Positive Staining For Ki-67 Research Articles

Clinical, Histologic, and Immunohistochemical Features of an Undifferentiated Renal Tubular Carcinoma in a Juvenile Olive Baboon (Papio anubis)

An undifferentiated renal tubular carcinoma was diagnosed in a juvenile male olive baboon (Papio anubis). The animal suddenly appeared depressed and refused to eat. During physical examination, a firm, palpable mass in the left abdominal area and flank pain were detected. Clinical pathology findings included mild anemia, hypoalbuminemia, hyponatremia, and mildly increased serum creatinine and urea concentrations. Radiographs revealed a large mass in the left abdominal area. Exploratory laparotomy disclosed a 10 cmx15 cm multilobulated mass involving the left kidney and adjacent organs. Because of a poor prognosis, the animal was humanely euthanized, and necropsy was performed. Tissue samples of the neoplasm were taken for histopathological examination. Immunohistochemical staining was done using vimentin, cytokeratin, S-100 protein, Ki-67, alpha-actin, and desmin-specific primary antibodies. Microscopically, elongated and irregular tubules were lined by 2 or more layers of atypical epithelial cells. Anisocytosis, anisokaryosis, and frequent mitotic figures were also observed. Following immunohistochemical staining, the cytoplasm of neoplastic cells was positive for cytokeratin, vimentin, and S-100 protein and negative for alpha-actin and desmin. Positive nuclear staining for Ki-67 was observed. The neoplasm was diagnosed as an undifferentiated renal tubular carcinoma.

Spontaneous recovery of myocardial function after ligation of Ameroid-stenosed coronary artery

Objectives. We aimed to assess the spontaneous healing of myocardial function after occlusion of a chronically stenosed coronary vessel in a porcine model. Design. Ischemia and infarction was produced by Ameroid constrictor placement and a subsequent ligation of the left circumflex artery. Cardiac MRI and 18FDG-PET were performed one and five weeks later. Ki67 staining was used to identify proliferating cells. Results. Restoration of perfusion defect was detected by MRI (p=0.0065), reduced systolic function of the lateral segment spontaneously recovered (p=0.03). There was also a suggestive raise in impaired ejection fraction (p=0.06). Left ventricular early diastolic filling and peak filling rate were substantially improved (p=0.039 and p=0.0078). Scar size reduced (p=0.03). On the 18FDG-PET, deranged metabolism was alleviated (p=0.03). Cardiomyocytes with positive Ki-67 staining were located principally in the non-infarcted myocardium as compared to the infarction or border areas (p=0.037). Conclusions. We demonstrated spontaneous functional healing of ischemic and infarcted left ventricle, suggesting border zone perfusion recovery. Scar reduction was detected. Different pattern of myocyte proliferation between infarction and non-ischemic myocardium was seen.

Intraperitoneally applied gentamicin increases collagen content and mechanical stability of colon anastomosis in rats

Anastomotic leakage remains a serious complication in colorectal surgery, and is being caused by a multitude of factors. Recent reports reveal changes of the extracellular matrix as risk factors as well as gentamicin as a potential agent to influence wound healing. This experimental study was initiated to investigate the influence of intraperitoneally applied gentamicin on colonic anastomotic wound healing and in particular on mechanical stability, overall collagen content and collagen type I/III ratio. Sixty Sprague Dawley rats were randomized to one of two groups. In each animal, a standard transverse colonic end-to-end anastomosis was performed. Immediately postoperative, either 5 ml gentamicin (1 ml/kg bodyweight) or NaCl 0.9% was applied intraperitoneally. On postoperative days 3, 5, and 14, ten of the animals in each group were sacrificed. Measurements of the anastomosis bursting pressure were performed on postoperative days 3 and 5. At each explantation time, the collagen per protein ratio, the collagen types I/III ratio, and both the expression of MMP-2, -9, and Ki67 were analyzed. None of the animals died. None of the rats exhibited clinical evidence of anastomotic leakage. The bursting strength in the gentamicin group was significantly elevated on postoperative day 5. Both the overall collagen content and the collagen type I/III ratio in the gentamicin group were significantly increased 3, 5, and 14 days postoperatively compared to the control group. The expression of MMP-9 was significantly elevated in the gentamicin group both 3 and 5 days postoperatively. In contrast, there were no significant differences in the expression of MMP-9 14 days postoperatively. All investigated samples demonstrated positive staining for MMP-2 and Ki67 without statistically significant differences at any term, respectively. The present data confirm that intraperitoneally applied gentamicin is able to enhance healing and stability of colonic anastomosis due to an increase of both the overall collagen content and collagen type I/III ratio.

Trpv4 induces collateral vessel growth during regeneration of the arterial circulation

The development of a collateral circulation (arteriogenesis), bypassing an arterial occlusion, is important for tissue survival, but it remains functionally defective. Micro array data of growing collateral vessels, exposed to chronically elevated fluid shear stress (FSS), showed increased transcription of the transient receptor potential cation channel, subfamily V, member 4 (Trpv4). Thus, the aim of this study was to investigate the role of the shear stress sensitive Trpv4 in transmitting this physical stimulus into an active growth response. qRT-PCR at different time points during the growth of collateral vessels after femoral artery ligature (FAL) in rats showed a strong positive correlation of Trpv4 transcription and the intensity of FSS. An increased protein expression of Trpv4 was localized in the FSS-sensing endothelium by means of confocal immunohistochemistry. Cultured porcine endothelial cells showed a dose-dependent expression of Trpv4 and an increased level of Ki67-positive cells upon treatment with 4alpha-Phorbol 12,13-didecanoate (4alphaPDD), a specific Trpv4 activator. This was also demonstrated by flow culture experiments. These results were confirmed by in vivo application of 4alphaPDD in rabbit hind limb circulation via an osmotic mini-pump after FAL. Trpv4 expression as well as Ki67-positive staining was significantly increased in collateral vessels. Finally, 4alphaPDD treatment after FAL led to a 61% (215.5 ml/min/mmHg versus 350 ml/min/mmHg) recovery of conductance when compared with the non-occluded artery. Cell culture and in vivo studies demonstrate that an FSS- or a 4alphaPDD-induced activation of Trpv4 leads to an active proliferation of vascular cells and finally triggers collateral growth. Trpv4, a well-known FSS-sensitive vasodilator, has hitherto not been implicated in active growth processes of collateral arteries. This new function may lead to new therapeutic strategies for the treatment of arterial occlusive diseases.

T17b murine embryonal endothelial progenitor cells can be induced towards both proliferation and differentiation in a fibrin matrix

Endothelial progenitor cells (EPC) may enhance blood vessel formation in a variety of clinical settings such as ischaemia and tumour angiogenesis as well as in tissue-engineered matrices. In the present study, we cultured a murine endothelial progenitor cell line, T17b, in vitro in cell culture as well as in an FDA-approved fibrin matrix and investigated cell proliferation, differentiation and secretion patterns of the angiogenic growth factor VEGF under hypoxia and differentiation. We show that T17b EPC remain viable for at least 8 days in the fibrin matrix where they proliferate and form clusters including lumen-like structures. Proliferation in fibrin clots overlayed with basal medium (BM) was confirmed morphologically and immunohistochemically by positive Ki67 staining, indicating mitotic activity. Significant cell proliferation and Ki-67 expression were absent when cells were incubated with dibutyryl-cAMP and retinoic acid (RA). Incubation with dibutyryl-cAMP and RA stimulated the expression of the EPC differentiation markers von Willebrand Factor (vWF) and VEGF receptor 2 (VEGFR-2), indicating successful differentiation in the fibrin clot. EPC differentiation induced by dibutyryl-cAMP and RA was confirmed in 2-D chamber slide cultures by positive vWF immunostaining, which was absent in BM controls. EPC chamber slides also displayed positive vWF staining when exposed to hypoxia under BM conditions, indicating EPC activation and differentiation could also be induced by hypoxia. Taken together, T17b EPC secrete increased levels of VEGF when submitted to either hypoxia or differentiation and can be differentiated into mature endothelial cells not only in cell and matrigel cultures but also in a fibrin matrix that is FDA approved for clinical application.

P.340 Plate-guided distractor for a segmental mandibular defect

To investigate the clinical and pathologic characteristics of primary intradural retroclival chordoma and improve the understanding of this rare disease.A retrospective study was conducted on six cases patients withof an intradural chordoma in the retroclival region who underwent surgery and were confirmed by pathology and imaging. Expression of brachyury, galectin-3, and Ki-67 in paraffin-embedded sections of the specimens was detected by streptavidin-peroxidase immunohistochemistry.Lesions were located in the subdural prepontine cistern. Computed tomography scan showed that the bone of the skull base was not destroyed, and findings on magnetic resonance imaging varied, resulting in a misdiagnosis of 50% of the cases in the preoperative imaging. All six cases were classified by their pathology into the classical subtype. They presented a strong positive staining for brachyury and galectin-3, and the Ki-67 labeling index was between 2.5% and 8.2%. Three cases presented no signs of recurrence or regrowth, whereas in the other three patients recurrence or regrowth occurred at 7∼14 months after initial surgery. Two patients died of this disease.Our study suggests that a positive staining for brachyury, galectin-3, and Ki-67 would be helpful for differential diagnosis, discriminating intradural retroclival chordoma from ecchordosis physaliphora and chordoid meningioma. Our study also shows that within intradural retroclival chordoma, there are significant prognostic differences. Tumors with an abundant blood supply, flake-like cellular arrangement, and a Ki-67 labeling index greater than 5% belong to a rapid-growth type and are prone to short-term recurrence and poorer prognosis.

Granular Cell Tumor of the Esophagus

Purpose: 24 y.o. female referred for 10-month history of emesis and abdominal pain underwent EGD revealing a white-yellow submucosal growth at 32 cm from the incisors measuring 1 cm in length and 0.5 cm in width. Pathology report read: “One fragment consists of pink-staining granular cells with small, rounded nuclei consistent with granular cell tumor (GCT). Cells are strongly positive for S-100 and vimentin. Features of malignancy are absent”. Methods: Abrikossoff first described “granular cell myoblastoma” in 1926. It was considered a myogenic tumor until Fisher and Wechsler's 1962 challenge. Using electron microscopy and Wallerian degeneration studies, they noted similarities between a myoblastoma cell and a degenerating Schwann cell. Today, most pathologists and oncologists acknowledge the neural origin of GCTs supported by positive IHC staining for CD68, Ki-67 (slightly), NKI/C3, NSE, nestin, p53 (slightly), S-100 protein, and vimentin. In addition, positive PAS stain with resistance to diastase digestion and nonreactivity to HHF35, desmin, and alpha-smooth muscle actin are characteristic. Results: GCTs comprise only 0.03% of all tumors, and GI GCTs are even more rare with the distal 1/3 of esophagus cited most frequently. Mean age of diagnosis is 45 years with equal M:F ratio. Dysphagia is the most common symptom correlating to >1 cm size. Classic appearance is a small, non-tender, broad-based, submucosal growth resembling a “molar tooth” when central depression exists. A rubbery or firm consistency is characteristic with pinktan, gray-white, or white-yellow coloration. EUS reveals a homogenous, hypo/isoechoic submucosal lesion. Approximately 200 cases have been documented. Conclusion: GCTs are notoriously benign, but no management guidelines exist. Presently, an esophageal GCT > 2 cm size, symptomatology, interval growth, recurrence, tissue invasion, or advanced histologic/sonographic features prompt resection. Preoperative EGD re-evaluation with confirmatory biopsy followed by EUS to address muscularis and lymphatic invasion are recommended. EMR, Nd:YAG laser ablation, and APC are safe, effective alternatives to surgery in the absence of muscularis or lymphatic involvement. Esophageal GCTs are not radiosensitive, but recurrence is uncommon.Figure: Cells with granular, pink-staining cytoplasm and small nuclei.

P.341 Poorly differentiated carcinoma: Regaud's lymphoepithelioma

To investigate the clinical and pathologic characteristics of primary intradural retroclival chordoma and improve the understanding of this rare disease.A retrospective study was conducted on six cases patients withof an intradural chordoma in the retroclival region who underwent surgery and were confirmed by pathology and imaging. Expression of brachyury, galectin-3, and Ki-67 in paraffin-embedded sections of the specimens was detected by streptavidin-peroxidase immunohistochemistry.Lesions were located in the subdural prepontine cistern. Computed tomography scan showed that the bone of the skull base was not destroyed, and findings on magnetic resonance imaging varied, resulting in a misdiagnosis of 50% of the cases in the preoperative imaging. All six cases were classified by their pathology into the classical subtype. They presented a strong positive staining for brachyury and galectin-3, and the Ki-67 labeling index was between 2.5% and 8.2%. Three cases presented no signs of recurrence or regrowth, whereas in the other three patients recurrence or regrowth occurred at 7∼14 months after initial surgery. Two patients died of this disease.Our study suggests that a positive staining for brachyury, galectin-3, and Ki-67 would be helpful for differential diagnosis, discriminating intradural retroclival chordoma from ecchordosis physaliphora and chordoid meningioma. Our study also shows that within intradural retroclival chordoma, there are significant prognostic differences. Tumors with an abundant blood supply, flake-like cellular arrangement, and a Ki-67 labeling index greater than 5% belong to a rapid-growth type and are prone to short-term recurrence and poorer prognosis.

Histopathological features and prognostic significance of the micropapillary pattern in lung adenocarcinoma

The micropapillary pattern is characterized by small papillary tufts with no fibrovascular core lying in spaces and has been reported as an aggressive variant of carcinoma in several organs. We investigated the histopathobiological properties of the micropapillary pattern with immunohistochemistry, serial sections, and electron microscopy in lung adenocarcinoma. We further analyzed its clinicopathological character and prognosis. The subjects included 383 adenocarcinoma cases, of which 184 (48%) were micropapillary pattern-positive and 199 (52%) were micropapillary pattern-negative. On histology, micropapillary tufts seemed to float in the alveolar space or spaces encased by connective tissues, whereas serial sections revealed that most tufts had continuity with other tufts and even with the main tumor. Positive staining for the adhesion molecules E-cadherin and Β-catenin suggested the preservation of tight adhesion, and electron microscopy showed the existence of intercellular junctions. Negative staining for laminin and loss of basement membrane as determined by electron microscopy suggest a loss of cell–matrix contact. Positive staining for Ki-67 indicates that cells constituting micropapillary tufts retained their proliferation potency. There were no CD34-positive cells in micropapillary tufts, and the loss of the vascular core was confirmed. In micropapillary pattern-positive cases, lymphatic invasion was identified significantly more frequently than in micropapillary pattern-negative cases (P<0.001), even at stageIA (without lymph node metastasis, N=197) (P<0.001). The 5-year and 10-year overall survival rates of the micropapillary pattern-positive stageIA group were 77.6 and 67.6%, respectively, which were significantly less than those of the micropapillary pattern-negative stageIA group (98.1 and 98.1%) (P=0.001). In conclusion, cells constituting the micropapillary pattern are likely to have acquired anchorage-independent growth and a potential for high malignancy.

Kidney injury molecule-1 expression in transplant biopsies is a sensitive measure of cell injury

Kidney injury molecule-1 (KIM-1) is a specific histological biomarker for diagnosing early tubular injury on renal biopsies. In this study, KIM-1 expression was quantitated in renal transplant biopsies by immunohistochemistry and correlated with renal function. None of the 25 protocol biopsies showed detectable tubular injury on histologic examination, yet 28% had focal positive KIM-1 expression. Proximal tubule KIM-1 expression was present in all biopsies from patients with histological changes showing acute tubular damage and deterioration of kidney function. In this group, higher KIM-1 staining predicted a better outcome with improved blood urea nitrogen (BUN), serum creatinine, and estimated glomerular filtration rate (eGFR) over an ensuing 18 months. KIM-1 was expressed focally in affected tubules in 92% of kidney biopsies from patients with acute cellular rejection. By contrast, there was little positive staining for Ki-67, a cell proliferation marker, in any of the groups. KIM-1 expression significantly correlated with serum creatinine and BUN, and inversely with the eGFR on the biopsy day. Our study shows that KIM-1 staining sensitively and specifically identified proximal tubular injury and correlated with the degree of renal dysfunction. KIM-1 expression is more sensitive than histology for detecting early tubular injury, and its level of expression in transplant biopsies may indicate the potential for recovery of kidney function.

Down‐regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens-Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP-Pax6 transgenes into these Pax6(-) clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium.

Prediction of aggressiveness of gastrointestinal stromal tumours based on immunostaining with bcl‐2, Ki‐67 and p53

While the use of fine needle aspiration (FNA) in the diagnosis of gastrointestinal stromal tumours (GISTs) is well-established, it can be difficult to predict the prognosis of GIST based on morphology alone. The objective of the current study was to determine if expression of bcl-2, Ki-67 and p53 correlated with the outcome of GISTs based on cytological material. Cell-blocks from 14 GISTs diagnosed by FNA were retrieved. Immunostaining was performed with antibodies against bcl-2, Ki-67 and p53. All cytological diagnoses were confirmed by positive immunostaining with c-kit and/or subsequent histological evaluation. Positivity for bcl-2, Ki-67 and p53 was defined as the presence of > or =10% cytoplasmic staining, > or =5% nuclear staining and > or =5% nuclear staining respectively. The 14 patients consisted of seven males and seven females with a mean age of 58 years. The average follow-up interval was 46 months. Six had a benign course and eight developed recurrences/metastases. Thirteen (93%) cases showed positive staining for bcl-2. Positive Ki-67 and p53 staining was noted in one (7%) and seven (50%) cases respectively. The difference in staining for p53 between aggressive and non-aggressive GISTs was statistically significant. No statistically significant difference was noted for bcl-2 staining or Ki-67 labelling index between the two groups. According to our observations, p53 immunostaining may be useful in predicting the outcome of GIST diagnosed by FNA; Ki-67 and bcl-2 are not useful as prognostic markers for GIST in FNA specimens.

Liver regeneration is suppressed in alcoholic cirrhosis: correlation with decreased STAT3 activation

Liver regeneration is suppressed in alcoholic patients; however, the underlying mechanisms are not fully understood. We examined liver regeneration and signal transducer and activator of transcription 3 (STAT3) activation (an important signal for liver regeneration) in cirrhotic livers from alcoholics, hepatitis C virus (HCV) infection, and alcoholic plus HCV infection. Liver regeneration and STAT3 activation were determined by immunohistochemistry analysis of Ki67 and STAT3 phosphorylation, respectively, in 20 alcoholic cirrhosis, 13 HCV cirrhosis, 13 alcoholic + HCV cirrhosis. Alcoholic or alcoholic plus HCV cirrhotic livers had significantly lower Ki67 + and phospho-STAT3 + (pSTAT3 +) hepatocytes and bile duct cells than HCV cirrhotic livers. The pSTAT3 positive staining did not correlate with liver injury (elevation of serum levels of aspartate transaminase [AST] and alkaline phosphatase [ALP]) but correlated positively with cell proliferation (Ki67 positive staining). In conclusion: liver regeneration is suppressed in alcoholic cirrhotic livers, which may be partly due to decreased STAT3 activation.

Evaluation of Epithelial Dysplasia at the Surgical Margins in Patients with Early Tongue Carcinoma — Immunohistochemical Study of Recurrence of Epithelial Dysplasia

Objective: The diagnosis of epithelial dysplasia at the surgical margins in oral cancer plays an important role in its prognosis and requires careful evaluation. Epithelial dysplasia is usually asymptomatic, but often associated with recurrence and malignant transformation.Patients and Methods: The patients included 9 men and 5 women (mean age, 54.6 years; range, 31 to 76 years) with early tongue carcinoma and tumour-cell-'free " surgical margins. Immunohistochemical analysis of p53 and Ki-67 expression was performed in order to evaluate epithelial dysplasia and pathological changes.Results: None of the patients had cancer recurrence, but 2 showed recurrence of epithelial dysplasia. The resection margins at initial surgery and at recurrence were positive for p53 and Ki-67 in both these patients.Conclusions: Epithelial dysplasia, with positive staining for p53 and Ki-67, has the potential for morphological changes, with possible recurrence of epithelial dysplasia or malignant transformation. Such patients will require careful follow-up for local recurrence. Examination of surgical margins for these markers is useful for the early detection of pathological changes.

Immunohistochemical Expression of p16 and Ki-67 Correlates With Degree of Anal Intraepithelial Neoplasia

Anal intraepithelial neoplasia (AIN) is a human papilloma virus related lesion. It has been shown that infection with high-risk human papilloma virus results in up-regulation of p16 and increased cellular proliferation. The objective of this study is to correlate p16 expression and cellular proliferation measured by Ki-67 staining with the degree of dysplasia in the anal canal and to determine the efficacy of these markers in diagnosing high-grade AIN. Seventy-five anal specimens from 55 patients (37 men; 18 women; mean age: 48 y; median: 44 y; range 25 to 96 y) were studied including 35 normal/reactive lesions, 23 low-grade AIN (AIN I and condyloma), and 17 high-grade AIN (AIN II and III). Immunostaining for p16 and Ki-67 was performed. Expression of p16 in AIN correlated with that of Ki-67 (P<0.001). High-grade AIN often demonstrated p16 staining in more than one-third of the thickness of the epithelium in a diffuse/continuous fashion. p16 expression in low-grade AIN was often restricted to the lower 1/3 of the epithelium and/or was focal and discontinuous. The expression of both p16 and Ki-67 correlated with the degree of dysplasia (P<0.01). When positive p16 staining was defined as the presence of diffuse/continuous staining in more than one-third of the thickness of epithelium, the sensitivity, specificity, and accuracy of p16 as a marker for diagnosing high-grade AIN were 76%, 86%, and 84%, respectively. When positive Ki-67 staining was defined as the presence of nuclear staining in more than 25% of the cells in more than one-third of the thickness of epithelium, the sensitivity, specificity, and accuracy of Ki-67 as a marker for diagnosing high-grade AIN were 71%, 84%, and 83% respectively. Both p16 and Ki-67 are reliable markers for diagnosing high-grade AIN.

Utility of SKP2 and MIB-1 in grading follicular lymphoma using quantitative imaging analysis

Follicular lymphoma is classified into grades (G)1, 2, and 3 based on the number of centroblasts in neoplastic follicles. However, the accuracy of manually counting these centroblasts is limited by certain cells (large centrocytes, follicular dendritic cells, and histiocytes) that could mimic centroblast morphology. The reproducibility of follicular lymphoma grading is dependent upon observer experience; therefore, significant variations occur. This study is to explore a more objective, reliable way of grading follicular lymphoma using a quantitative imaging system in conjunction with immunostains with antibodies to proliferation markers MIB-1 and S-phase kinase-associated protein 2 (SKP2). Fifty-eight follicular lymphomas (G1, n = 23; G2, n = 18; and G3, n = 17) were studied on formalin-fixed, paraffin-embedded sections. Positive nuclear staining of both Ki-67 and SKP2 was recorded using the quantitative Clarient ACIS II system (Aliso Viejo, CA, USA). Ten high-power fields (x400) from randomly selected neoplastic follicles were counted by a pathologist blinded to the previously assigned morphologic grade. The results show that the percentages of Ki-67+ and SKP2+ cells significantly differ among the different grades of follicular lymphoma. A higher grade of follicular lymphoma is associated with a higher percentage of Ki-67+ and SKP2+ cells. The overall SKP2+% cells are substantially lower than Ki-67+% cells in the same grade of follicular lymphoma. Statistical significance is observed in Ki-67+ cells between follicular lymphoma G1 and follicular lymphoma G3 and between G1 and G2. In contrast, statistical significance is noted in SKP2+% cells between follicular lymphoma G1 and follicular lymphoma G3 and between follicular lymphoma G2 and follicular lymphoma G3. The findings suggest that the SKP2 expression has better discrimination with grades of follicular lymphoma than Ki-67 expression. Compared with traditional methods, quantitation of SKP2 expression using a quantitative image analysis system might be a useful and objective approach in grading follicular lymphoma.

Expression Patterns of Ki-67 and Telomerase Activity in Middle Ear Cholesteatoma

Keratinocytes in cholesteatoma demonstrate uncoordinated hyperproliferation, migration, and invasion properties. There is a controversy regarding the impact of Ki-67 and telomerase activities on cellular proliferation in cholesteatoma. We studied expression of Ki-67 protein and telomerase activity in cholesteatoma and its relationship with clinical findings. The expression level of Ki-67 protein was examined by immunohistochemical analysis of 51 cholesteatomas and 6 skin tissues obtained from patients during ear surgery. Telomerase activity was determined in 23 samples of cholesteatomas and 6 skin samples by polymerase chain reaction-based telomeric-repeat amplification protocol assay. The presence of Ki-67 protein was observed in 21 (41.2%) of 51 samples of acquired cholesteatoma. The average Ki-67 labeling index in the cholesteatoma group was 28.9 +/- 9.2 and was higher than that in the skin group (18.2 +/- 6.1). Telomerase activity was detected in 2 (8.7%) of 23 samples of cholesteatoma (21 of them were Ki-67 staining positive and 2, negative) and in 3 (50%) of 6 of control skin samples (p < 0.05). This study showed increased expression of Ki-67 in cholesteatoma, whereas there was no significant difference in rate of Ki-67 positive staining between skin and cholesteatoma (p = 0.066). Telomerase activation is a rare event in cholesteatoma. We assume that the absence of telomerase may lead to generation dysfunctional telomeres what in turn may impair the proliferative capacity of cholesteatoma.

Prognostic Significance of Surgical Margin, Ki-67 and Cyclin D1 Protein Expression in Grade II Canine Cutaneous Mast Cell Tumor

The prognosis for canine cutaneous mast cell tumor (CCMT) is thought to be correlated with histopathological grading. However, the wide variety of histopathologic types of grade II is one of the most troublesome and difficult points for prognosis. The objective of this study is to determine the prognostic value of surgical margin, ki-67 and cyclin D1 protein expression in grade II tumor. Surgically resected specimens of solitary grade II CCMT from 48 dogs with follow-up periods over 360 days (median was 1080 days) were used in this study. The expression of cyclin D1 and ki-67 proteins was determined by morphometrically using slides stained immunocytochemically, and the correlations among the results, survival rate, and recurrence and/or metastasis rate of each dog were analyzed statistically. The recurrence and/or metastasis and mortality rate in the incomplete surgical excision group within 30 months postoperatively were higher than that of the complete surgical excision group. In the incomplete surgical excision group, dogs with low positive staining of ki-67 had a significantly better survival, but the recurrence and metastasis rate and ki-67 positivity failed to show a significant correlation. Only a small number of cases showed cyclin D1-positive tumor cells, but most of them had a poor outcome with a high recurrence rate. In grade II CCMT, incomplete excision induced a relatively high metastasis rate and poor prognosis. Ki-67 positivity is a marker for the estimation of overall survival in incomplete surgical excision cases. Cyclin D1 positivity was low and may not have a prognostic role.

Distinct Phenotype of Chronic Lymphocytic Leukaemia (B-CLL) Cells Engrafting in NOD/SCID Mice.

We have previously reported reliable engraftment of CD19/CD5/CD23/CD45 positive human B-CLL cells in the spleens of NOD/SCID mice (NSS-CLL cells) following transplantation of human peripheral blood-derived B-CLL (PB-CLL) cells. On histology, in the majority of the samples NSS-CLL cells formed focal aggregates which also contained various amounts (range:0–50%) of human CD45/CD3+ T cells (ASH 2005; #52). We now have further characterized these NSS-CLL cells. In our model 1x10^8 MNC are injected i.v. plus i.p. and engraftment in the spleen and other organs is analyzed after 4 (−8) weeks by flow cytometry and immunohistochemistry. In 22 CLL samples investigated in a total of 64 NOD/SCID mice 1.6±0.4x106 NSS-CLL cells representing 10.5±2.3% of total spleen cells were recovered with recovery highly reproducible on repetitive experiments (3.0 vs. 2.0; 1.6 vs. 1.5; 0.2 vs. 1.1; 0.0 vs. 0.0x106 NSS-CLL cells; n=4). In contrast to the low proliferative status reported for human PB-CLL, considerable proliferation was observed in NSS-CLL cells. Following the application of BrdU (1mg/6g d5 i.p., 1mg/ml added to drinking water d6–13; 20–27) BrdU positive NSS-CLL cells ranged from 5.3–25.0% (n=5). Immunohistological analysis showed positive staining for the proliferation marker Ki67 in 18.5±3.5% of NSS-CLL cells. In addition, markers related to proliferation/activation of B-CLL proliferation center cells such as survivin (mean MFI: 5.5±0.8 vs. 2.3±0.2; p=0.028; n=6) and CD100 (semaphorin 4d; mean MFI: 3.3±0.5 vs. 1.1±0.2; p=0.043; n=5) were significantly up-regulated in NSS-CLL vs. PB-CLL cells. When NSS-CLL cells were characterized utilizing additional adhesion/migration, differentiation and/or activation markers, only a subset of NSS-CLL cells (15.7±8.2%, n=6) expressed chemokine coreceptor (CCR) 7, while PB-CLL cells uniformly showed high CCR7 expression. Also CCR5, a migration marker in the context of non-Hodgkin lymphomas, consistently was demonstrated on a fraction of NSS-CLL cells (8.1±3.0%; n=6), whereas here no CCR5 expression was found on PB-CLL cells. No expression of human CD10, CD11c, CD127 or CD49d was detected in B-CLL cells derived from either PB- or NSS-CLL cells, while both sources, as expected, uniformly stained positive for CD40. In summary, NSS-CLL exibit a distinct phenotype as compared to PB-CLL cells and show considerable similarity to B-CLL cells in the proliferation centers of involved human tissues. For these cells not only proliferation but also increased expression of survivin and CD100 as well as down-regulation of CCR7 have been described. Therefore, we think that our model may represent a novel tool to further elucidate biological properties of human B-CLL and, in addition, may serve to devise and evaluate novel treatment strategies.

Cellular Immunostaining of Angiotensin-Converting Enzyme in Human Coronary Atherosclerotic Plaques

The aim of this study was to determine the cellular localization of angiotensin I-converting enzyme (ACE) in the atherosclerotic plaque and its correlation with inflammation and cellular proliferation. Angiotensin I-converting enzyme inhibitors reduce the incidence of vascular events; therefore, tissue ACE may play a determinant role in the pathophysiology of the atherosclerotic plaque. Histology and immunocytochemistry of de novo coronary plaques retrieved with directional coronary atherectomy from 141 patients were analyzed: 87 with stable angina, 39 with subacute unstable angina, and 15 with acute unstable angina. Compared with stable patients, unstable patients showed more thrombotic lesions (72% vs. 27%, p < 0.0001), smaller areas of fibrous plaque (2.3 +/- 1.2 mm2 vs. 2.8 +/- 1.1 mm2, p = 0.02), higher cellular proliferative score (0.78 +/- 0.9 vs. 0.27 +/- 0.6, p = 0.003), larger content of ACE-stained cells (26.3 +/- 23% vs. 12.6 +/- 15%, p = 0.005) and larger areas of inflammation as identified by CD68 immunostaining (29.5 +/- 22% vs. 20.2 +/- 19%, p = 0.02). A significant linear correlation was found between CD68- and ACE-stained areas (mm2) among unstable patients (r = 0.6, p = 0.0001), but it was absent among stable patients (r = 0.006, p = 0.9). Co-localization of ACE, CD68, and alpha-actin was confirmed by double immunostaining. Patients with Ki-67-positive staining as an index of cell proliferation showed also significantly larger areas of ACE immunoactivity (p = 0.004). Our data demonstrate ACE immunoactivity in inflammatory and proliferative cells of coronary atherosclerotic plaques. In particular, patients with unstable angina showed larger areas of ACE immunoactive tissue and proliferating cells compared with stable patients. These observations support a role of the enzyme in the pathophysiology of coronary unstable plaques and suggest potentially different effects of ACE inhibitors according to clinical presentation.