Complex phenotypic heterogeneity of combined hepatocellular-cholangiocarcinoma with a homogenous TERT promoter mutation.

To clarify the mechanism underlying the development and poor prognosis of combined hepatocellular-cholangiocarcinoma (cHCC-CCA), we characterized liver cancer driver mutations and poor prognostic markers in both the HCC and intrahepatic CCA (iCCA) components of a cHCC-CCA tumor. The telomerase reverse transcriptase (TERT) promoter mutation C228T was quantified by digital polymerase chain reaction using DNA from multiple microdissected cancer components of a single cHCC-CCA nodule. The protein expression of cancer-related markers, including TERT, was examined by serial thin-section immunohistochemistry and double-staining immunofluorescence. TERT promoter mutation and TERT protein expression were detected in all cancer components but not in noncancer regions. TERT promoter mutation frequencies were similar among components; those of TERT protein-positive cancer cells were higher in iCCA and mixed components than in HCC. The frequencies of Ki67- and p53-positive cells were similarly higher in iCCA and mixed components than in HCC. However, double-positive cells for the three proteins were unexpectedly rare; single-positive cells dominated, indicating phenotypic microheterogeneity in cancer cells within a component. Interestingly, HCC and CCA marker protein immunohistochemistry suggested dedifferentiation of HCC and transdifferentiation from HCC to iCCA in HCC and iCCA components, respectively. Such phenotypic intercomponent heterogeneity and intracomponent microheterogeneity were detected in a tumor nodule of cHCC-CCA uniformly carrying the early HCC driver mutation. Moreover, poor prognostic markers were randomly expressed without a regular pattern, consistent with the poor prognosis.

Different effects of crizotinib treatment in two non-small cell lung cancer patients with SDC4::ROS1 fusion variants.

The possibility of stratifying patients according to differences in ROS proto-oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well-controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse-transcription-PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)-1, ROS-1, Ki67, and phosphorylated extracellular signal-regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

Correlations between class I glucose transporter expression patterns and clinical outcomes in non-small cell lung cancer.

BACKGROUND: Glucose transporters (GLUTs) are highly expressed in various cancers. However, the implications of these variable expression patterns are unclear. This study aimed to clarify the correlation between class I GLUT expression patterns and clinical outcomes in non-small cell lung cancer (NSCLC), including their potential role in inflammatory signaling. METHODS: Biopsy tissues from 132 patients with NSCLC (92 adenocarcinomas [ADC] and 40 squamous cell carcinomas [SQCC]) were analyzed. mRNA expression levels of class I GLUTs (solute carrier 2A [SLC2A]1, SLC2A2, SLC2A3, and SLC2A4) and inflammation-related molecules (toll-like receptors TLR4, RelA/p65, and interleukins IL8 and IL6) were measured. Cellular localization of GLUT3 and GLUT4 was investigated using immunofluorescence. RESULTS: Single, combined, and negative GLUT (SLC2A) expression were observed in 27/92 (29.3%), 27/92 (29.3%), and 38/92 (41.3%, p < 0.001) of ADC and 8/40 (20.0%), 29/40 (72.5%, p < 0.001), and 3/40 (7.5%) of SQCC, respectively. In ADC, the single SLC2A3-expressed group had a significantly poorer prognosis, whereas the single SLC2A4-expressed group had a significantly better prognosis. The combined expression groups showed no significant difference. SLC2A expression was not correlated with SQCC prognosis. SLC2A4 expression correlated with lower IL8 expression. GLUT3 and GLUT4 expressions were localized in the tumor cytoplasm. CONCLUSIONS: In lung ADC, single SLC2A3 expression correlated with poor prognosis, whereas single SLC2A4 expression correlated with better prognosis and lower IL8 expression. GLUT3 expression, which is increased by IL8 overexpression, may be suppressed by increasing the expression of GLUT4 through decreased IL8 expression.

Direct molecular evidence for both multicentric and monoclonal carcinogenesis followed by transdifferentiation from hepatocellular carcinoma to cholangiocarcinoma in a case of metachronous liver cancer.

Frequent recurrence is a major issue in liver cancer and histological heterogeneity frequently occurs in this cancer type. However, it has remained elusive whether such cancers are multicentric or monoclonal. To elucidate the clonal evolution of hepatocellular carcinoma (HCC) recurrence and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) development, the somatic mutation frequency and signatures in a patient with triple occurrence of liver cancer every three years were examined, with samples designated as #1HCC, #2HCC and #3cHCC-CCA, respectively. A total of four tumor regions, including HCC (#3HCC) and intrahepatic CCA (#3iCCA) components of #3cHCC-CCA, and three nontumor regions (#1N, #2N and #3N) were precisely dissected from formalin-fixed paraffin-embedded tissues of each surgical specimen. DNA was extracted and subjected to tumor-specific somatic mutation determination. Of note, five nonsynonymous single-nucleotide variants (SNVs), namely those of KMT2D, TP53, DNMT3A, PKHD1 and TLR4, were identified in #3cHCC-CCA. All five SNVs were detected in both #3HCC and #3iCCA and #2HCC but not in #1HCC. The telomerase reverse transcriptase (TERT) promoter mutation C228T, but not C250T, was observed in all tumors. Digital PCR of C228T also indicated the presence of the TERT promoter mutation C228T in nontumorous liver tissues (#1N, #2N and #3N) at a frequency of 0.11-0.83% compared with normal liver and blood samples. These results suggest the following phylogenetic evolution of three metachronous liver cancers: #1HCC was not related to #2HCC, #3HCC and #3iCCA; both #3HCC and #3iCCA arose from #2HCC. From the above, three novel findings were deduced: i) Both multicentric occurrence and intrahepatic metastasis may be involved in liver cancer in a three-year interval; ii) transdifferentiation from HCC to iCCA is a possible pathogenic mechanism of cHCC-CCA; and iii) a nontumorous, noncirrhotic liver may contain a preneoplastic region with a cancer driver mutation in the TERT promoter.

Roles of Ki-67 and p16 as biomarkers for unknown primary head and neck squamous cell carcinoma.

PURPOSE: Treatment guidelines have not been established for unknown primary head and neck squamous cell carcinoma (SCC). For these patients, chemoradiotherapy (CRT) can provide a better prognosis than that for patients with other head and neck cancers. The presence of HPV in the tumor is associated with a better outcome. However, not all patients with HPV-positive unknown primary head and neck SCC experience good treatment outcomes in actual clinical settings. METHODS: We thus retrospectively determined the Ki-67 proliferation index and p16 expression status to assess the associations of these parameters with treatment outcomes of patients with unknown primary head and neck SCC. RESULTS: The subjects were 13 patients who underwent CRT after surgery or excision biopsy between 1999 and 2016. The 2- and 5-year overall survival (OS) rate was 76.9% and 68.4%, respectively. The prognostic factor was age. There was no significant difference in survival between patients with a high Ki-67 vs. low Ki-67 or between patients with p16-positive vs. p16-negative metastases OS. However, all p16-positive patients with low Ki-67 showed good locoregional control. CONCLUSIONS: The combination of ki67 expression and p16 expression status may allow prediction of local control more accurately than p16 expression status alone.

Contradictory intrahepatic immune responses activated in high-load hepatitis C virus livers compared with low-load livers.

We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCV-infected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (CD8+ T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (CD4+ T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma.

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

p62 Regulates the Proliferation of Molecular Apocrine Breast Cancer Cells.

p62, also called sequestosome 1 (SQSTM1), is a multifunctional signaling molecule that affects cell proliferation. Recently, we found accumulation of p62 in apocrine carcinoma of the breast, however, the biological role of p62 expression in apocrine carcinoma still remains unclear. To investigate whether p62 might contribute to tumor cell proliferation in apocrine carcinomas, we used the MDA-MB-453 (androgen receptor-positive, HER2-type) and MFM223 (androgen receptor-positive, triple-negative type) breast cancer cell lines as models of molecular apocrine carcinoma. Both MDA-MB-453 and MFM223 showed strong and d high p62 protein expression than MCF7 cells (androgen receptor-negative, luminal A type). Knockdown of p62 resulted in significant reduction of the cell proliferative activity in both MDA-MB-453 (P<0.01) and MFM223 (P<0.05). In conclusion, p62 could contribute to cell proliferation and represent a therapeutic target in apocrine carcinoma.

Immunohistochemical co-expression status of cytokeratin 5/6, androgen receptor, and p53 as prognostic factors of adjuvant chemotherapy for triple negative breast cancer.

Triple negative breast cancer (TNBC) is immunohistochemically characterised by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2). TNBC is known for its poor prognosis and high recurrence probability. There is no effective targeted treatment for TNBC, but only adjuvant chemotherapies. There are two TNBC subtypes, basal-like and non-basal-like, which are defined based on positive cytokeratin (CK) 5/6 and/or epidermal growth factor receptor (EGFR) expression. In particular, CK5/6 expression is reported to correlate with TNBC recurrence. TNBC lacks ER-α expression, but some TNBCs are known to express the androgen receptor (AR). Moreover, although p53 accumulation is detected in various malignant tumors, its influence on adjuvant chemotherapy for patients with TNBC remains unclear. The aim of this study was to assess the combined immunohistochemical expression of CK 5/6, AR, and p53 as a potential prognostic marker of adjuvant chemotherapy for patients with TNBC. The expression of CK5/6, AR, and p53 in formalin-fixed and paraffin-embedded (FFPE) surgical sections from 52 patients with TNBC was analysed by immunohistochemistry (IHC) and the co-expression patterns in individual cells were investigated by immunofluorescent (IF) staining. Low AR expression was correlated with high clinical stage (P < 0.05) and low nuclear grade (P < 0.05). The expression of CK5/6 and p53 did not correlate with clinicopathological features. Patients who needed adjuvant chemotherapy presented the worst prognosis. In particular, when the IHC expression pattern was CK5/6 (-), AR (-), and p53 (+), the disease free survival (DFS) and overall survival (OS) were the worst. On the other hand, patients with AR (+) and p53 (-) TNBC presented a good prognosis. The analysis of the co-expression status of these three markers showed that no cells presented both AR and CK5/6 expression. Furthermore, TP53 mRNA expression was higher in patients with AR-negative TNBC (P < 0.05) and in patients with the worst prognosis (P < 0.05) than in the other patients. These results suggested that, in patients with CK5/6-negative TNBC, AR expression correlated with good prognosis, but p53 accumulation correlated with poor prognosis. The present IHC markers allowed us to predict the post-surgery prognosis of patients with TNBC. In conclusion, TNBCs are heterogeneous. Patients with the CK5/6 (-), AR (-), and p53 (+) TNBC subtype, evaluated by IHC, presented the worst prognosis. These IHC markers will be helpful to follow patients with TNBC.

Increased phosphorylation of ERK1/2 is associated with worse chemotherapeutic outcome and a poor prognosis in advanced lung adenocarcinoma.

Constitutive activation of extracellular signal-regulated kinase (ERK)1/2 pathway, that is activated by various stimuli including growth factors and oncogenic driver mutations, is observed in various cancers. However, the difference of the activated levels of the pathway is still unclear in clinical significances. The aim of this study was to investigate the effect of different ERK1/2 pathway activation, assessed by the expression levels of phosphorylated (p) ERK1/2, on the prognosis of advanced lung adenocarcinoma patients. Paraffin-embedded lung biopsy samples were obtained from 85 lung adenocarcinoma patients. Correlation between pERK1/2 expression levels that were assessed by immunohistochemistry (IHC) analysis and oncogenic driver mutation status, clinicopathological factors, outcome from standard anticancer therapies, and prognosis was investigated. Varying levels of pERK1/2 expression were observed in 68 (80.0 %) patients. The overall survival was significantly reduced in patients with higher pERK1/2 expression in comparison to those with lower expression levels (P = 0.03). In particular, higher pERK1/2 expression levels correlated with worse performance status and worse clinical outcome. Thus, the IHC analysis of pERK1/2 expression levels may predict patient prognosis in advanced lung adenocarcinoma. Inhibition of ERK1/2 pathway activated by various signals may improve the effects of standard chemotherapies and the clinical condition of patients with advanced cancer.

Overexpression of PGC1α and accumulation of p62 in apocrine carcinoma of the breast.

Apocrine carcinoma is categorized as a special type of breast carcinoma because of its specific morphological features. To clarify the characteristics of apocrine carcinoma from the point of view of the mitochondrial profile, we conducted a comparative study between apocrine and non-apocrine carcinomas. The expressions of mitochondrial related factors (PGC1α, Nrf1, Nrf2, mtTFA and COX4) were examined in a testing set of breast cancer tissue. Apocrine carcinomas showed a clear tendency towards higher mRNA expression levels of PGC1α than non-apocrine carcinomas. The expression of the selected factor, PGC1α, as well as that of p62 was further examined. The results revealed that apocrine carcinomas showed a higher immunohistochemical positivity rate for PGC1α (21.3% vs. 3.2%; P = 0.008), and that the mRNA expression level of PGC1α was significantly higher in apocrine carcinoma than in non-apocrine carcinoma (P = 0.007). The immunohistochemical positivity rate for p62 protein was also higher in apocrine carcinomas (44.7% vs. 21.0%; P = 0.015), although no significant difference in the p62 mRNA expression level was detected between the two types of carcinoma (P = 0.633). In conclusion, this study revealed that apocrine carcinoma overexpressed PGC1α contributing to mitochondrial biogenesis, and also p62 protein accumulation.