A novel nomogram for the preoperative prediction of sentinel lymph node metastasis in breast cancer.

BACKGROUND OR PURPOSE: A practical noninvasive method to identify sentinel lymph node (SLN) status in breast cancer patients, who had a suspicious axillary lymph node (ALN) at ultrasound (US), but a negative clinical physical examination is needed. To predict SLN metastasis using a nomogram based on US and biopsy-based pathological features, this retrospective study investigated associations between clinicopathological features and SLN status. METHODS: Patients treated with SLN dissection at four centers were apportioned to training, internal, or external validation sets (n = 472, 175, and 81). Lymph node ultrasound and pathological characteristics were compared using chi-squared and t-tests. A nomogram predicting SLN metastasis was constructed using multivariate logistic regression models. RESULTS: In the training set, statistically significant factors associated with SLN+ were as follows: histology type (p < 0.001); progesterone receptor (PR: p = 0.003); Her-2 status (p = 0.049); and ALN-US shape (p = 0.034), corticomedullary demarcation (CMD: p < 0.001), and blood flow (p = 0.001). With multivariate analysis, five independent variables (histological type, PR status, ALN-US shape, CMD, and blood flow) were integrated into the nomogram (C-statistic 0.714 [95% CI: 0.688-0.740]) and validated internally (0.816 [95% CI: 0.784-0.849]) and externally (0.942 [95% CI: 0.918-0.966]), with good predictive accuracy and clinical applicability. CONCLUSION: This nomogram could be a direct and reliable tool for individual preoperative evaluation of SLN status, and therefore aids decisions concerning ALN dissection and adjuvant treatment.

Hormone receptors analysis in Chinese patients with dermatofibrosarcoma protuberans.

BACKGROUND AND OBJECTIVES: Dermatofibrosarcoma protuberans (DFSP) is a relatively rare skin tumor. Clinical observations indicated that DFSP has a more aggressive behavior during pregnancy, which suggest there might be a hormonal influence on this tumor. We evaluated the expression of estrogen receptor (ER) and progesterone receptor (PR) in DFSP patients. METHODS: In our present case series, patients with histopathological-confirmed DFSP at a single institution were identified. The clinical, pathological, and immunohistochemical data were gathered for each patient. Expression of ER and PR were determined on formalin-fixed, paraffin-embedded tissue sections using immunohistochemistry. Some objective clinical and pathological indicators were then selected to compare between ER and PR status. RESULTS: Immunoreactivity revealed none of these tumors stained positively for ER, while eight tumors (28.6%) stained positively for PR. There was a statistically significant difference in the distribution of tumor location between the PR-positive/negative groups. CONCLUSIONS: This finding suggests that progesterone may have potential effects in growth of DFSPs. Further studies are needed to fully address this question.

[Clinicopathologic features and expression of epidermal growth factor receptor and vascular endothelial growth factor in adrenocortical tumors].

OBJECTIVE: To study the clinicopathologic features and expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in adrenocortical tumors. METHODS: Forty-two cases of adrenocortical tumors operated at the Beijing Union Medical College Hospital during the period from July, 2001 to July, 2010 were retrospectively reviewed. Immunohistochemical study for EGFR and VEGF was carried out. The clinical information and follow-up data were analyzed. RESULTS: The cases included 21 adrenocortical carcinomas (ACC) and 21 adrenocortical adenomas (ACA). Nine patients suffered from primary aldosterone syndrome, including 8 cases with ACA and 1 case with ACC. The average tumor size, tumor weight, and duration between disease onset and diagnosis in the 21 cases of ACC were 11.7 cm, 542 g and 8.5 months, respectively. This was in contrast to 3 cm, 9.8 g and 45.6 months, respectively in cases of ACA. Histologically, the WEISS score in all the 21 cases of ACA was ≤ 2 (average = 0.9). None of the ACC cases had score less than 4 (average = 6.6). The presence of sinus invasion correlated with tumor metastasis (P < 0.01). Immunohistochemical study showed that EGFR was expressed in 61.9% of ACC patients (13/21), whereas EGFR staining was mostly negative in ACA (except for weak staining in 5 cases and moderate staining in 1 case). The difference of EGFR expression between ACC and ACA was statistically significant (P = 0.030). On the other hand, the positive rate of VEGF in ACC was 71.4% (15/21), including 28.6% (6/21) with strong expression and 28.6% (6/21) with moderate expression. In contrast, the expression rate of VEGF in ACA was 30.0% (7/21), including 14.3% (3/21) with moderate expression. The difference of VEGF expression between ACC and ACA was statistically significant (P = 0.013). There was correlation between VEGF expression and venous invasion (P = 0.028). The average duration of survival in patients with ACC was shorter than that in ACA. The tumor weight in ACC also correlated with prognosis. CONCLUSIONS: Tumor size, weight and presence of endocrine symptoms may help in the differential diagnosis between ACC and ACA. A WEISS score of ≥ 3 highly suggests ACC. The presence of sinus invasion is associated with metastasis. EGFR or VEGF expression may also be important in differentiating ACC from ACA.

[Analysis of chromosomal abnormalities in pancreatic cancer by spectral karyotyping].

OBJECTIVE: to investigate the chromosomal characteristics of pancreatic ductal adenocarcinomas by spectral karyotyping. METHODS: cytogenetic aberrations of pancreatic cancer cell line P2 established from a Chinese patient was investigated by spectral karyotyping (SKY). Chromosomal alterations were further evaluated in 10 cases of pancreatic cancer and 10 cases of chronic pancreatitis by two color fluorescence in situ hybridization (FISH) by using EGFR/CEP7 probe and paraffin embedded tissue samples. RESULTS: hypotriploid and 26 chromosomal aberrations were revealed in cell line P2. Recurrent chromosomal numerical alterations included loss of chromosome 4, 9, 18, 19, 22, Y, 10p, 15p, 8p, 6q and 12p, with gain of chromosome 7 and 12q. Frequent chromosomal structural abnormalities included der(9;15)(q10;q10), der(10)(3;10)(?;q26) and der(12)(8;12)(?;p13). Four of 10 cases showed EGFR copy number changes by FISH. CONCLUSIONS: highly complex chromosomal rearrangements occur in pancreatic cancers. Additional studies of more cases are needed, including FISH analysis of EGFR copy number changes, to reach a conclusion.

[HER2 status in breast cancer of Chinese women: a study of 1170 cases using fluorescence in-situ hybridization].

OBJECTIVE: To assess the prevalence of HER2 amplification according to HER2 and chromosome 17 copy numbers and HER2 FISH (fluorescence in-situ hybridization) ratio in breast cancer occurring in Chinese women. METHODS: Eleven hundreds and seventy cases of breast cancer occurring in Chinese women, who would be treated by trastuzumab and/or relevant chemotherapy based on HER2 status, were enrolled into the study. The formalin-fixed and paraffin-embedded tumor tissues were tested by FISH (PathVysion, Vysis). RESULTS: Among the 1170 cases of breast cancer studied, 408 cases (34.87%) were FISH-negative, whereas 762 cases (65.13%) were FISH-positive, including 87 cases (87/762, 11.42%) with highly amplified HER2 gene (signals arranged in aggregates). As for the remaining 675 FISH-positive cases, 159 cases (23.56%) showed low amplification (HER2/CEP17 ratio = 2 to 4), 422 cases (62.52%) showed moderate amplification (ratio = 4 to 10) and 94 cases (13.93%) showed high amplification (ratio > 10) for HER2 gene. Only 14 of the 1170 cases (1.20%) had indeterminate results (ratio between 1.8 and 2.2), including 1.23% (5/408) borderline FISH-negative (ratio between 1.8 and 2.0) and 1.18% (9/762) borderline FISH-positive (ratio between 2.0 and 2.2). Our data showed that 73.00% (854/1170) of cases were chromosome 17 aneusomy, including 22.65% (265/1170) hypodisomy (chromosome 17 copy number per cell < or = 1.75), 38.38% (449/1170) low polysomy (chromosome 17 copy number per cell 2.26 to 3.75) and 11.97% (140/1170) high polysomy (chromosome 17 copy number per cell > or = 3.76). The frequency of chromosome 17 polysomy was 50.34%. In the FISH-positive subgroup, 23.88% (182/762) was disomy (chromosome 17 copy number per cell between 1.76 and 2.25), 24.15% (184/762) hypodisomy, 39.37% (300/762) low polysomy and 12.60% (96/762) high polysomy. The frequency of chromosome 17 polysomy in the FISH-positive subgroup was 51.97%. In the FISH-negative subgroup, 32.84% (134/408) were disomy, 19.85% (81/408) hypodisomy, 36.52% (149/408) low polysomy and 10.78% (44/408) high polysomy. The frequency of chromosome 17 polysomy in the FISH-negative subgroup was 47.30%. On the other hand, HER2 monoallelic deletion (HER2/CEP17 < or = 0.7) was observed in 2.39% of cases. Chromosome 17 monosomy was detected in 5.00% (38/762) and 4.41% (18/408) of HER2-positive and HER2-negative groups, respectively. A HER2 ratio of < 1.5 was noted in 32.30% of all cases (including 92.65% of HER2-negative cases), compared with 9.23% (108/1170) with ratio between 1.5 and 2.2. CONCLUSIONS: The results show that a high amplification of HER2 gene is detected by FISH. Moderate amplification of HER2 gene and chromosome 17 polysomy are commonly seen in breast cancer patients in China Mainland. These findings may carry significant clinical and pathogenetic implication.

[Fluorescence in-situ hybrydization detection of 18q21 LOH in human pancreatic ductal carcinoma and chronic pancreatitis].

OBJECTIVE: To investigate 18q21 LOH in human pancreatic ductal adenocarcinomas and chronic pancreatitis by fluorescence in-situ hybrydization (FISH) technique, and to analyze the relationship between 18q21 LOH and clinicopathologic characteristics. METHODS: RP11-729G3 and RP11-850A17, the regions on 18q21, were selected as the target fragments, the region RP11-621L6, close to the centromere of chromosome 18, was selected as the reference fragment. The specific BAC clones were used to isolate and purify the corresponding genomic DNA, which were labeled with biotin or DIG by nick translation into dual color probes. 18q21 LOH was assessed by dual-color FISH in 30 cases of pancreatic ductal adenocarcinoma and 10 cases of chronic pancreatitis. All samples were 10% formalin fixed and paraffin embedded. The relationship between 18q21 LOH and clinicopathologic characteristics was analyzed. RESULTS: Among 30 cases of pancreatic ductal adenocarcinoma, 25 cases showed LOH at the region RP11-729G3 (83.3%), and 26 cases showed LOH at the region RP11-850A17 (86.6%). Among these, 25 cases with LOH at both regions, 1 case showed LOH only at the region of RP11-850A17. No LOH was found in 10 cases of chronic pancreatitis. CONCLUSIONS: 18q21 LOH is a high-frequency event in human pancreatic ductal adenocarcinomas. LOH at the regions RP11-729G3 and RP11-850A17 demonstrates a high concordance. 18q21 may play an important role during pancreatic carcinogenesis and tumor progression. 18q21 LOH may be used as a diagnostic marker for pancreatic ductal adenocarcinoma.

[Status of gene mutation and copy number of EGFR in 290 cases of non-small cell lung carcinoma].

OBJECTIVE: To investigate EGFR mutations and gene copy number status in non-small cell lung carcinomas in the Chinese patients. METHODS: Using formalin fixed and paraffin embedded tissue samples, EGFR mutations were investigated in 290 cases of non-small cell lung carcinomas by microdissection and scorpions amplification refractory mutation system. The status of EGFR gene copy number was investigated by FISH. Furthermore, the relationship between EGFR mutations and gene copy number, and the relationship between EGFR gene status and clinicopathological variables of non-small cell lung carcinoma were analyzed. RESULTS: The overall mutation rate of EGFR was 41.7% (121/290). The mutation rates in adenocarcinoma, large cell carcinoma and squamous carcinoma were 48.4%, 16.7% and 0, respectively. Ninety-two of 121 cases with mutations had exon 19 deletion and L858R mutation, and 6 tumors contained both types of the mutation. The overall FISH positive rate of EGFR was 51.2% (107/209). FISH positive rates in adenocarcinoma, large cell carcinoma and squamous carcinoma were 52.1%, 75.0% and 11.1%, respectively. Therefore, EGFR mutations mainly occurred in the adenocarcinoma, and was significantly correlated with EGFR high copy number. CONCLUSIONS: There are higher EGFR mutation rate and FISH positive rate in non-small cell lung carcinoma in Chinese patients. Combined analysis of EGFR mutation and gene copy number by FISH may provide a superior approach in selecting patients who may benefit anti-EGFR target therapy.

[Detection of epidermal growth factor receptor gene mutations in non-small cell lung cancers by real-time polymerase chain reaction using scorpion amplification refractory mutation system].

OBJECTIVE: To investigate mutations of EGFR gene in non-small cell lung cancers (NSCLC) using scorpions amplification refractory mutation system (Scorpions ARMS) is in comparing the detection sensitivity with that by PCR-direct sequencing method, and in addition to study the correlation between the mutations and the clinicopathological characteristics of the patients. METHODS: Tumor cells were collected by microdissection from paraffin embedded tumor specimens and adjacent normal lung tissues of 82 NSCLC patients. The genomic DNA was extracted. Mutations of EGFR gene (exons 18, 19, 20 and 21) were detected by PCR-direct sequencing and Scorpions ARMS methods respectively. RESULTS: Somatic mutations were identified involving the tyrosine kinase domain of the EGFR gene in 42 of 82 cases with a mutation detection rate of 51.2% by Scorpions ARMS assay. In-frame deletions of exon 19 occurred in 20 patients and point mutation occurred at codon 858, 861 (exon 21) in 18 and 1 patients respectively. Two patients had insertions mutations and 1 patient had point mutation occurring at codon 768 (exon 20). Among the 58 informative cases analyzed by PCR-direct sequencing, 25 mutations (detection rate of 30.5%) were identified. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred at codon 858, 861 (exon 21) in 10 and 1 patients respectively. In addition, 1 patient had point mutation at codon 768 (exon 20). Overall, Scorpions ARMS assay was more sensitive in detecting mutations of EGFR than PCR-direct sequencing. CONCLUSIONS: A higher incidence of somatic mutations of EGFR gene was detected in NSCLC of Chinese patients. Mutations were more common in female, non-smoking patients with adenocarcinoma and bronchioloalveolar carcinoma histology. Scorpions ARMS method is quicker, more sensitive and accurate in detecting the EGFR gene mutations and should provide important therapeutic and prognostic information to the clinicians.

[Analysis of 13q14 chromosomal instability in soft tissue tumors by fluorescence in-situ hybridization].

OBJECTIVE: To investigate the genetic status of 13q and its role in the oncogenesis and progress of soft tissue tumors. METHODS: Forty-one soft tissue tumors, including 9 benign tumors, 9 tumors of malignant potential and 23 sarcomas, were studied by fluorescence in-situ hybridization (FISH) using dual color probes. The probes were generated from BAC clones RP11-685I15, RP11-352N7 and RP11-505F3, corresponding to Rb, RFP2, KCNRG and KLF5 genes respectively. RESULTS: Loss of heterozygosity (LOH) of RP11-685I15 were found in 8/41 cases, LOH of RP11-352N7 was seen in 4/41 cases and LOH of RP11-505F3 was present in 3/41 cases. LOH of all 3 loci were detected in 2 cases. LOH of RP11-61K9, an internal control locus, was detected in 2 cases. One case of malignant peripheral nerve sheath tumor showed amplification at all 3 loci. Amplification of RP11-505F3 was seen in another 2 cases. CONCLUSIONS: A significant percentage of soft tissue tumors exhibited chromosomal instability, reflected by an increase of LOH at tumor-suppressing gene loci. The incidence of 13q abnormality was different in various types of soft tissue tumors, indicating that alterations of Rb, RFP2, KCNRG and KLF5 tumor suppressing genes may play diverse roles in different types of soft tissue tumor.

[Protein overexpression and gene copy number of EGFR and HER2 in colorectal carcinoma].

OBJECTIVE: To investigate the protein expression and gene copy number of EGFR and HER2, and the correlation between the two markers in colorectal carcinomas in Chinese. METHOD: Total 42 samples of paraffin-embedded colorectal carcinomas in tissue microarray format were studied by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for EGFR and HER2 protein expression and gene copy number status, respectively. RESULTS: Among 42 cases evaluated, EGFR scores were 0 in 18 cases, 1+ in 10 cases, 2+ in 5 cases and 3+ in 9 cases. HER2 expression was negative in 39 tumors, 1+ in 1 tumor, 2+ in 1 tumor and 3+ in 1 tumor. For FISH assessing EGFR, 18 (42.9%) cases showed no apparent copy number changes, including 14 (33.3%) cases of disomy and 4 (9.5%) cases of low trisomy, 24 (57.1%) cases showed increased gene copy numbers including high trisomy in 3/42 (7.1%), low polysomy in 9/42 (21.4%) and high polysomy in 12/42 (28.6%) cases. Gene amplification of EGFR is not detected. Four of 42 patients (9.5%) had increased HER2 gene copy number, including 3 patients with high polysomy and 1 patient with gene amplification. Significant association was not seen between EGFR protein expression and the gene copy number, nor between two markers and tumor differentiation. There was a highly significant concordance between the gene amplification and IHC 3+ for HER2 similar to that of breast cancer. CONCLUSIONS: Protein expression and/or increased gene copy number of EGFR is common in colorectal carcinomas but unrelated to pathological features in this cohort. HER2 protein overexpression and/or gene amplification are rare.

Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies.

BACKGROUND: The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ can be of recipient origin after transplantation. In this study, we tested whether endothelial chimerism correlated with the graft rejection and cold ischemia. METHODS: We studied the biopsy samples from 34 renal transplants of female recipients who received the kidney from a male donor for the presence of endothelial cells of recipient origin. We examined the tissue sections of renal biopsy samples by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes using a biotinylated Y chromosome probe and digoxigenin labelled X chromosome probe, and then analyzed the relationship between the endothelial cell chimerism and the rejection and cold ischemia. RESULTS: Endothelial chimerism was common and irrespective of rejections (P > 0.05). The cold ischemic time of chimerism group was longer than no chimerism group ((14.83 +/- 4.03) hours vs (11.27 +/- 3.87) hours, P < 0.05). CONCLUSIONS: There is no correlation between the percentage of recipient endothelial cells in vascular endothelial cells and the type of graft rejection. The endothelium damaged by ischemic injury might be repaired by the endothelial cells from the recipient.

[Topoisomerase IIalpha and HER2/neu gene alterations and their correlation in pancreatic ductal adenocarcinomas].

OBJECTIVE: To investigate the changes of topoisomerase IIalpha (TOP2A) and HER2/neu genes in pancreatic ductal adenocarcinomas of Chinese patients, and to determine their roles during carcinogenesis and tumor progression. METHODS: Expressions of TOP2A and HER2/neu proteins were detected by using immunohistochemistry, while gene amplifications of TOP2A and HER2/neu were assessed by using multi-color fluorescence in situ hybridization (FISH). All the samples were of paraffin embedded and 10% formalin fixed tissue, including 26 cases of pancreatic ductal adenocarcinomas with adjacent non-neoplastic pancreatic tissues, 10 cases of chronic panreatitis, and 10 cases of normal pancreas. The correlation between TOP2A and HER2/neu gene status was analyzed. RESULTS: By immunohistochemistry, the nuclear positive index of TOP2A in pancreatic ductal adenocarcinomas varied from 0.5% to 70%, and the positive rate of HER2/neu in pancreatic ductal adenocarcinomas was 46.2% (12/26). By FISH, 9/10 TOP2A amplified adenocarcinomas showed TOP2A and HER2/neu gene coamplification, while one case with HER2/neu gene amplification adenocarcinoma showed no TOP2A amplification. No expression of TOP2A, HER2/neu proteins and no amplification of TOP2A and HER2/neu gene were detected in adjacent non-neoplastic pancreatic tissues, chronic pancreatitis tissues and normal pancreas. No relationship was found between protein expression and gene amplification of TOP2A and HER2/neu (P > 0.05). TOP2A gene amplification was significantly correlated with HER2/neu gene amplification (P < 0.01). CONCLUSIONS: Protein expression of TOP2A and HER2/neu are not associated with the gene amplification. There is a significant correlation between TOP2A amplification and HER2/neu gene amplification. Co-amplification of TOP2A and HER2/neu may play an important role in the carcinogenesis and progression of pancreatic carcinoma. Evaluation of the status of TOP2A and HER2/neu may be helpful to achieve target therapy of pancreatic carcinoma.

[Relationship between chromosome 8 alterations and Gleason score in prostatic adenocarcinoma].

OBJECTIVE: To study the gain of chromosome 8 and c-myc gene and lipoprotein lipase gene status in prostatic adenocarcinoma of Chinese patients, and to analyze the relationship between chromosome 8 alterations and Gleason score of prostatic cancer. METHODS: Formalin-fixed, paraffin-embedded prostatic biopsy tissues from 34 Chinese patients with untreated prostatic adenocarcinoma were studied by three-color fluorescence in situ hybridization (FISH) using ProVysion(TM) probe kit. The materials included 1 case with Gleason score 5, 10 cases with Gleason score 6, 14 cases with Gleason score 7, 4 cases with Gleason score 8 and 5 cases with Gleason score 9. The relationship between Gleason score and chromosome 8 aneusomy, c-myc and lipoprotein lipase gene copy number, including gene amplification or deletion, were analyzed. RESULTS: Seventeen (50%) of the 34 cases studied had gain of chromosome 8, while 21 cases (61.8%) had gain of c-myc gene copy number, 15 cases (44.1%) had lipoprotein lipase gene monosomy, 23 cases (67.6%) had c-myc gene amplification, 21 cases (61.8%) had deletion of lipoprotein lipase gene and 16 cases (47.1%) had lipoprotein lipase gene deletion coupled with c-myc gene amplification. In general, at least one type of chromosome 8 alteration was identified in 85.3% of cases (29/34). Gain of chromosome 8 was strong significant associated with Gleason score (P = 0.0006). A positive correlation between increased c-myc copy number and high Gleason score was also noted (P = 0.0035). On the other hand, loss of lipoprotein lipase gene was negatively correlated with high Gleason score (P = 0.0383). In addition, the association of c-myc gene amplification with high Gleason score was noted after age adjustment (P = 0.0462). CONCLUSIONS: Alterations in chromosome 8 are common in prostatic adenocarcinoma occurring in Chinese patients. There is a correlation between Gleason score and gain of chromosome 8, increased c-myc gene copy number, c-myc gene amplification and lipoprotein lipase gene deletion. C-myc gene amplification accompanied by lipoprotein lipase gene deletion is also a common occurrence in prostatic cancer. Our data suggest that chromosome 8 alterations may play some roles in the development and progression of prostatic adenocarcinoma.

[Analysis of HER2 gene status in breast cancer with HER2 protein overexpression].

OBJECTIVE: To study the HER2 gene status (by fluorescence in situ hybridization (FISH) in breast cancer with HER2 protein overexpression, the correlation between gene amplification and protein overexpression, as well as the rate and significance of chromosome 17 aneusomy. METHODS: One hundred and twenty archival cases of breast cancer with formalin-fixed and paraffin-embedded tumor tissues with 2+ (42 cases) and 3+ (78 cases) HER2 protein overexpression by immunohistochemistry (IHC, HercepTest, Dako) were tested by FISH (PathVysion, Vysis) for HER2 gene status. The rate of chromosome 17 aneusomy was also analyzed. RESULTS: Amongst the 42 samples with IHC 2+, HER2 gene amplification was identified in 32 cases (76.19%), which included 11 cases with low amplification (ratio 2 approximately 4), 20 cases with moderate amplification (ratio 4 approximately 10) and 1 case with high amplification (ratio>10). Amongst the 78 samples with IHC 3+, HER2 gene amplification was identified in 71 cases (91.03%), which included 9 cases with low amplification, 48 cases with moderate amplification and 14 cases with high amplification. Chromosome 17 aneusomy was found in 83 cases (83/120, 69.17%), in which 14 cases (11.67%) showed hypodisomy (chromosome 17 copy number per cellor=3.76). Amongst the 42 cases with IHC 2+, 25 samples (59.52%) had chromosome 17 aneusomy, including 3 cases with hypodisomy, 18 cases with low polysomy and 4 cases with high polysomy. Amongst the 78 cases with IHC 3+, 58 samples (74.36%) had aneusomy 17, including 11 cases with hypodisomy, 34 cases with low polysomy and 13 cases with high polysomy. Most of IHC 2+ and FISH-positive cases had low or moderate HER2 gene amplification, while most of the IHC 3+ and FISH-positive cases had moderate or high gene amplification (P=0.0003). Six of the 7 samples with IHC 3+ and FISH-negativity had chromosome 17 aneusomy and 5 of the 10 samples with IHC 2+ and FISH-negativity had such aneusomy. CONCLUSIONS: A high concordance rate is noted between IHC 3+ and FISH positive results. The rate of FISH positive in IHC 2+ patients was higher than reported in other studies. Low or moderate HER2 gene amplification in IHC 2+ and moderate or high gene amplification in IHC 3+ occurs quite frequently. Chromosome 17 aneusomy (including hypodisomy, low polysomy and high polysomy) is also a relatively common phenomenon in our cohort with HER2 overexpression, with predominance of low polysomy.

[EGFR and HER2 gene expression status and their correlation in non-small cell lung cancer].

OBJECTIVE: To explore epidermal growth factor receptor (EGFR) and HER2 gene status, to assess the correlation between EGFR and HER2 gene status, and to investigate the role of copy number increase and amplification of EGFR gene and HER2 gene in the tumorigenesis and disease progression of non-small-cell lung cancer. METHODS: Using Path Vysion kit and LSI EGFR SpectrumOrange/CEP7 Spectrum Green probes, EGFR gene and HER2 gene status were evaluated by fluorescence insitu hybridization (FISH) using formalin-fixed, paraffin-embedded samples from 31 patients with non-small-cell lung cancer, including 20 adenocarcinomas, 2 squamous cell carcinomas, 2 large cell carcinoma, 4 bronchoalveolar carcinomas and 3 adenosquamous carcinomas. The correlation between EGFR and HER2 gene status was analyzed. RESULTS: Six of thirty-one carcinomas showed EGFR gene amplification. Of 25 cases without EGFR gene non-amplification, four tetrasomy and 5 polysomy were detected. Overall, 15 out of 31 carcinomas demonstrated either EGFR gene copy number increase or gene amplification (15/31). HER2 gene amplification was seen in 2 of the 31 cases. Four trisomy, one tetrasomy and nine polysomy cases were found in 29 tumors that had no HER2 gene amplification. Overall, 16 of 31 cases showed either HER2 gene copy number increase and/or amplification (16/31). Synchronous EGFR and HER2 gene numerical changes, i.e. gene copy number increase and gene amplification, were found in 12 of 31 cases (12/31), and almost all such patients had either clinical stage III or IV tumor. EGFR gene numerical changes significantly correlated with HER2 gene abnormality (chi(2)(Adj) = 7.3045, P = 0.0069). CONCLUSIONS: EGFR or HER2 copy number increase is much more frequent than gene amplification in no-small-cell lung cancer. Our data based on gene alterations indicate, for the first time, that there is a significant correlation between EGFR alterations and HER2 abnormalities. Both genes are involved in the tumorigenesis and development of lung cancer. EGFR/HER2 dimer is one of the predominant heterodimerization types in lung cancer. The interactions between EGFR and HER2 may play a rule in the progression of non-small-cell lung cancer.

[K-ras mutation analysis in ovarian serous borderline and malignant tumors].

OBJECTIVE: To investigate the roles of K-ras gene in the tumorigenesis of ovarian serous borderline and malignant tumors. METHODS: Fifty one tissue samples of ovarian serous tumors, including 18 conventional serous borderline tumors, 11 micropapillary serous borderline tumors, 12 invasive micropapillary serous carcinomas, and 10 conventional serous carcinomas were investigated for the presence of K-ras mutation. DNA was extracted after microdissection of the tumor tissue, the exon 1 of K-ras gene was amplified by PCR, and the presence of mutation at the codons 12 and 13 was evaluated by direct sequencing analysis. RESULTS: GGT to GTT mutation at codon 12 of the K-ras gene was found in one conventional serous borderline tumors, resulting in valine to glycine substitution. All other 50 cases showed no K-ras mutation. All tumors had a wild-type codon 13. CONCLUSIONS: Mutations of K-ras at codons 12 and 13 in ovarian serous tumors are very rare in this series of patients, suggesting a difference present between the Chinese and Caucasian populations. K-ras mutations may play a less important role in the tumorigenesis of ovarian serous tumor of the Chinese patients.

Chromosome 1q loss of heterozygosity frequently occurs in sporadic insulinomas and is associated with tumor malignancy.

The pathogenesis of sporadic insulinomas is not clear, and there are no reliable genetic determinants that are useful to distinguish malignant and benign forms of this tumor. It was reported that 1q LOH might contribute to pathogenesis in gastrinomas and was correlated with tumor progression. However, little data are available on 1q LOH in sporadic insulinomas. In our study, we determine whether 1q LOH occurs in sporadic insulinomas and is associated with tumor malignancy by performing 1q allelotyping with 17 markers in 40 tumors and pair normal DNA. Thirty-five (88%) insulinomas had 1q LOH. Of the 35 insulinomas with 1q LOH, 14 (40%) had 1q21.3-23.2 LOH over a 7.5 cM region (SRO-1), whereas LOH in 21 tumors (60%) occurred at 1q31.3 over an 11.4 cM area (SRO-2). Of 24 tumors without MEN1 LOH, 20 had either SRO-1 or SRO-2 LOH (83%), whereas in 16 tumors with MEN1 LOH, 9 were shown to have LOH at either SRO-1 or SRO-2 (56%) (p = 0.065). This result suggests that LOH at 2 SRO might be MEN1 gene independent and may contribute to the pathogenesis in a subset of insulinomas without MEN1 gene LOH. The presence of 1q21.3-23.2 LOH is significantly associated with malignancy of insulinomas (p = 0.014). The high frequency of LOH at 1q 21.3-23.2 and 1q31.3 suggests these 2 areas may harbor putative tumor suppressor genes that may play an important role in the tumorigenesis of a subset of insulinomas. LOH at 1q21.3-23.2, which was associated with tumor malignancy, could be one of the genetic markers for identifying malignancy in sporadic insulinomas.

[Evaluation of HER2 gene expression status in breast cancer by fluorescence in-situ hybridization].

OBJECTIVE: To deduce the protocol, scoring criteria and interpretive guidelines for assessment of HER2 gene expression status by fluorescence in-situ hybridization (FISH) and to compare the results with those obtained by immunohistochemistry. METHODS: The HercepTest kit from Dako Cytomation was employed for immunohistochemistry. FISH for HER2 gene expression status was performed using PathVysion DNA probe kit on the archival paraffin-embedded sections of breast cancer tissues from 28 Chinese female patients with immunohistochemical staining scores of (3 +), (2 +), (1 +) and 0. RESULTS: Ten of the 12 patients with score (3 +) by immunohistochemistry were positive for HER2 by FISH, with 2 cases being polysomy. Two other cases with FISH-negative were also shown to be polysomy. Seven of the 10 patients with score (2 +) by immunohistochemistry showed HER2 gene amplification, with 1 case being polysomy. Two of the remaining 3 cases, which were FISH-negative, were shown to be polysomy. All the patients with scores (1 +, number = 3 ) or 0 ( number = 3) by immunohistochemistry failed to show amplification. One case of polysomy was noted in either group. CONCLUSIONS: Immunohistochemistry is useful as an initial screening tool for HER2 expression status. Because of the obvious discrepancies between protein expression and gene amplification, patients with score (2 +) by immunohistochemistry should undergo FISH testing as well. FISH is also required in selected examples with score (3 +) immunohistochemical results, especially in those with false-positive immunohistochemistry due to chromosome 17 aneuploidy.

[Frequent loss of heterozygosity at MEN-1 gene and chromosome 22q in insulinomas and its significance].

OBJECTIVE: To detecte whether loss of heterozygosity (LOH) at the MEN-1 locus as well as 22q occurs in sporadic insulinoma and if LOH can be used as a genetic marker to differentiate malignant and benign insulinomas. METHODS: MEN-1 gene and 22q allelotyping were performed by PCR with microsatallite markers in DNA from microdissected normal and tumor tissues from archived or frozen insulinomas (8 malignant and 32 benign, from 38 patients). The significance was calculated using t test and Cochran-Mantel-Haenszel Statistics, P < 0.05 was considered significant. RESULTS: Sixteen of the 40 insulinomas (40.0%) had MEN-1 LOH and 22q LOH was shown in 30 of the 40 tumors (75.0%). Eleven of the 30 tumors (37.0%) with 22q LOH had 22q12 LOH over a 3 cm region, whereas LOH in 14 tumors (47.0%) occurred at 22q13.3. Eight tumors with D22S 280 locus (22q12) LOH were shown without MEN-1 LOH while 14 of the 30 tumors without D22S280 LOH had MEN-1 LOH (0% vs 47%, P = 0.016). Six of the 14 tumors (43.0%) with 22q13.3 LOH were malignant, whereas 2 of 26 tumors without 22q13.3 LOH (8.0%) are malignant (P = 0.0088). CONCLUSION: MEN-1 gene LOH may contribute to a proportion of insulinomas, and 22q LOH occurs frequently in insulinomas. D22S 280 (22q12) LOH may independently contribute to the tumorogenesis of sporadic insulinoams, whereas detecting 22q13.3 LOH in insulinomas can be a potential genetic marker to distinguish malignancy from benign tumors.