNECs, we discuss an integrative and practical diagnostic approach to aid in discriminating challenging cases.     

Loss of nectin‐3 expression as a marker of tumor aggressiveness in pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PanNETs) are rare, and prediction of aggressive characteristics, such as recurrence and metastasis and prognosis of PanNETs remain difficult. Nectins are cell adhesion molecules that regulate the formation of adherens and tight junctions. In this study, we investigated the clinicopathological significance of nectin‐3 expression in patients with PanNETs. Immunohistochemical analysis of nectin‐3 expression was performed on 78 cases of PanNET. Low nectin‐3 expression in the membrane (positive ratio ≤25%) was observed in 62 cases (79.5%) and was significantly correlated with larger tumor size (>20 mm; P = 0.003), G2/G3 tumors (P = 0.025), higher Ki67 labeling index (≥3%; P = 0.009), lymphatic involvement (P = 0.047), advanced pT‐factor (T2–T4; P = 0.003), lymph node metastasis (P = 0.006), advanced Union for International Cancer Control/American Joint Committee on Cancer‐stage (Stage II–IV; P = 0.001), advanced ENETS stage (Stage IIa–IV; P = 0.001), nonfunctioning tumors (P = 0.002), and a shorter disease‐free survival (P = 0.019). However, there was no significant correlation between nectin‐3 expression in the membrane and/or cytoplasm and the clinicopathological parameters. The present results suggest that decreased nectin‐3 expression in the membrane is associated with increased tumor aggressiveness of PanNETs. Clinically, immunohistochemical analysis of nectin‐3 may help predict tumor aggressiveness for PanNETs.     

Apparent diffusion coefficients in prostate cancer: correlation with molecular markers Ki‐67, HIF‐1α and VEGF

Prostate cancer (PCa) is the second most common cancer in men. The Gleason score (GS) and biomarkers play important roles in the diagnosis and treatment of patients with PCa. The purpose of this study was to investigate the relationship between the apparent diffusion coefficient (ADC) and the molecular markers Ki‐67, hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in PCa. Thirty‐nine patients with 39 lesions, who had been diagnosed with PCa, were enrolled in this study. All patients underwent diffusion‐weighted magnetic resonance imaging (DW‐MRI) (b = 800 s/mm²). The expression of Ki‐67, HIF‐1α and VEGF was assessed by immunohistochemistry. Statistical analysis was applied to analyze the association between ADC and prostate‐specific antigen (PSA), GS and the expression of Ki‐67, HIF‐1α and VEGF. The group differences in ADC among different grades of Ki‐67, HIF‐1α and VEGF were also analyzed. The mean ± standard deviation of ADC was (0.76 ± 0.27) × 10^?3 mm²/s. ADC correlated negatively with PSA and GS (p < 0.05). The Ki‐67 staining index (SI), HIF‐1α expression and VEGF expression in PCa were correlated inversely with ADC, controlling for age (r = –0.332, p < 0.05; r = ?0.662, p < 0.0005; and r = ?0.714, p < 0.0005, respectively). ADC showed a significant difference among different grades of Ki‐67 (F = 9.164, p = 0.005), HIF‐1α (F = 40.333, p < 0.0005) and VEGF (F = 22.048, p < 0.0005). In conclusion, ADC was correlated with PSA, GS, and Ki‐67, HIF‐1α and VEGF expression in patients with PCa. ADC may be used to evaluate tumor proliferation, hypoxia and angiogenesis in PCa.     

Usefulness of Ki‐67 (MIB‐1) immunostaining in the diagnosis of pulmonary sclerosing hemangiomas

Pulmonary sclerosing hemangioma (PSH) is an uncommon lung neoplasm with a clinical outcome that is generally benign. However, differentiating PSH from pulmonary carcinoma is sometimes difficult as both lesions share similar histopathologic and immunohistochemical features. In this study, we investigated the usefulness of Ki‐67 (MIB‐1) immunostaining in the diagnosis of PSH. We compared the staining pattern for Ki‐67 (MIB‐1) in 29 cases of typical PSH and 79 cases of pulmonary non‐small cell carcinoma (NSCLC) using an immunohistochemical method on formalin‐fixed paraffin‐embedded tissues. In all studied PSH cases, we noted cell membrane and cytoplasmic staining for Ki‐67 (MIB‐1), but this was not observed in any of the NSCLC cases. The Ki‐67 proliferation index was lower in PSH than in the NSCLC cases (mean, 1.1% vs mean, 5.5%; p < 0.001). These findings suggest that cell membrane and cytoplasmic staining for Ki‐67 (MIB‐1), as well as the Ki‐67 proliferation index, may be useful for distinguishing PSH from pulmonary carcinoma.     

High‐grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication—a comprehensive clinical,radiographic, pathologic,and genomic analysis

High‐grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication (HGNET BCOR ex15 ITD) is a recently proposed tumor entity of the central nervous system (CNS) with a distinct methylation profile and characteristic genetic alteration. The complete spectrum of histologic features, accompanying genetic alterations, clinical outcomes, and optimal treatment for this new tumor entity are largely unknown. Here, we performed a comprehensive assessment of 10 new cases of HGNET BCOR ex15 ITD. The tumors mostly occurred in young children and were located in the cerebral or cerebellar hemispheres. On imaging all tumors were large, well‐circumscribed, heterogeneous masses with variable enhancement and reduced diffusion. They were histologically characterized by predominantly solid growth, glioma‐like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. They demonstrated sparse to absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. While BCOR exon 15 internal tandem duplication was the solitary pathogenic alteration identified in six cases, four cases contained additional alterations including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2 and STAG2. While the limited clinical follow‐up in prior reports had indicated a uniformly dismal prognosis for this tumor entity, this cohort includes multiple long‐term survivors. Our study further supports inclusion of HGNET BCOR ex15 ITD as a distinct CNS tumor entity and expands the known clinicopathologic, radiographic, and genetic features.     

Immunohistological expression of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 in consecutive biopsies during chemoradiotherapy in patients with rectal cancer

The aim of this study was to describe the dynamics of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 during chemoradiotherapy (CRT) of rectal cancer, and to investigate the fluctuation of these biomarkers in relation to pathological response to CRT. The study included 86 patients with rectal adenocarcinoma receiving preoperative CRT (>50.4 Gy and Uracil/Tegafur). Immunohistological expressions of HIF‐1α, GLUT‐1, Bcl‐2 and Ki‐67 were investigated in biopsies taken before treatment, after 2, 4 and 6 weeks of CRT and in specimens from the operation. Decreasing expressions of HIF‐1α, Bcl‐2 and Ki‐67 were observed during CRT, whereas GLUT‐1 overall was unchanged. No significant changes of the markers were observed in the interval between CRT and surgery. A significant association was observed between the presence of residual carcinoma after 6 weeks of treatment and pathological response to CRT, but no association was seen between the fluctuations of any of the markers and response to CRT. This unique material containing specimens before, after and during CRT for rectal cancer demonstrated biological dynamics in HIF‐1α, Bcl‐2 and Ki‐67, but not GLUT‐1, expression during CRT, and a significant association was seen between the presence of residual carcinoma after 6 weeks of treatment and pathological response to CRT.     

Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry

In order to evaluate the suitability of Ki-67 and proliferating cell nuclear antigen (PCNA) for determination of proliferative activity, the immunohistochemically determined nuclear expression of these antigens in canine non-neoplastic and neoplastic tissues was compared with the results of in vivo bromodeoxyuridine (BrdU) labelling, which - by measurement of the fraction of S-phase cells - is considered as the standard in the analysis of proliferative activity. The samples investigated consisted of non-neoplastic mammary and lymphoid tissues, and of benign and malignant (primary/metastatic) mammary tumours, and malignant lymphomas. Great regional heterogeneity prevented determination of an overall labelling index (LI) in normal lymphoid tissues. In the remaining combined group of samples, LI values were significantly ranked in the order PCNA>Ki-67>BrdU. However, the correlation of Ki-67 or PCNA as compared to BrdU LI values was only moderate in the combined group [approximately 0.5, Spearman rank test] as well as in most subgroups, whilst it was very poor in the group of primary mammary cancers. These observations indicate that Ki-67 or PCNA LIs as markers of proliferation do not evenly match in vivo BrdU labelling.     

Cytopathologic differential diagnosis of low‐grade urothelial carcinoma and reactive urothelial proliferation in bladder washings: a logistic regression analysis

Conventional cytomorphologic assessment is the first step to establish an accurate diagnosis in urinary cytology. In cytologic preparations, the separation of low‐grade urothelial carcinoma (LGUC) from reactive urothelial proliferation (RUP) can be exceedingly difficult. The bladder washing cytologies of 32 LGUC and 29 RUP were reviewed. The cytologic slides were examined for the presence or absence of the 28 cytologic features. The cytologic criteria showing statistical significance in LGUC were increased numbers of monotonous single (non‐umbrella) cells, three‐dimensional cellular papillary clusters without fibrovascular cores, irregular bordered clusters, atypical single cells, irregular nuclear overlap, cytoplasmic homogeneity, increased N/C ratio, pleomorphism, nuclear border irregularity, nuclear eccentricity, elongated nuclei, and hyperchromasia (p ? 0.05), and the cytologic criteria showing statistical significance in RUP were inflammatory background, mixture of small and large urothelial cells, loose monolayer aggregates, and vacuolated cytoplasm (p ? 0.05). When these variables were subjected to a stepwise logistic regression analysis, four features were selected to distinguish LGUC from RUP: increased numbers of monotonous single (non‐umbrella) cells, increased nuclear cytoplasmic ratio, hyperchromasia, and presence of small and large urothelial cells (p = 0.0001). By this logistic model of the 32 cases with proven LGUC, the stepwise logistic regression analysis correctly predicted 31 (96.9%) patients with this diagnosis, and of the 29 patients with RUP, the logistic model correctly predicted 26 (89.7%) patients as having this disease. There are several cytologic features to separate LGUC from RUP. Stepwise logistic regression analysis is a valuable tool for determining the most useful cytologic criteria to distinguish these entities.     

Intramuscular low-grade myxoid neoplasm with recurrent potential (cellular myxoma) of the lower extremity: case report with cytohistologic correlation and review of the literature

"Low-grade myxoid neoplasm with recurrent potential" (cellular myxoma) is a term recently used to describe a subset of soft tissue lesions with histology intermediate between intramuscular myxoma and low-grade myxofibrosarcoma or myxoid malignant fibrous histiocytoma (MFH), while resembling a deeper counterpart of superficial angiomyxoma. Their distinctive biological behavior is characterized by the potential to recur locally, in contrast to intramuscular myxoma, while having no potential to advance in grade or metastasize when compared to low-grade myxofibrosarcoma. We present a cytohistological correlation for an intramuscular location of such a tumor in the lower extremity of a 49-yr-old male.