Hidekazu Kameshima

Immunotherapeutic benefit of α-interferon (IFNα) in survivin2B-derived peptide vaccination for advanced pancreatic cancer patients

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA‐A24‐restricted antigenic peptide, survivin‐2B80–88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin‐2B80–88 plus incomplete Freunds adjuvant (IFA) and α‐interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme‐linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin‐2B80‐88 peptide‐specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor‐prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905. (Cancer Sci 2013; 104: 124–129)

Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients

We previously identified a human leukocyte antigen (HLA)‐A24‐restricted antigenic peptide, survivin‐2B80‐88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin‐2B80‐88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin‐2B80‐88 plus incomplete Freund’s adjuvant (IFA); and (ii) survivin‐2B80‐88 plus IFA and a type‐I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin‐2B80‐88 plus IFA was not significantly different from that with survivin‐2B80‐88 alone, treatment with the vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin‐2B80‐88 peptide‐specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme‐linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single‐cell clone separation by cell sorting of peptide‐specific CTL showed that each CTL clone was indeed not only peptide‐specific but also cytotoxic against human cancer cells in the context of the expression of both HLA‐A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin‐2B80‐88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers. (Cancer Sci 2011; 102: 1181–1187)

Establishment of quantitative reverse transcription–polymerase chain reaction assays for human telomerase-associated genes

Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to the ends of chromosomes. Recently, several telomerase-associated genes have been cloned, making it possible to study the expression of these genes. Quantitative comparisons of the expression of these genes and of telomerase activity might help clarify the regulation of telomerase activity. Therefore, we established the validity of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the human telomerase catalytic subunit (hTERT) mRNA and telomerase associated protein (TEP1) mRNA using the TaqMan fluorogenic detection system. Using this assay, we quantitated hTERT mRNA and TEP1 mRNA expression in two human pancreatic cancer cell lines, AsPC-1 and PANC-1. Our results indicated that the levels of hTERT mRNA and TEP1 mRNA expression in AsPC-1 were 1.50 and 2.31 times higher than in PANC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the quantities of hTERT and TEP1 mRNAs in clinical specimens. Taken together, our results indicate that it is possible to measure the expression of the major telomerase genes subunits. Furthermore it is possible to apply this technique to determine the amount of other types of mRNA.

Heat-shock protein-73 protects against small intestinal warm ischemiareperfusion injury in the rat

Abstract Background: The protective effects of heat-shock protein (hsp) in rat small intestinal warm ischemia-reperfusion (I/R) injury are poorly understood. Methods: Hsp-73 expression was induced in rat small intestine with use of sodium arsenite injected (6 mg/kg) through a catheter cannulated into the left common carotid artery 24 hours before ischemia (group 1). In the control group an equal volume of phosphate-buffered saline solution was injected (group 2). To block the induction of hsp-73 expression, sodium arsenate and quercetin (5 mg/kg) were injected (group 3). Results: The mean peak plasma levels of tumor necrosis factor-α and cytokine-induced neutrophil chemoattractant after reperfusion were lower in group 1 than in group 2. The tissue myeloperoxidase activity after reperfusion was lower in group 1 than in group 2. The mean peak plasma level of interleukin-10 after reperfusion was higher in group 1 than in group 2. The induction of hsp-73 expression reduced the synthesis of nitric oxide and the magnitude of the small intestinal warm I/R injury. The results in group 3 were similar to those in group 2. Conclusion: Hsp-73 protects against small intestinal warm I/R injury by inhibiting the synthesis of inflammatory cytokines and the activation of neutrophils and by accelerating the synthesis of anti-inflammatory cytokines. (Surgery 1999;125:385-95.)

Helicobacter pylori Infection: Augmentation of Telomerase Activity in Cancer and Noncancerous Tissues

Abstract. Telomerase adds hexameric repeats of 5′-TTAGGG-3′ to the ends of chromosomal DNA (telomere) and has been implicated in cell immortalization and cellular senescence. The aim of this study was to measure quantitatively the telomerase activity and human telomerase RNA component (hTR) content in gastric cancer and to examine the relation between these values and histologic factors including Helicobacter pylori as a risk factor for gastric cancer. Telomerase activity was measured by a modified telomeric repeat amplification protocol in cancerous and noncancerous tissues (intestinal metaplasia, chronic gastritis, normal mucosa) from 27 gastric cancer patients; hTR expression was examined by the quantitative reverse transcriptase-polymerase chain reaction using fluorescent probes. Telomerase activity was higher in cancers (total product generated: 33.7) than in noncancerous tissues. Telomerase activity was higher in intestinal metaplasia (16.7) and chronic gastritis (10.6) than in normal mucosa (3.5). In patients with intestinal-type gastric cancer, telomerase activity was higher in intestinal metaplasia with H. pylori infection than in that without infection. hTR expression was not correlated with telomerase activity. H. pylori infection may influence telomerase activity in cancer and noncancerous tissues.

Role of Human Telomerase Reverse Transcriptase and Telomeric‐repeat Binding Factor Proteins 1 and 2 in Human Hematopoietic Cells

Telomerase, an enzyme that adds hexameric repeats of 5′‐TTAGGG‐3′, termed telomeres, to the ends of chromosomal DNA, has been implicated in cellular immortalization and cellular senescence. Recently several relevant genes have been cloned, including those encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase‐associated protein‐1 (TEP1). Also important are genes encoding human telomeric‐repeat binding factor proteins (TRF) 1 and 2. We compared 10 human malignant hematopoietic cell lines, 19 samples from patients with acute leukemia and normal granulocytes and monocytes to study telomerase activity and expression of these various genes using a reverse transcription‐polymerase chain reaction (RT‐PCR). In all 10 malignant cell lines with telomerase activity, hTR, hTERT mRNA, and TEP1 mRNA were expressed, while in normal monocytes and granulocytes without telomerase activity, expression of hTR, but not hTERT mRNA was detected. TEP1 mRNA was expressed in normal monocytes, but not granulocytes. Expression of TRF1 and TRF2 mRNAs was greater in the normal cells than in human malignant hematopoietic cell lines and in 16 samples of patients with acute leukemia. When differentiation of the malignant hematopoietic cell line HL‐60 was induced using tumor‐necrosis‐factor 471 and all‐trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation. Of the three telomerase components, only hTERT mRNA expression showed changes paralleling telomerase activity, becoming undetectable with differentiation. In contrast, initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. Not only hTERT, but also TRF1 and TRF2 are important regulators of telomerase activity that represent potential targets for gene therapy against cancer.

Limitations of urinary telomerase activity measurement in urothelial cancer.

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10(6) cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37 degrees C and 25 degrees C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37 degrees C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25 degrees C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

Expression of Telomerase-associated Genes: Reflection of Telomerase Activity in Gastric Cancer?

Abstract. Telomerase activation is a characteristic of immortalized tumor cells but not of normal cells. Telomerase activity has been detected in approximately 85% of malignant tumors, and assaying for telomerase activity is thought to be useful for diagnosing cancer. Three telomerase-associated molecules [human telomerase RNA component (hTR), telomerase-associated protein (TEP1), and human telomerase reverse transcriptase (hTERT)] have been cloned. We semiquantitatively measured telomerase activity and the expression of these genes in cancerous and noncancerous regions of gastric cancer patients. We also investigated whether the expression of these genes correlated with telomerase activity. Telomerase activity in cancerous regions was significantly higher than in noncancerous regions, but there was no correlation between telomerase activity and the expression of these genes. Furthermore, no clear difference was observed between cancerous and noncancerous regions. These data indicate that the level of three telomerase-associated genes (i.e., hTR, TEP1 mRNA, hTERT mRNA), do not reflect telomerase activity, and the RNA levels of these genes are not useful markers for diagnosing gastric cancer.

Evaluating the utility of N1,N12-diacetylspermine and N1,N8-diacetylspermidine in urine as tumor markers for breast and colorectal cancers.

BACKGROUND Among natural polyamines, the concentrations of the diacetylated form of spermine and spermidine increase in the urine of patients with cancer. We evaluated the utility of urinary N(1),N(12)-diacetylspermine (DiAcSpm) and N(1),N(8)-diacetylspermidine (DiAcSpd) as tumor markers for breast and colorectal cancers. METHODS Urinary DiAcSpm and DiAcSpd concentrations were measured by an enzyme-linked immunosorbent assay. Urine and serum samples were collected from 33 and 28 patients with colorectal and breast cancers, respectively. The sensitivity of urine samples to DiAcSpm and DiAcSpd concentrations was compared with serum concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen CA 15-3 in breast cancer patients and with serum concentrations of CEA and CA 19-9 in colorectal cancer patients, respectively. RESULTS In breast cancer patients, the sensitivity of DiAcSpm and DiAcSpd was 46.4% and 14.2%, respectively, which was higher than that of CEA and CA 15-3. In patients with colorectal cancer, the sensitivity of DiAcSpm and DiAcSpd was 69.6% and 36.3%, respectively. CEA was the second sensitive marker and CA 19-9 was the least sensitive marker in these patients. CONCLUSION DiAcSpm is a highly sensitive tumor marker. DiAcSpm can serve as a powerful tool in settings such as initial screening for cancers in routine health examination.

Pigmented mammary Paget's disease mimicking melanoma: report of three cases

Pigmented mammary Pagets disease (PMPD) is a rare subtype of mammary Pagets disease. The differential diagnosis of PMPD and melanoma is difficult clinically and sometimes histopathologically. Here we present three cases of PMPD with a variable-sized lesion. All cases showed an irregular-shaped black-brown macule, one of which was accompanied by nipple retraction. Dermoscopically, all cases showed reticular pigmentation with or without irregular black dots, regression structures and streaks, which were indistinguishable from those of melanoma. In all but one of the cases, preoperative examinations confirmed the presence of a subcutaneous mammary lesion. All patients underwent a total mastectomy with the histopathological results indicating invasive ductal carcinoma. These cases emphasize how difficult it is to distinguish PMPD from melanoma. Dermoscopic features also mimic those of melanoma, but the reticular pigmentation seen in all cases could be a feature specific to PMPD. For suspicious cases, histopathological assessment using immunohistochemistry is highly recommended.