Feasibility study of personalized peptide vaccination for metastatic recurrent triple-negative breast cancer patients.

INTRODUCTION: Since treatment modalities for metastatic recurrent triple-negative breast cancer (mrTNBC) are limited, a novel treatment approach including immunotherapy is required. We have developed a novel regimen of personalized peptide vaccination (PPV), in which vaccine antigens are individually selected from a pool of different peptide candidates based on the pre-existing host immunity. Herein we conducted a phase II study of PPV for metastatic recurrent breast cancer patients to investigate the feasibility of PPV for mrTNBC. METHODS: Seventy-nine patients with metastatic recurrent breast cancer who had metastases and had failed standard chemotherapy and/or hormonal therapy were enrolled. They were subgrouped as the mrTNBC group (n = 18), the luminal/human epidermal growth factor receptor 2 (HER2)-negative group (n = 41) and the HER2-positive group (n = 18), while the remaining two patients had not been investigated. A maximum of four human leukocyte antigen (HLA)-matched peptides showing higher peptide-specific immunoglobulin G (IgG) responses in pre-vaccination plasma were selected from 31 pooled peptide candidates applicable for the four HLA-IA phenotypes (HLA-A2, -A24, or -A26 types, or HLA-A3 supertypes), and were subcutaneously administered weekly for 6 weeks and bi-weekly thereafter. Measurement of peptide-specific cytotoxic T lymphocyte (CTL) and IgG responses along with other laboratory analyses were conducted before and after vaccination. RESULTS: No severe adverse events associated with PPV were observed in any of the enrolled patients. Boosting of CTL and/or IgG responses was observed in most of the patients after vaccination, irrespective of the breast cancer subtypes. There were three complete response cases (1 mrTNBC and 2 luminal/HER2-negative types) and six partial response cases (1 mrTNBC and 5 luminal/HER2-negative types). The median progression-free survival time and median overall survival time of mrTNBC patients were 7.5 and 11.1 months, while those of luminal/HER2-negative patients were 12.2 and 26.5 months, and those of HER2-positive patients were 4.5 and 14.9 months, respectively. CONCLUSIONS: PPV could be feasible for mrTNBC patients because of the safety, immune responses, and possible clinical benefits. CLINICAL TRIAL REGISTRATION NUMBER: UMIN000001844 (Registration Date: April 5, 2009).

Usefulness of endoscopic breast-conserving surgery for breast cancer.

PURPOSE: We compared the safety, invasiveness and cosmetic outcomes between endoscopic breast-conserving surgery (endoscopic group) and surgery under direct vision (direct vision group) for treating breast cancer. METHODS: We compared 100 cases of endoscopic surgery with 150 cases of direct vision surgery. The safety was evaluated in terms of the blood loss, length of the operation and presence or absence of complications, whereas the degree of invasiveness was assessed using preoperative and postoperative leukocyte counts, neutrophil counts, interleukin (IL-6) levels and fever. The cosmetic outcome was assessed on the basis of a breast evaluation by the medical staff and the patient's subjective satisfaction. RESULTS: In both groups, serious postoperative complications were absent. No significant differences were observed in the leukocyte counts, neutrophil counts, IL-6 level or fever between the groups. An evaluation of the cosmetic outcomes by the staff showed a more favorable breast size, breast shape and scar condition in the endoscopic group. A significantly higher level of patient satisfaction was also observed in the endoscopic group. Postoperative local recurrence was absent. CONCLUSIONS: The endoscopic approach showed comparable safety and invasiveness, and provided better postoperative cosmetic outcomes than direct vision surgery. Our results suggest that endoscopic breast-conserving surgery is a potentially useful surgical method for the treatment of breast cancer.

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways.

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor ß and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.

[Liver arterial infusion chemotherapy with adjuvant trastuzumab for the simultaneous treatment of liver and breast cancer-a case report].

A 62-year-old woman being treated for chronic hepatitis C and high blood pressure was shown by computed tomography to have tumors in the lateral and medial segments of her liver, and in her right breast. The tumor in the lateral segment of the liver was excised, the tumor in the medial segment of the liver was treated with microwave coagulation therapy, and the breast tumor was treated with simple mastectomy and sentinel lymph-node biopsy. Based on pathological features, the liver tumors were classified as moderately differentiated liver cell carcinoma, and the breast tumor as estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor-2-positive ductal carcinoma. Hepatic arterial infusion chemotherapy using fluorouracil and cisplatin with trastuzumab as an adjuvant was administered to treat both cancers simultaneously. Twelve months after the operation, neither of the cancers had relapsed. This case suggests that when the breast cancer is human epidermal growth factor receptor-2-positive, trastuzumab should be administered as adjuvant therapy.

[Docetaxel in combination with epirubicin as the first-line chemotherapy for advanced and recurrent breast cancer: a multicenter phase II study].

This study examined the efficacy and tolerability of docetaxel(DOC)in combination with epirubicin(EPI)as the first-line treatment for patients with advanced and recurrent breast cancer. A total of 56 female patients with metastatic breast cancer not previously treated for metastatic disease received DOC(60mg/m²)and EPI(60mg/m2)on day 1 every 3 weeks. The patient characteristics included a median age of 53 years. Advanced disease was present in 86% of patients, and recurrent disease was found in 14%; 3 or more metastatic sites had been diagnosed in 38% of patients, and 59% patients were ER+. The median number of courses administered was 6. The median dose intensity was 18. 7mg/m²week for DOC and EPI, and the relative dose intensities were 93. 5%and 93. 3%, respectively. The clinical responses included a complete response in 5%, a partial response in 54%, and stable disease in 33% of patients, with a disease control rate of 92%. The progression-free survival was 78. 3%, and the overall survival was 91. 9% at 1 year. Grade 3/4 toxicities included neutropenia in 82%, leukopenia in 71%, febrile neutropenia in 16%, anorexia in 9%, and anemia in 7%of the patients. Neither congestive heart failure nor toxic death occurred. The D and E combination with doses of 60mg/m2 is an active and generally well-tolerated regimen that can be used as first-line chemotherapy for patients with metastatic breast cancer.

[A case of breast meningeal carcinomatosis caused by trastuzumab treatment as adjuvant chemotherapy].

A 52-year-old woman underwent modified radical mastectomy and axillary lymph node resection for right breast cancer (stage IIB). Afterwards FEC therapy (5-FU 500 mg/m/2, epirubicin 75 mg/m2, cyclophosphamide 500 mg/m2) x 4, docetaxel therapy (60 mg/m2) x 4 and radiation of the illness side collarbone, upper and lower lymph nodes were enforced for adjuvant therapy after the operation. Furthermore, administration of aromatase inhibitor (anastrozole) and trastuzumab was started due to the postoperative pathological diagnosis of hormone receptor-positive and HER2 (score 3+). This became an urgent hospital admission because of the sudden escape power from impaired consciousness due to the articulation disorders and limb weakness when trastuzumab was administered nine times. It was diagnosed by MRI examination and the cerebrospinal fluid cytology as meningeal carcinomatosis of breast cancer, and she died on the 31st recurrence of disease. A serious relapse may be caused in a case of fast-progressing breast cancer like this while being administered trastuzumab as an adjuvant treatment.

YB-1 prevents apoptosis via the mTOR/STAT3 pathway in HER-2-overexpressing breast cancer cells.

Evaluation of: Lee C, Dhillon J, Wang MY et al.: Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res. 68 (21), 8661-8666 (2008). The transcription factor Y-box binding protein (YB)-1 is highly expressed in breast cancer cells and is strongly linked with breast cancer patient prognosis. In this paper, siRNA knockdown of YB-1 was used to investigate breast cancer cell proliferation. Six breast cancer cell lines that either overexpress HER-2 or were triple negative demonstrated growth inhibition following YB-1 knockdown. In particular, YB-1 knockdown induced apoptosis in BT-474-m1 and Au565 cells. Knockdown of YB-1 also decreased phosphorylation of STAT3S727, ERK1/2T202/Y204, mTORS2448 and total mTOR expression. When STAT3 was knocked down by siSTAT3, apoptosis was induced and constitutively active phosphorylated STAT3 was found to rescue YB-1-induced apoptosis. Furthermore, YB-1 knockdown remarkably suppressed colony formation in a soft agar assay, while delayed tumor formation was observed in mice. YB-1 knockdown inhibited cell growth and it is thought to involve induction of apoptosis via the mTOR/STAT3 intracellular signaling pathway. YB-1 is a promising molecular target for HER-2-overexpressing or triple-negative breast cancer cells.

[A case of triple negative chest wall recurrent breast cancer treated with capecitabine and docetaxel combination therapy (XT therapy)].

A 59-year-old woman underwent modified radical mastectomy for left breast cancer 9 years earlier. This time, a chest wall recurrence was found. A chest CT showed a chest wall tumor and lymph node metastases. PET images showed increased uptake in chest wall tumor and lymph nodes. The serum tumor markers have also elevated. Open biopsy of chest wall tumor was performed, and the tumor was diagnosed as invasive ductal carcinoma[ER(-), Pg R (-), HER2 score(0)]. Combination chemotherapy with capecitabine and docetaxel was initiated. After 7 courses of treatment, a marked response has been seen. Capecitabine and docetaxel combination therapy are considered useful for treatment of triple negative recurrent breast cancer.

Preclinical and clinical studies of novel breast cancer drugs targeting molecules involved in protein kinase C signaling, the putative metastasis-suppressor gene Cap43 and the Y-box binding protein-1.

Breast cancer is a common cause of tumors in women. The development of effective adjuvant therapies using drugs such as anthracyclines, taxanes, and aromatase inhibitors has improved the survival of breast cancer patients. Molecular cancer therapeutics are also attracting attention, and targeted molecular therapies, such as trastuzumab, have already contributed to effective new treatments for breast cancer. Other candidate targeted molecular therapies for breast cancer, including erlotinib, gefitinib, lapatinib, bevacizumab, and celecoxib, are currently undergoing clinical evaluation, and promising results are expected. The current review provides an up-to-date summary of the preclinical and clinical development of these drugs for breast cancer. In particular, we focus on therapies targeting protein kinase C (PKC) signaling, the putative metastasis-suppressor gene Cap43/N-myc downstream-regulated gene 1 (NDRG1)/differentiation-related gene-1 (Drg-1), and the Y-box binding protein-1 (YB-1). The PKC signaling pathway is widely considered to be a promising target for the development of novel therapeutics. Cap43 expression is significantly modulated by estrogen and/or anti-estrogens in breast cancer cells that are positive for estrogen receptor-alpha (ER-alpha). Cap43 is therefore of particular interest as a molecular indicator of the therapeutic efficacy of anti-estrogenic agents in breast cancer. The nuclear expression of YB-1 plays an essential role in the acquisition of malignant characteristics by breast cancer cells, through epidermal growth factor receptor 2 (HER2)-Akt-dependent pathways. Basic research investigating the key selective molecular changes that sustain breast cancer growth and progression, as demonstrated for PKC, Cap43, and YB-1, is allowing the development of specific targeted molecular diagnostics and therapeutics.

Improved detection of breast cancer on FDG-PET cancer screening using breast positioning device.

OBJECTIVE: The aim of this study was to investigate the detection rate of breast cancer by positron emission tomography cancer screening using a breast positioning device. METHODS: Between January 2004 and January 2006, 1,498 healthy asymptomatic individuals underwent cancer screening by fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) at our institution; 660 of 1498 asymptomatic healthy women underwent breast PET imaging in the prone position using the breast positioning device to examine the mammary glands in addition to whole-body PET imaging. All subjects that showed abnormal (18)F-FDG uptake in the mammary glands were referred for further examination or surgery at our institution or a local hospital. Our data were compared with the histopathological findings or findings of other imaging modalities in our institution and replies from the doctors at another hospital. RESULTS: Of the 660 participants, 7 (1.06%) were found to have breast cancers at a curable stage. All the seven cancers were detected by breast PET imaging, but only five of these were detected by whole-body PET imaging; the other two were detected by breast PET imaging using the breast positioning device. CONCLUSIONS: In cancer screening, prone breast imaging using a positioning device may help to improve the detection rate of breast cancer. However, overall cancer including mammography and ultrasonography screening should be performed to investigate the false-negative cases and reduce false-positive cases. The effectiveness of prone breast PET imaging in cancer screening should be investigated using a much larger number of cases in the near future.

[A case of recurrent breast cancer with bone marrow metastasis treated with weekly paclitaxel therapy].

A 44-year-old woman with bone marrow metastasis from breast cancer was treated with weekly paclitaxel therapy. She underwent radical mastectomy for right breast cancer (T2N1M0, Stage II B) in April 2003, and was then treated with hormonal therapy (leuprorelin). In November 2005, she received radiation for bone metastasis in thoracic and lumbar vertebrae, and bisphosphonate therapy was performed. Additional hormonal therapy (tamoxifen) was administered for progressive bone metastasis. However, in September 2006, pancytopenia was recognized and bone marrow metastasis was diagnosed by bone biopsy. Disseminated intravascular coagulation (DIC) developed, so she was given weekly paclitaxel therapy with blood transfusion and G-CSF injection. Improvement of pancytopenia and tumor markers was recognized temporarily, and but 3 months later the tumor markers increased again. Four months after introduction of chemotherapy, she died of gastrointestinal hemorrhage.

17Beta-estradiol induces down-regulation of Cap43/NDRG1/Drg-1, a putative differentiation-related and metastasis suppressor gene, in human breast cancer cells.

PURPOSE: Cap43 is known as a nickel- and calcium-inducible gene. In the present study, we examined whether 17beta-estradiol (E2) could affect the expression of Cap43 in breast cancer. EXPERIMENTAL DESIGN: Real-time PCR, immunoblotting, and immunocytochemistry were used to examine the expression of Cap43 and estrogen receptor-alpha (ER-alpha) in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cell lines were transfected with ER-alpha cDNA to establish cells overexpressing ER-alpha. Immunohistochemistry was used to evaluate the expression of the Cap43 protein in breast cancer patients (n = 96), and the relationship between Cap43 expression and clinicopathologic findings was examined. RESULTS: Of the eight cell lines, four expressed higher levels of Cap43 with very low levels of ER-alpha, whereas the other four expressed lower levels of Cap43 with high ER-alpha levels. Treatment with E2 decreased the expression of Cap43 dose-dependently in ER-alpha-positive cell lines but not in ER-alpha-negative lines. Administration of antiestrogens, tamoxifen and ICI 182780, abrogated the E2-induced down-regulation of Cap43. Overexpression of ER-alpha in both ER-alpha-negative cell lines, SK-BR-3 and MDA-MB-231, resulted in down-regulation of Cap43. Immunostaining studies showed a significant correlation between Cap43 expression and the histologic grade of tumors (P = 0.0387). Furthermore, Cap43 expression was inversely correlated with the expression of ER-alpha (P = 0.0374). CONCLUSIONS: E2-induced down-regulation of Cap43 seems to be mediated through ER-alpha-dependent pathways in breast cancer cells both in culture and in patients. Cap43 has potential as a molecular marker to determine the therapeutic efficacy of antiestrogenic anticancer agents in breast cancer.

HER2 overexpression increases sensitivity to gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, through inhibition of HER2/HER3 heterodimer formation in lung cancer cells.

Gefitinib (Iressa), an epidermal growth factor receptor targeting drug, has been clinically useful for the treatment of patients with non-small cell lung cancer (NSCLC). Gefitinib is currently being applied in clinical studies as either a monotherapy, or as part of a combination therapy against prostate, head and neck, gastric, breast, and colorectal tumors. However, success rates vary between different tumor types, and thus it is important to understand which molecular target(s) are responsible for limiting the therapeutic efficacy of the drug. In this study, we ask whether expression of HER2 affects sensitivity to gefitinib in human lung cancer cells. We established two clones, LK2/HER2-32 and LK2/HER2-57, by transfecting HER2 cDNA into LK2, a NSCLC line with a low expression level of HER2. We observed no mutations in exons 18, 19, and 21 of EGFR gene in LK2, LK2/mock- and two HER2-trasfectants when we observed in-frame deletion mutations (E746-A750) adjacent to K745 in a gefitinib-sensitive NSCLC cell line, PC9. These LK2/HER2-32 and LK2/HER2-57 were much more sensitive to the cytotoxic effects of gefitinib than the parental LK2 lines. Treatment with 0.5 to 1 micromol/L gefitinib specifically blocked Akt activation in both HER2-transfectant lines, but not in the parental LK2 cells. Extracellular signal-regulated kinase-1/2 activation, however, was not blocked by gefitinib up to 10 micromol/L in either the parent or transfectant lines. Gefitinib was also shown to induce cell cycle arrest in the G1-S phase, and an accompanying increase of p27Kip1 was observed. LK2/HER2 transfectants showed constitutive formation of HER2/HER3 heterodimer, which were seen to associate with a regulatory subunit of phosphoinositide-3-kinase, p85alpha, when active. Treatment of LK2/HER2 cells with gefitinib markedly decreased the formation of HER2/HER3 heterodimers, HER3 basal phosphorylation, and the association of p85alpha with HER3. This study is the first to show that under basal growth conditions, HER2 sensitizes low-EGFR NSCLC cell lines to growth inhibition by gefitinib.

[Conventional chemotherapy combined with the repetitive immune cell transfer for patients with refractory advanced gastric cancer].

In order to take advantage by both the anticancer effects and reconstruction of antitumor immunity, we compared the feasibility of a combination of CTL transfer and chemotherapy (ChT) for patients (pts) with malignant ascites due to carcinomatous peritonealitis of refractory gastric cancer to that of ChT only and/or cellular immunotherapy after failing ChT. A total of 22 pts, 8 underwent only conventional ChT (Group A), 6 performed cellular IT after failing ChT (Group B) and 8 underwent combination therapy (Group C), were enrolled in this retrospective study. ChT was based on conventional conditioning regimen with a standard dose for gastric cancer cases: S-1 (80-120 mg/body) plus paclitaxel (60-80 mg/m2), or CPT-11 (70-80 mg/m2) plus CDDP (80 mg/m2). Autologous tumor cells stimulated with T lymphocytes (AuTL), a kind of CTL, were generated ex vivo from peripheral blood lymphocytes over a two-week co-culturing process with autologous tumor cells separating from the ascites. IT was performed for pts of Group B and C. AuTLs were administered twice prior to ChT for pts of Group C, and were injected 1 x /2 weeks directly into the peritoneal cavity. The treatment was repeated at least three cycles with one-week interval. The mean survival period of Group A, B and C was 8.4, 5.2 and 11.3 months, respectively, and 1 pt in Group A and 3 pts in Group C survived over one year. Adverse events related to both of the ChT and AuTL transfer at all doses were minimal. Ascites had decreased or disappeared in 8 pts in this study. Lymphocytes of ascites were evaluated for cytokine production and subset of CD4+CD25+ T cell before the treatment, and after 3 treatments. The group C pts had increased IFN-gamma and IL-12 production with no TGF-beta1 responses by their ascites after 3 treatments. In contrast, the group A and B had no IFN-gamma, IL-12 or TGF-beta1 responses. These data show that combination therapy of CTL transfer and ChT is a feasible option for patients with refractory peritoneal carcinomatous of gastric cancer without serious adverse events. Although it depends on each mechanism of IT and ChT, a more stringent evaluation of CTL transfer combined with ChT for refractory gastric cancer should be performed.

Erratum to immediate breast volume replacement using a free dermal fat graft after breast cancer surgery: multi-institutional joint research of short-term outcomes in 262 Japanese patients.

[This corrects the article on p. 179 in vol. 4, PMID: 26005649.].

Activation of protein kinase C delta induces growth arrest in NPA thyroid cancer cells through extracellular signal-regulated kinase mitogen-activated protein kinase.

Protein kinase C (PKC) is a family of serine-threonine kinases that regulate many cell processes. To study the role of PKCdelta in thyroid cancer cells, we used a replication-deficient adenovirus (PKCdeltaAdV), to tightly control PKCdelta expression. In NPA cells, activation of wild-type (WT) PKCdelta with phorbol 12-myristate 13-acetate (PMA) induced an arrest in cell growth at G(1) phase, which was itself inhibited by the PKCdelta inhibitor rottlerin. Furthermore, overexpression of a dominant negative PKCdelta did not induce G(1) arrest. These findings strongly suggested that PKCdelta induced cell growth arrest in NPA cells. We investigated the mechanism of G1 arrest by examining G(1)-related proteins and mitogen-activated protein kinase (MAPK) by Western blotting. After activation of WTPKCdelta with PMA, cyclin E expression and retinoblastoma protein (Rb) phosphorylation decreased; the expression of p27(Kip1) increased and the phosphorylation of extracellular signal-regulated kinase (ERK) MAPK decreased. These results indicated that the activation of PKCdelta induced cell growth arrest in NPA cells, through an ERK MAPK-p27(Kip1)-cyclin E-pRb pathway. PKCdelta may therefore be an effective molecular target for novel therapy in thyroid cancer.

Regulation of vascular smooth muscle proliferation and migration by beta2-chimaerin, a non-protein kinase C phorbol ester receptor.

The proliferation and migration of vascular smooth muscle cells (SMC) are important aspects of atherogenesis. Activated growth factor signaling in injured vessels subsequently promotes a number of intracellular events resulting in the phenotypic modulation of SMC. Here, we investigated the role of beta2-chimaerin, a non-protein kinase C phorbol ester receptor with Rac-GTPase-activating protein activity, in growth factor-stimulated SMC. The endogenous expression of beta2-chimaerin was detected in cultured human SMC by reverse transcription-polymerase chain reaction and immunohistochemistry. Next, the overexpression of HA-tagged wild-type human beta2-chimaerin was attempted using cultured rat SMC with a recombinant adenovirus (Adv-beta2-Chim). Adv-LZ encoding beta-galactosidase (LacZ) was used as the control. The proliferation of SMC stimulated by platelet-derived growth factor (PDGF-BB, 10 ng/ml), as measured by cell-counting and 5-bromo-2'deoxyuridine incorporation assay, was suppressed by infection with Adv-beta2-Chim (50-200 MOI), but not with control viruses. PDGF-induced SMC migration was inhibited by approximately 25% after infection with Adv-beta2-Chim (200 MOI) using a modified Boyden's chamber assay with a fibronectin-coated membrane. Confocal microscopy revealed that PDGF stimulation altered the sub-cellular localization of beta2-chimaerin. The administration of 12-O-tetradecanoyl phorbol 13-acetate also induced changes in the sub-cellular localization of beta2-chimaerin, which was not affected by a presence of the PKC inhibitor (GF109203X). Finally, PDGF-induced Rac1 activation was found to be inhibited in the Adv-beta2-Chim-infected cells. Thus, we demonstrated that beta2-chimaerin regulates the proliferation and migration of SMC downstream of growth factor signaling pathway via the regulation of Rac1 activity. The signaling mediated by beta2-chimaerin may play a role in the regulation of SMC phenotypes, thereby implicating human atherogenesis.

Mutated p53 gene encodes a nonmutated epitope recognized by HLA-B*4601-restricted and tumor cell-reactive CTLs at tumor site.

Mutations of p53 gene occur in approximately 50% of human cancers, and accumulated p53 protein may be an appropriate target molecule to use for cancer immunotherapy. Indeed, mutated or nonmutated p53-derived peptides can induce HLA class I-restricted and tumor cell-reactive CTLs in vitro. However, to our knowledge, evidence that p53-derived peptides are truly recognized by CTLs at tumor sites has not yet been obtained. This study revealed that a mutated p53 gene encoded a nonmutated nonapeptide recognized by a HLA-B46-restricted and tumor cell-reactive CTL line that was established from T cells infiltrating a colon cancer lesion with the p53 mutation. This p53 peptide, at amino acid positions 99-107, had the ability to induce HLA-B46-restricted and peptide-specific CTLs reactive to tumor cells with the p53 mutation from the peripheral blood mononuclear cells of cancer patients, but not from those of healthy donors. These peptide-induced CTLs did not react to either HLA-B46(+) tumor cells without the p53 mutation or to HLA-B46(+) phytohemagglutinin-blastoid cells. These results provide a scientific basis for the development of p53-directed specific immunotherapy for HLA-B46(+) cancer patients.

Cooperative cell-growth inhibition by combination treatment with ZD1839 (Iressa) and trastuzumab (Herceptin) in non-small-cell lung cancer.

An important recent advance in anticancer therapy was the development of molecular-targeting drugs, such as the epidermal growth-factor receptor (EGFR)-targeting drug ZD1839 (Iressa) and the HER2-trageting anti-HER2 monoclonal antibody trastuzumab (Herceptin). ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-small-cell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. In culture ZD1839 inhibited the growth of four NSCLC cell lines (A549, NCI-H23, NCI-H727, and NCI-H661) that expressed various levels of EGFR, HER2, HER3, and HER4. A significant cytotoxic effect was observed when ZD1839 was combined with trastuzumab in A549 cells. However, this combination had no apparent effect in NCI-H23 cells. Significant G(1)-phase arrest, increased p27 expression and decreased cyclin E or D1 levels were detected in A549 cells treated with ZD1839 and trastuzumab. No significant effects were detected in NCI-H23 cells examined. The combination treatment significantly inhibited the phosphorylation of EGFR, HER2, retinoblastoma, extracellular signal-regulated kinase-1/2, and protein kinase B/Akt in A549 cells, but not in NCI-H23 cells. Our results indicated that increased levels of constitutive EGFR/HER2 heterodimers were formed in A549 cells in the presence of ZD1839, whereas no heterodimer formation was detected in NCI-H23 cells. We therefore suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.

Antineoplaston induces G(1) arrest by PKCalpha and MAPK pathway in SKBR-3 breast cancer cells.

Antineoplastons such as A10 include naturally occurring peptides and amino acid derivatives that control the neoplastic growth of cells. The mechanism underlying this antitumor effect was investigated using the breast cancer cell line, SKRB-3. Cells treated with A10 were monitored for any changes in cell cycle, expression of protein kinase C (PKC), or intracellular signal transduction, particularly phos-phorylation of mitogen-activated protein kinase (MAPK). The A10 markedly inhibited SKBR-3 proliferation due to arrest in the G(1) phase. A10 down-regulated the expression of PKCalpha protein, resulting in inhibition of extracellular signal-regulated kinase (ERK) MAPK phosphorylation. This increased the expression of p16 and p21 protein, with resultant inhibition of Rb phosphorylation, leading to G(1) arrest. This study has defined a pathway in which A10 arrested SKBR-3 cells in the G(1) phase via PKCalpha and MAPK. Our findings indicate that the antineoplaston A10 antitumor effect could be utilized as an effective therapy for breast cancer patients.