Mi Jung Kwon

Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue

KRAS, BRAF, and PIK3CA mutation testing before administration of anti-epidermal growth factor receptor therapy of metastatic colorectal cancer (CRC) has become important. However, considerable uncertainty exists regarding which detection method can be applied in a reproducible, sensitive, and simple manner in the routine diagnostic setting. We compared the detection rates of KRAS, BRAF, and PIK3CA mutations in 92 routine formalin-fixed, paraffin-embedded CRC specimens by 2 discrete methods: direct sequencing and peptide nucleic acid (PNA)-mediated PCR. The detection rates for KRAS, BRAF, and PIK3CA mutations by direct sequencing were 20.7%, 3.3%, and 1.1%, respectively. PNA-mediated PCR clamping significantly increased the percentages of KRAS, BRAF, and PIK3CA mutations by up to 7.6%, 1.2%, and 5.4%, respectively, compared to the detection rate of regular PCR followed by direct sequencing (p=0.039, p=0.250, and p=0.031, respectively). The tumor volume of discordant cases was not significantly different from concordant cases (56.2±28.7% vs. 67.6±17.9%, p=0.41), which implies that there is a minor population of mutant alleles in the heterogeneous tumor population. The PNA-mediated PCR clamping method is highly sensitive and is efficiently applicable to the detection of KRAS, BRAF, and PIK3CA mutations in a clinical setting.

MET is a potential target for use in combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple‐negative/basal‐like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p < 0.001). High expression of MET in breast cancer resulted in poor overall survival (p = 0.001) and disease‐free survival (DFS, p = 0.010). MET expression was relatively high in TNBC cell lines, and the silencing of MET via small interfering RNA reduced cell proliferation and migration. We observed reduced TNBC cell viability after treatment with the MET inhibitor PHA‐665752. In the most drug‐resistant cell line, MDA‐MB‐468, which showed elevated epidermal growth factor receptor (EGFR) expression, silencing of EGFR resulted in increased sensitivity to PHA‐665752 treatment. We confirmed that PHA‐665752 synergizes with the EGFR inhibitor erlotinib to decrease the viability of MDA‐MB‐468 cells. TNBC patients coexpressing MET and EGFR showed significantly worse DFS than that in patients expressing EGFR alone (p = 0.021). Our findings strongly suggest that MET may be a therapeutic target in TNBC and that the combined therapy targeting MET and EGFR may be beneficial for the treatment of TNBC/BLBC patients.

Expression of Histone Deacetylases HDAC1, HDAC2, HDAC3, and HDAC6 in Invasive Ductal Carcinomas of the Breast

Purpose DNA deacetylation by histone deacetylase (HDAC) is an important mechanism involved in the oncogenic tumorigenesis of breast cancer. Previous studies have reported an association of the estrogen receptor (ER) with HDACs and demonstrated the efficacy of HDAC inhibitors for the treatment of breast cancers via in vitro experiments. In this study, we examined the association of HDAC expression with clinicopathological parameters and disease-specific survival. Methods Immunohistochemical (IHC) analysis of HDAC1, HDAC2, HDAC3, and HDAC6 was performed using tissue microarrays in 300 invasive ductal carcinomas. IHC scoring was determined by multiplication of the intensity (0 to 3) and the proportion (0 to 4) of staining, and we classified tumors into low- and high-HDAC expression groups. Results High expression of HDAC1 was correlated with the molecular subtype (p=0.001) and human epidermal growth factor 2 (HER2) amplification (p=0.012). High expression of HDAC6 was correlated with a younger age (p<0.001), ER expression (p=0.025), progesterone receptor expression (p=0.034), molecular subtype (p=0.023), and HER2 amplification (p=0.011). High HDAC1 expression was correlated with luminal A tumors (p=0.001), while high HDAC6 expression was more common in luminal B tumors (p=0.023). Although the expression of HDACs did not exhibit prognostic significance in the entire cohort, high expression of HDAC1 and HDAC6 was associated with improved overall survival (OS) in patients with ER-positive tumors (p=0.017 and p=0.029, respectively), and high expression of HDAC2 was correlated with improved OS in ER-negative tumors (p=0.048) on univariate analysis. Furthermore, high HDAC6 expression was associated with improved disease-free survival (p=0.048) on multivariate analysis. Conclusion HDAC1 expression is significantly correlated with the molecular subtypes of tumors, with the highest expression being observed in luminal A tumors. HDAC6 is a significantly correlated with ER expression and the molecular subtype, thereby supporting the estrogen regulatory property of HDAC6. HDAC1 and HDAC6 expression are good prognostic factors for ER-positive tumors.

Favorable clinical outcomes of pemetrexed treatment in anaplastic lymphoma kinase positive non-small-cell lung cancer.

INTRODUCTION The development of anaplastic lymphoma kinase (ALK) inhibitor has just followed the recent discovery of ALK rearrangement in lung cancer, therefore not much is yet known about the clinical course and treatment outcomes to chemotherapy in ALK-positive patients. The purpose of this study was to investigate the clinical characteristics and treatment outcomes in patients with ALK-positive NSCLC treated with conventional chemotherapy during pre-ALK inhibitor period. PATIENTS AND METHODS We retrospectively screened 381 consecutive NSCLC patients without known epidermal growth factor receptor (EGFR) or KRAS mutation who were diagnosed between 2007 and 2008 at a single center, and identified ALK rearrangements by fluorescence in situ hybridization. Additional 44 ALK-positive patients who were identified since 2009 by central lab for participation on clinical trial were included for the analysis of clinical outcomes. RESULTS Of the 381 tumors screened, 21 (5.6%) showed ALK rearrangements, with twenty adenocarcinomas and one pleomorphic carcinoma. Of 65 ALK-positive patients including additional 44 ALK-positive patients, 32 patients received pemetrexed as a second- or further-line therapy, in whom the response rate was 34.4% (11/32), median progression-free survival (PFS) was 4.0 months (range: 0-22.0 months) and median overall survival (OS) was 50.8 months (95% confidence interval [CI]: 38.7-62.8). CONCLUSIONS The prevalence of ALK rearrangement was 5.6% among EGFR and/or KRAS wild-type/unknown NSCLC population. Pemetrexed, given as a second- or further-line therapy, showed favorable clinical outcomes in ALK-positive NSCLC patients.

Frequent hepatocyte growth factor overexpression and low frequency of c-Met gene amplification in human papillomavirus–negative tonsillar squamous cell carcinoma and their prognostic significances ☆

Human papillomavirus (HPV) is an important prognostic factor for tonsillar squamous cell carcinoma (TSCC). HPV-positive and HPV-negative TSCCs are considered distinct in terms of prognosis and sensitivity to chemo/radiotherapy. However, to date, no study has thoroughly evaluated the individual prognostic factors for these 2 disease subgroups. Hepatocyte growth factor (HGF)-Met signaling pathway can be a predictive marker for prognosis or therapy response, especially in HPV-negative TSCC. We therefore investigated the prognostic values of HGF and c-Met expression in TSCC according to HPV status. Immunohistochemical analyses of HGF and c-Met protein expression and silver in situ hybridization of c-Met gene copy number were performed in 79 formalin-fixed, paraffin-embedded specimens. In HPV-negative TSCC, HGF overexpression, regional lymph node category, and ipsilateral cervical nodal metastasis predicted decreased overall survival (OS) (P = .017, P = .024, and P = .003, respectively). The latter 2 were also independent prognostic factors for progression-free survival (P = .023 and P = .002, respectively). In HPV-positive TSCC, heavy alcohol consumption and advanced primary tumor category were predictive of progression-free survival, whereas no independent prognostic factor for OS was identified. HGF overexpression had a significant effect on OS in HPV-negative TSCC but not in HPV-positive TSCC. HPV-negative/HGF-high expression tumors exhibited the worst survival outcomes, whereas HPV-positive/HGF-low expression tumors had the most favorable prognosis. c-Met expression and c-Met gene amplification were not associated with survival outcomes in TSCC patients. In conclusion, HGF may be a potential prognostic marker in HPV-negative TSCC, whereas c-Met exhibited limited clinical significance in TSCC.

TWIST1 promoter methylation is associated with prognosis in tonsillar squamous cell carcinoma.

Tonsillar squamous cell carcinomas (TSCC) frequently present with locally advanced diseases and cervical metastases, which are associated with poor prognoses. Epithelial-mesenchymal transition (EMT) is critical for tumor invasiveness and metastatic potential. Recent studies have shown that TWIST1-inducing EMT is overexpressed and hypermethylated in several cancers, indicating disease progression. The aim of the present study was to determine the clinical and prognostic significance of TWIST1 hypermethylation and EMT-related protein expression in TSCC. Methylation levels of TWIST1 promoter were analyzed by quantitative real-time methylation-specific polymerase chain reaction. Immunohistochemical analyses of TWIST1, Snail, and SMAD nuclear interacting protein-1 (SNIP1) were performed in 65 formalin-fixed, paraffin-embedded blocks of surgically resected specimens. TWIST1 promoter hypermethylation was found in 27.7% (18/65) of TSCCs. TWIST1 promoter hypermethylation was associated with poor differentiation (P = .012). Contralateral cervical lymph node metastasis was more frequently observed in TWIST1-methylated tumors (P = .029). High protein expressions of TWIST1, Snail, and SNIP1 were observed in 14 TSCC specimens (21.5%), 21 TSCC specimens (32.3%), and 38 TSCC specimens (58.5%), respectively. SNIP1 expression correlated significantly with TWIST1 methylation (P = .001), whereas TWIST1 protein expression did not. Contralateral cervical lymph node metastasis was an independent risk factor of the decreased overall survival rate (P = .002). TWIST1 methylation (P = .031) and pN stage (P = .037) were independent factors of poor prognoses affecting disease-free survival. TWIST1 promoter hypermethylation may be a useful molecular marker for predicting prognoses and contralateral cervical lymph node metastases in patients with TSCC.

Mutated IDH1 Is a Favorable Prognostic Factor for Type 2 Gliomatosis Cerebri

The prognostic significance of IDH1 mutations has been demonstrated in gliomas. It is unclear whether IDH1 mutation is also a prognostic factor in gliomatosis cerebri (GC). Primary GCs can be grouped into type 1 GCs, which have the classical diffuse growth pattern without mass formation, and type 2 GCs, which form neoplastic masses in addition to classic diffuse lesions. In this study, the prognostic relevance of IDH1/2 mutations in 74 GCs (43 type 1 and 31 type 2) was evaluated. We detected 33 (44.6%) IDH1 mutations, including R132H and R132S, by bidirectional Sanger sequencing. No mutations were detected in IDH2. The percentage of 2‐year overall survival for wild‐type IDH1 patients was 46 vs. 72% for patients with IDH1‐mutated tumors. Mutations of IDH1 were strongly correlated with both increased overall survival (OS) and progression‐free survival (PFS) in patients with type 2 GCs, and IDH1 mutations were also an independent prognostic factor predicting increased OS and PFS in type 2 GC patients in multivariate analysis. However, IDH1 mutations did not correlate with survival outcomes in patients with type 1 GCs. Finally, the subgroup of GC, which has IDH1 wild‐type and additional solid component showed the worst prognosis.

Pituicytoma with unusual histological features.

Pituicytoma is a rare low‐grade glial neoplasm that originates in the neurohypophysis or infundibulum. Because of its rare occurrence, the morphology and differentiation of pituicytoma have not been fully clarified. Here, we report a case of pituicytoma with unusual histological features mimicking ependymoma, but exhibiting the diverse morphology and differentiation of pituicytoma. The 1.4 cm‐sized suprasellar mass was incidentally found in the magnetic resonance image of a 42‐year‐old Korean woman who had had a traffic accident. Four years later, she presented with symptoms of hypopituitarism and the follow‐up images revealed slight enlargement of the mass. After gross total resection, microscopic examination revealed oval to elongated cells with abundant eosinophilic cytoplasm arranged in a perivascular pseudorosette pattern and short interlacing fascicles. Pleomorphic tumor cells and Herring bodies were diffusely distributed within the tumor. Neither Rosenthal fibers nor eosinophilic granular bodies were identified. The tumor cells were immunohistochemically positive for glial fibrillary acidic protein, vimentin and S‐100 protein, but negative for synaptophysin and adenohypophyseal hormones. The epithelial membrane antigen and CD99 were expressed with a paranuclear dot‐like or membranous pattern in some tumor cells. Ultrastructural examination revealed that the tumor cells with intermediate filaments were closely apposed with intercellular junctions and frequent basal lamina production.

P2X7 Receptor Expression in Coexistence of Papillary Thyroid Carcinoma with Hashimoto's Thyroiditis

Background This study was aimed at investigating the relation of P2X7 receptor (P2X7R) expression with the clinicopathological features of papillary thyroid carcinoma (PTC) coexisting with Hashimotos thyroiditis (HT). Methods We examined 170 patients (84, PTC with HT; 86, PTC without HT). P2X7R expression was examined by immunohistochemical methods. The staining intensity and patterns were evaluated and scored using a semi-quantitative method. Results The PTC with HT group was more likely to contain women and had less extrathyroid extension, lymph node (LN) metastasis, lymphovascular invasion, and recurrence than the PTC without HT group. Patients positive for P2X7R had significantly higher frequencies of lymphovascular invasion, extrathyroid extension, LN metastasis, and absence of HT. As shown by multivariate analysis, the expression of P2X7R was significantly higher if HT was absent and extrathyroid extension was present. In the PTC with HT group, the expression of P2X7R was significantly higher in patients with tumor multifocality, lymphovascular invasion, and extrathyroid extension. In the PTC without HT group, the expression of P2X7R was significantly higher in women and those having tumor multifocality. Conclusions Coexistence of PTC with HT is associated with good prognostic factors, and P2X7R expression in PTC was correlated with poor prognostic factors and the absence of HT.

Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs.