Use of flow cytometry in the analysis of stage III squamous cell carcinoma of the oesophagus and its association with MIB-1.

AIMS: To examine the prognostic and pathobiological importance of DNA content in oesophageal squamous cell carcinomas in Hong Kong Chinese subjects; to evaluate its association with the immunohistochemical proliferative marker MIB-1. METHODS: Paraffin wax embedded tumour tissue and adjacent normal tissue (control tissue) samples from 45 resected stage III oesophageal squamous cell carcinomas were studied using flow cytometric analysis. The DNA content and the clinicopathological data of these patients were analysed together with the MIB-1 labelling index. RESULTS: DNA aneuploidy was present in 14 (31%) of the 45 cases. However, the DNA content did not correlate significantly with the age, sex, or survival of the patients, nor the length, location, differentiation and MIB-1 labelling index of the oesophageal carcinomas. The synthetic (S) phase fraction of diploid tumours bore no relation to the patients' survival or MIB-1 score. CONCLUSIONS: Flow cytometry was not as useful as the MIB-1 labelling index in predicting the biological characteristics of the tumours and the prognosis of patients with oesophageal squamous cell carcinomas. This study does not support the routine use of DNA flow cytometric analysis in oesophageal cancers.     

CD117 expression in operable oesophageal squamous cell carcinomas predicts worse clinical outcome

Aims

To investigate the aberrant expression of CD117 in oesophageal squamous cell carcinoma (SCC) and its prognostic significance.

Methods and results

Immunohistochemical staining for CD117 was performed on tissue microarray and routine tissue sections from 157 oesophageal SCC patients and 10 normal oesophageal epithelia adjacent to tumour. The positive rate of CD117 expression was 29.9% in oesophageal SCC tissues, whereas no CD117 expression was detected in the 10 normal oesophageal epithelia. CD117 expression was significantly associated with T stage (P < 0.001), distant metastasis (P = 0.015), lymph node metastasis (P = 0.019), and clinical stage (P = 0.021). Progression‐free survival in the patients with CD117‐positive tumours was shorter than that in the patients with CD117‐negative tumours (P = 0.010). In univariate analyses, CD117 expression was the most significant factor for overall survival of oesophageal SCC patients (P < 0.001), followed by lymph node metastasis (P = 0.001), T stage (P = 0.002), clinical stage (P = 0.006), distant metastasis (P = 0.020), and histological grade (P = 0.027). Multivariate analyses verified that CD117 expression was an independent prognostic marker for oesophageal SCC patients (P = 0.002). In addition, CD117 expression predicted poorer survival in patients without distant metastases.

Conclusions

CD117 expression in operable oesophageal SCC may be a valuable prognostic marker, and detection of its expression in clinical samples may be useful in defining a subclass of oesophageal SCCs with extremely poor clinical outcome, which may require a specially targeted treatment modality.     

Detection of telomerase in hepatocellular carcinomas using a PCR ELISA assay: comparison with hTR expression

BACKGROUND: While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected in many immortal cell lines and various cancers. AIM: To investigate telomerase expression in hepatocellular carcinoma, and to assess the expression of the RNA component of telomerase, hTR. METHODS: 39 hepatocellular carcinomas were studied using a telomerase polymerase chain reaction (PCR) enzyme linked immunosorbent assay, which does not require radioactive PCR amplification and yields a semiquantitative measurement. Expression of hTR was also assessed by a non-radioactive in situ hybridisation procedure. The correlations between these two markers and the clinicopathological data were analysed. RESULTS: Telomerase activity was detected in 23 of 39 hepatocellular carcinoma specimens (59%). Comparison of hepatocellular carcinoma with and without telomerase expression, or with high and low telomerase (10 cases v 13 cases), showed no differences in the principal clinicopathological data. Although median survival was lower in the group with detectable telomerase activity than in that with undetectable activity (510 v 720 days) the difference was not significant (log-rank test, p = 0.08). hTR expression was detected in 11 of 14 cases of hepatocellular carcinoma tested (78%) and in four of 12 samples of adjacent non-cancerous tissue (33%). Five tumours and four non-cancerous tissues were positive for hTR, whereas no telomerase activity was detected in these. CONCLUSIONS: The presence of telomerase activity in hepatocellular carcinomas is confirmed. No correlation was observed between clinicopathological data and telomerase expression in hepatocellular carcinoma, but survival seemed better in the absence of telomerase expression. hTR seems to be more widely expressed than telomerase.     

Different amplification patterns of the human telomerase RNA gene in invasive cervical carcinomas and cervical intraepithelial neoplasia grade III

The aim of this study was to compare the amplification patterns of the human telomerase RNA gene (hTERC) in invasive cervical carcinomas (ICC) and cervical intraepithelial neoplasia grade III (CIN III) and to define their potential clinical implications. Cervical liquid-based cytological (LBC) specimens were collected from 53 squamous cell carcinomas (SCC), 14 CIN III, and 20 normal controls. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization (FISH) using a dual-color probe containing the hTERC probe and the control, chromosome 3 centromere-specific probe (CSP3). Nucleus with abnormal FISH pattern for hTERC was observed in 0.94-90.65% of SCC cells and in 0-85.59% of CIN III cells. Using the threshold of 5.89%, the occurrence of hTERC amplification in SCC and CIN III was similar (90.6% vs. 85.7%, P = 0.630). However, the median percentage of cells with extra gains of hTERC (hTERC:CSP3 > 1) in SCC was higher than in CIN III (64.3% vs. 31.7%, P = 0.001). Among those cells, the 3:2 signal pattern was the leading pattern for both SCC and CIN III; high-level amplification of hTERC was more common in SCC than in CIN III (60.9% vs. 48.9%, P = 0.002). In SCC, it was not found that extra gains of hTERC were associated with any clinicopathological parameters. Thus, hTERC amplification was common in cervical exfoliated cells from SCC and CIN III. More complex amplification patterns of hTERC were present in ICC. Clinical usefulness of hTERC amplification in LBC samples was limited in ICC.     

Eosinophilia is a favorable prognostic marker for oral cavity and lip squamous cell carcinoma

Eosinophils are frequently encountered with squamous cell carcinomas (SCC) and it has been proposed that tumor‐associated tissue eosinophilia (TATE) could be of prognostic significance in oral SCC. The aim was to evaluate TATE in 83 oral cavity and 16 lip SCCs as well as the best possible use of TATE as a prognostic marker. The number of eosinophils was counted per high power fields (HPF, ×400) in three different representative areas of the tumor and its stroma. The degree of TATE was analyzed in relation to clinicopathological features of tumors and patients’ survival (follow‐up mean 40.7 months) using Fisher's exact test. TATE was detected in 58 (70%) oral and 8 (50%) lip SCC samples. The median number of eosinophils between oral and lip SCC was different (p = 0.028) but TATE was similar per HPF (p = 0.085). Totally, 6% of lip and 21% of oral SCC patients died during the follow‐up. The patients with the higher TATE had significantly better survival than the patients with the lower TATE (p = 0.0136). The best cut‐off value predicting the survival was 4 eosinophils/HPF. TATE is a prognostic marker for oral and lip SCC: more than 4 eosinophils/HPF may predict more favorable prognosis.     

A rare case of nasal Schneiderian (inverted) papilloma associated with basaloid squamous cell carcinoma

Sinonasal inverted papilloma (IP) is a benign, locally aggressive epithelial neoplasm. In less than 9% of cases it is associated with malignancies, typically conventional squamous cell carcinomas (SCC), while other histological variants have been less frequently reported. We describe the third case of basaloid squamous cell carcinoma (BSCC) arising in nasal IP.An 81-year-old female patient presented with a pinkish irregular lesion on the nasal septum. Biopsy was consistent with IP and carcinoma in situ. Two surgical procedures were needed to obtain radical excision. Histology on the surgical specimen revealed BSCC. Seven months after surgery, there was no evidence of disease recurrence.Although IP is more frequently associated with conventional SCC, other malignancies should be considered. The histological differential diagnosis should be supported by immunohistochemistry. The generally-recommended treatment for sinonasal BSCC is complete surgical resection, although this may be a problem in multifocal distributions, as in the present case.     

Association of Kruppel-like factor 4 expression with the prognosis of esophageal squamous cell carcinoma patients

Objective: To investigate the association of Kruppel-like factor 4 (KLF4) expressions with the prognosis of esophageal squamous cell carcinoma (SCC) patients. Methods: Ninety-eight cases of esophageal carcinoma patients were enrolled. The expression of KLF4 in the esophageal SCC and normal esophageal mucosa tissues were examined by immunohistochemistry. The correlations between the expression of KLF4 protein and patients’ clinical characteristics and prognosis were analyzed. Results: We observed higher expressed KLF4 in normal esophageal mucosa tissues than esophageal SCC tissues, with positive rate of 82.7% (81/98) and 43.8% (43/98) respectively. In patients with lymphatic metastasis, the positive rate of KLF4 was 24.4% (10/41), whereas it was 57.9% (33/57) in patients without lymphatic metastasis, and the difference was significant (x² = 10.871, P = 0.001). The positive rates of KLF4 were 62.5% (5/8), 53.1% (26/49) and 29.3% (12/41) in stage I, II and III patients, respectively. There were no correlations between the expression of KLF4 and gender, age, tumor size, location, differentiation grade and infiltration depth. The 5-year survival rates and median survival times were 48.8% and 25.5%, and 55 and 26 months for the patients with KLF4 positive and negative expression, respectively. There were significant differences between the patients with KLF4 positive expression and negative expression in the 5-year survival rates and median survival times (x² = 5.747 and 4.493, P = 0.017 and 0.034). Conclusion: KLF4 might act as a tumor suppressor in esophageal SCC and the expression status of KLF4 could be considered as a prognosis predictor for esophageal SCC patients.     

Molecular pathology and potential therapeutic targets in esophageal basaloid squamous cell carcinoma

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma. Emerging studies show that genetic alterations are more frequent in BSCC than in conventional SCC, and some of which led to the identification of potential therapeutic targets in esophageal BSCC. Approximately half of the esophageal BSCC cases harbor either an EGFR mutation or amplification, and these occur in a mutually exclusive fashion. Therefore, the application of tyrosine kinase inhibitors may be beneficial to esophageal BSCC patients. This tumor is partly characterized by the activation of the Wnt and Hedgehog (HH) signaling pathways. Wnt signaling is activated by SFRP2 promoter hypermethylation and HH signaling is activated by the frequent mutations in PTCH1. Increasing evidence shows that the Wnt signaling pathway is involved in cross-talk with other developmental pathways, including the HH pathway. Therefore, pharmaceutical therapy targeting both the HH and Wnt pathways would be quite effective in patients with esophageal BSCC with highly malignant potential. In this review, we discuss the pathology, prognostic factors, genetic alterations and potential therapeutic targets in BSCC of esophagus.     

Primary squamous cell carcinoma of the thyroid gland: an entity with aggressive clinical behaviour and distinctive cytokeratin expression profiles

AIMS: Primary squamous cell carcinoma of the thyroid gland is uncommon. This study aims to identify the clinicopathological features and the pattern of expression of cytokeratins and oncoproteins in this tumour. METHODS AND RESULTS: Histological slides from Chinese patients with thyroid cancer treated in our institution from 1980 to 1999 were reviewed. Patients with primary squamous cell carcinoma of the thyroid were identified and their clinical records were analysed. The expression of cytokeratins (CKs), p53 and p21 in these cases were also studied by an immunohistochemical method. Four women (mean age 71 years) with squamous cell carcinoma of thyroid were found. The main presenting features were signs and symptoms of airway obstruction in three patients and neck swelling in one. The tumours were located at the right lobe (n=2), left lobe (n=1) or in both lobes of the thyroid (n=1). One patient died shortly after admission and the other three died within 4 months after thyroidectomy. The p53 protein was positive in 50% (2/4) of the tumours and p53+ tumours were poorly differentiated. The tumours were negative for p21. CK19 was expressed in all the tumours while CK7 expression was noted in 3/4 of the tumours. One carcinoma showed focal positivity to CK18. The tumours were negative for CKs 1, 4, 6, 10/13 and 20. The pattern of cytokeratin expression in squamous cell carcinoma of the thyroid gland was different from carcinoma showing thymus-like differentiation (CASTLE) of the thyroid gland and oesophageal squamous cell carcinoma. CONCLUSIONS: Squamous cell carcinoma of the thyroid has aggressive clinical behaviour and characteristic CK expression pattern. p53 over-expression in these tumours was associated with tumour differentiation.     

Correlation between cathepsin D expression and p53 protein nuclear accumulation in oesophageal squamous cell carcinoma

AIM: The lysosomal protease cathepsin D has been reported to be associated with tumour progression in malignant tumours. Expression of the gene encoding cathepsin D is known to be stimulated by oestrogen in mammary cancer cells. Recent experiments revealed that a p53 DNA binding site is located in the promoter region of the cathepsin D gene. This fact indicates that cathepsin D expression may correlate with p53 protein expression. The purpose of this study is to evaluate the expression patterns of the cathepsin D and p53 proteins in oesophageal squamous cell carcinoma (SCC). METHODS: In 154 patients with oesophageal SCC, expression of the cathepsin D and p53 proteins was measured in tumours by means of immunohistochemistry using monoclonal antibodies against cathepsin D (clone, 1C11) and p53 (clone, BP53-12). RESULTS: Cathepsin D was detected in tumour cells, although it was not found in normal oesophageal epithelium adjacent to carcinoma. High cathepsin D expression (positive tumour cells > 10%) was detected in 76 of 154 cases (49%) and high p53 nuclear expression (positive tumour cells > 50%) was detected in 70 cases (46%). High cathepsin D expression was significantly associated with invasive tumour growth (p = 0.002), poor prognosis (p = 0.049), and nuclear accumulation of p53 protein (p = 0.001). Overexpression of both p53 and cathepsin D was seen in 45 of the 154 cases (29.2%). In addition, there was a positive correlation between the cathepsin D index (percentage of cathepsin D positive tumour cells) and Ki-67 labelling index (percentage of Ki-67 positive tumour cells) in 154 oesophageal SCCs (rho = 0.257; p = 0.009). However, in multivariate survival analysis, cathepsin D expression by the tumours was not an independent prognostic factor in patients with oesophageal SCC (p = 0.236). CONCLUSIONS: The expression of cathepsin D by cancer cells may play an important role in the invasive growth of oesophageal SCC. Overexpression of both p53 and cathepsin D was seen frequently in tumours; p53 gene abnormalities may correlate with cathepsin D overexpression in oesophageal SCC.     

Metallothionein expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patient survival

Theocharis S, Klijanienko J, Giaginis C, Rodriguez J, Jouffroy T, Girod A, Point D, Tsourouflis G & Sastre‐Garau X
(2011) Histopathology 59 , 514–525 Metallothionein expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patient survival Aims: Metallothionein (MT) has been implicated in several aspects of cancer pathobiology, such as differentiation, proliferation, apoptosis and invasion. The aim of the present study was to evaluate the clinical significance of MT expression in mobile tongue squamous cell carcinoma (SCC). Methods and results: MT protein expression was assessed immunohistochemically on 49 mobile tongue SCC specimens, and was analysed in relation to clinicopathological characteristics, and overall and disease‐free patient survival. All of the examined mobile tongue SCC cases showed MT positivity in tumour cells; however, neither MT overexpression nor staining intensity was significantly associated with clinicopathological parameters. MT cellular distribution was significantly associated with histopathological grade of differentiation and depth of invasion (P = 0.0188 and P = 0.0484, respectively). MT staining intensity was identified as a significant predictor of overall patient survival at both univariate (P = 0.0377) and multivariate (P = 0.0472) levels. Twenty‐seven (55.10%) of the examined SCC cases showed MT positivity in squamous tongue epithelium adjacent to the tumour, the MT positivity being correlated with depth of invasion (P = 0.0281), vascular invasion (P = 0.0194), and the existence of lymph node metastases (P = 0.0194). Conclusions: MT may be implicated in the development and progression of mobile tongue SCC and could be considered as a useful clinical marker for patient management and prognosis.     

MIB-1 proliferative activity in invasive breast cancer measured by image analysis.

AIMS: To determine cell proliferation in infiltrating breast carcinomas. METHODS: Using the MIB-1 monoclonal antibody, the proliferation index was measured in paraffin wax sections of 871 breast cancers. The MIB-1 proliferation index was compared with other markers of disease progression: size, lymph node status, histotype, oestrogen and progesterone receptor status, expression of p53 and Neu, and DNA ploidy. All parameters were measured using image analysis. In 347 tumours, the MIB-1 and Ki-67 proliferation indexes were compared. Follow up data were available for 170 cases (median 66.5 months). RESULTS: Of the tumours, 314 (36%) had a high proliferation index. The MIB-1 proliferation index was correlated directly with size, nodal status, overexpression of p53 and Neu, and the DNA index; and inversely with oestrogen and progesterone receptor status. The correlation between MIB-1 and Ki-67 proliferation indexes was statistically significant. In patients with pT1 tumours, a low proliferation index correlated with a longer relapse-free interval and overall survival; node negative patients with a low proliferation index had a longer overall survival. CONCLUSIONS: The MIB-1 proliferation index is a reliable, practical and useful method of measuring proliferative activity and is an important predictor of clinical behaviour.     

Prognostic implication of telomerase activity in patients with brain tumors

Telomerase adds telomeric repeats to the ends of telomeres to compensate for their progressive loss. A favorable prognosis is associated with low or no telomerase in some tumors. The authors investigated whether telomerase activity is associated with survival of patients with brain tumors. Sixty-two consecutive patients with brain tumors underwent surgery, and their surgical specimens were investigated. The patients were pathologically categorized as group I (aggressive group) and group II (non-aggressive group). Telomerase activity was examined by the telomeric repeat amplification protocol (TRAP) assay. The median time was calculated in association with overall survival and progression-free survival in each group. The significant difference was noted in telomerase activity between high-grade gliomas and lowgrade gliomas (p=0.022). Telomerase activity was significantly associated with the median overall survival and progression-free survival in all tumors of the aggressive group. On the other hand, the median overall survival in the non-aggressive group was not dependent on telomerase activity, while the median progression-free survival was. Our data suggests that telomerase is an important prognostic indicator of survival in patients with brain tumors.     

Expression of cell cycle-regulatory proteins, MIB-1, p16, p53, and p63, in squamous cell carcinoma of conjunctiva: not associated with human papillomavirus infection

Squamous cell carcinoma (SCC), the most common primary malignant tumor of the conjunctiva, has a variable clinical presentation and immunohistochemical profile. Abundant cell cycles exist, including MIB-1 (Ki67 antigen), p16, p53, and p63, within the conjunctiva SCC. This investigation first reports the expressions of cell cycle markers in SCC. A retrospective study was conducted between December 1976 and June 2004, comprising 13 consecutive patients with conjunctiva SCC who were treated with surgical excision. Detailed clinical parameters were also reviewed. Overexpression of MIB-1, p16, p53, and p63 genes were studied by immunohistochemistry. Genechip containing 39 subtypes was used to elucidate human papillomavirus (HPV). The study group contained 13 (100%) men, with a mean age of 68±18 years and follow-up period of 20±17 months. The sample included four (33%) SCC located in the left eye and two (17%) recurrent SCC. Overexpression of the p53 and p63 was considerably higher than that of the p16 (P<0.01). HPV DNA was not detected in any of the 13 cases. This work first examined the immunohistochemical overexpression of cell cycle (MIB-1, p16, p53, and p63) in SCC. This investigation then showed that the expression of cell cycles in SCC was associated with key tumor clinicopathological features. This approach can help distinguish the potential roles of cell cycle in the development of SCC.     

Oesophageal mesenchymal tumours: clinicopathological features and absence of Epstein-Barr virus

BACKGROUND: Recent studies have suggested that the Epstein-Barr virus (EBV) is associated with smooth muscle tumours (leiomyoma and leiomyosarcoma) in patients with human immunodeficiency virus and in organ transplant recipients. Leiomyoma is the most common mensenchymal tumour found in the oesophagus. AIM: To report a single institution experience on oesophageal mesenchymal tumours and to determine whether EBV is associated with these tumours. METHODS: 40 sporadic oesophageal mesenchymal tumours were studied and their diagnosis confirmed on pathological review and immunohistochemical studies. Formalin fixed, paraffin was embedded tissues from these tumours were analysed for EBV using in situ hybridisation for two messenger RNA (mRNA) probes, EBER and BamH1 W. RESULTS: The oesophageal mesenchymal tumours comprised 36 leiomyomas, two undifferentiated stromal tumours, and two gastrointestinal autonomic nerve tumours (GANTs). Median age of the patients with leiomyoma (26 men, 10 women) was 62 years (range 30 to 85) and 81% of them had an asymptomatic lesion. The median longitudinal size was 1.2 cm. Multiple leiomyomas were seen in 11% of the patients and calcification was noted in one tumour. Coexisting squamous cell carcinoma was found in one third of cases. The stromal tumours were small, asymptomatic, and located in the lower third of the oesophagus, while the GANTs were large, symptomatic, and found in the upper third of the oesophagus. EBV mRNAs were not detected in all these tumours. CONCLUSIONS: The clinicopathological features of oesophageal leiomyoma, undifferentiated stromal tumour, and GANT were different. Some oesophageal leiomyomas were associated with oesophageal squamous cell carcinomas. EBV is not associated with sporadic oesophageal mesenchymal tumours.     

PROGNOSTIC VALUE OF MIB-1 IN NEUROENDOCRINE TUMOURS OF THE LUNG

The spectrum of neuroendocrine lung tumours ranges from highly aggressive small cell carcinomas (SCLC) to carcinoid tumours (CD) of low malignant potential. Between these two extremes, the ‘well-differentiated neuroendocrine carcinomas’ (WDNEC) form a transitional group with uncertain biological behaviour. This study investigated the prognostic value of the proliferation marker MIB-1 (paraffin Ki-67) in 59 neuroendocrine lung tumours (32 SCLC, 13 WDNEC, 14 CD) by immunostaining of routinely processed paraffin sections. Morphometric evaluation was done by semi-automatic image analysis. The results were compared with survival data (mean follow-up: 42 months). The proliferation rates of the tumours as determined by MIB-1 immunoreactivity (MIB-1-PR) were significantly different between the tumour types (SCLC>WDNEC>CD) and showed a strong inverse correlation with survival time. In CD, the percentage of MIB-1-labelled nuclei never exceeded 1·1 per cent; higher values would therefore favour the diagnosis of WDNEC over that of CD. Among WDNEC, MIB-1 was able to differentiate a subgroup with excellent prognosis (MIB-1-PR: 0·3–3·4 per cent) from another subgroup with a death rate of 50 per cent (MIB-1-PR: 7·3–20·3 per cent). Within each tumour type, all patients without distant metastases at diagnosis survived when MIB-1-PR was ⩽9·4 per cent, suggesting a potential threshold for prognosis. Although the status of metastases was the dominant prognostic factor in these neoplasms, MIB-1 was able to provide additional prognostic information allowing the definition of prognostically different subgroups of patients. In conclusion, MIB-1 and the status of metastases are complementary prognostic indicators and are best used in combination to characterize the biological behaviour of neuroendocrine lung tumours.     

Expression of Bcl-2 and Amplification of c-myc Are Frequent in Basaloid Squamous Cell Carcinomas of the Esophagus

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare, poorly differentiated variant of typical esophageal squamous cell carcinoma (SCC) characterized by high proliferative activity and frequent spontaneous apoptoses. In the present study, we investigated the expression of the apoptosis-suppressing protein Bcl-2 in 23 BSCC of the esophagus and 23 stage-matched typical esophageal SCC by means of immunohistochemistry. In addition, amplification of the apoptosis- and proliferation-inducing gene c-myc was determined by means of differential polymerase chain reaction. Bcl-2 expression was found significantly more often in BSCC than in SCC (86.9% vs. 17.4%, P < 0.0001). Amplification of c-myc was nearly twice as common in BSCC as in SCC (47.8% vs. 26.1%, not significant). Bcl-2 protein expression together with c-myc amplification was detected in 43.5% of the BSCC but in none of the typical SCC (P < 0.0001). Taken together, our findings indicate that the molecular pathogenesis of esophageal BSCC differs from that of typical SCC and frequently involves coactivation of c-myc and Bcl-2.