Kazushige Ishida

Analysis of the anti-tumor effect of cetuximab using protein kinetics and mouse xenograft models

BackgroundThe binding of EGFR and its ligands leads to autophosphorylation of receptor tyrosine kinase as well as subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. An EGFR inhibitor, cetuximab binds to EGFR and consequently blocks a variety of cellular processes. KRAS/BRAF mutations are known to be associated with a low response rate to cetuximab. In the present study, to clarify the anti-tumor mechanisms of cetuximab, we evaluated the KRAS/BRAF status, phosphorylation level of the EGFR pathway, and the tumor suppression effect in vivo, using a human colon cancer cell line HT29, which exhibited the highest EGFR expression in response to the cetuximab therapy among the 6 colorectal cancer cell lines tested.FindingsThe conventional growth suppression assay did not work efficiently with cetuximab. EGF, TGF-α, and IGF activated the EGFR/MAPK cell signaling pathway by initiating the phosphorylation of EGFR. Cetuximab partially inhibited the EGFR/MAPK pathway induced by EGF, TGF-α, and IGF. However, cetuximab exposure induced the EGFR, MEK, and ERK1/2 phosphorylation by itself. Mouse xenograft tumor growth was significantly inhibited by cetuximab and both cetuximab-treated and -untreated xenograft specimens exhibited phosphorylations of the EGFR pathway proteins.ConclusionsWe have confirmed that cetuximab inhibited the EGFR/MAPK pathway and reduced tumor growth in the xenografts while the remaining tumor showed EGFR pathway activation. These results suggest that: ( i ) The effect of cetuximab in growth signaling is not sufficient to induce complete growth suppression in vitro; ( ii ) time-course monitoring may be necessary to evaluate the effect of cetuximab because EGFR signaling is transmitted in a minute order; and ( iii ) cetuximab treatment may have cells acquired resistant selectively survived in the heterogeneous cancer population.

A compensatory role of NF-κB to p53 in response to 5-FU-based chemotherapy for gastric cancer cell lines.

Despite of remarkable improvement of postoperative 5-FU–based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU–based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.

Molecular marker identification for relapse prediction in 5-FU-based adjuvant chemotherapy in gastric and colorectal cancers.

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.

Evaluation of chemosensitivity prediction using quantitative dose–response curve classification for highly advanced/relapsed gastric cancer

BackgroundThe use of standard chemotherapy regimens has changed the application of chemosensitivity tests from all chemotherapy-eligible patients to those who have failed standard chemotherapy, which includes patients with highly advanced, relapsed, or chemoresistant tumors.MethodsWe evaluated a total of 43 advanced primary and relapsed gastric cancers for chemosensitivity based on drug dose response curves to improve the objectivity and quality of quantitative measurements. The dose response curves were classified based on seven expected patterns. Instead of a binary chemosensitivity evaluation, we ranked drug sensitivity according to curve shapes and comparison with the peak plasma concentration (ppc) of each drug.ResultsA total of 193 dose response curves were obtained. The overall informative rate was 67.4%, and 85.3% for cases that had a sufficient number of cells. Paclitaxel (PXL)and docetaxel tended to show a higher rank, while cisplatin (CIS) and 5-fluorouracil (5-FU) tended to show resistance, particularly among the 20 cases (46.5%) that had recurrent disease after receiving chemotherapy with CIS and S-1 (5-FU). As such, we speculate that the resistant pattern of the chemosensitivity test suggests that cells with acquired drug resistance were selected by chemotherapy. Indeed, we observed a change in the chemosensitivity pattern of a sample before and after chemotherapy in terms of PXL sensitivity, which was used after primary chemotherapy.ConclusionsThese results suggest that: (i) the dose–response pattern provides objective information for predicting chemosensitivity; and (ii) chemotherapy may select resistant cancer cell populations as a result of the therapy.

Well-differentiated neuroendocrine tumor of the breast with recurrence due to needle tract seeding

Dear Editor, Recently, percutaneous imaging-guided core needle biopsy (CNB) ensuring a high degree of diagnostic accuracy and minimal invasiveness has been widely practiced as an alternative to surgical biopsy [1]. However, the procedure itself can, very rarely, contribute to disease recurrence [1, 2]. The WHO classifies mammary carcinomas with neuroendocrine (NE) features as a special tumor entity, representing <1 % of invasive breast carcinomas, and recognizes three subtypes: (i) NE tumor (NET), well-differentiated; (ii) NE carcinoma, poorly differentiated; and (iii) invasive carcinoma with NE differentiation [3, 4]. Herein, we describe the first case of a mammary NE cancer (well-differentiated NET) showing intramammary relapse related to needle implantation. A 60-year-old postmenopausal Japanese woman presented with slight skin retraction in the upper inner portion of the right breast. Ultrasonography revealed an irregular, hypoechoic right breast tumor with an echogenic halo and posterior attenuation. Systemic CT detected no other suspicious lesions. We performed ultrasound-guided 16-G automated CNB of the breast mass after obtaining informed consent, and the histological diagnosis was (ductal) carcinoma. The cut surface of the lumpectomy specimen contained a poorly delimited, grey-whitish and focally brownish-red, solid tumor, measuring 12×10 mm in size. Histologically, this tumor was composed of invasive growing carcinoma cells in solid and/or trabecular clusters with a highly vascular fibrovascular stroma and focal hemorrhage (Fig. 1). Carcinoma cells were polygonal or, occasionally, spindle-shaped with finely granular cytoplasm and ovoid nuclei with a finegranular chromatin pattern (Fig. 1a). Six mitotic figures were counted in 10 high-power fields. In situ carcinoma components were locally observed near invasive cancer nests. Neither lymphatic nor vascular infiltration was detected. Lateral, including nipple-side, margins were negative for cancer. On the other hand, we noted small foci of scarring with fat necrosis and hemosiderin-laden macrophages in the subcutaneous tissues away from the main tumor (Fig. 2a) and identified sporadic epithelial clusters showing cancer cell morphologies within and around the scar tissues (Fig. 2b, c). No metastases were identified in the four excised right axillary lymph nodes. T. Kawasaki (*) :A. Sato :M. Suzuki : R. Sugimoto :Y. Mue : N. Uesugi :K. Ishida : T. Sugai Department of Molecular Diagnostic Pathology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan e-mail: tomonori@yamanashi.ac.jp

A Phase I Study of Capecitabine Combined with CPT-11 in Metastatic Breast Cancer Pretreated with Anthracyclines and Taxanes

Objective: A dose escalation study of biweekly irinotecan (CPT-11) combined with capecitabine was performed to determine the maximum tolerated dose (MTD) and recommended dose (RD) for metastatic breast cancer (MBC) previously treated with anthracyclines and taxanes. Methods: Escalating doses of CPT-11 (80-120 mg/m2) were administered on days 1 and 15. Capecitabine was administered at a fixed dose of 1,657 mg/m2/day for 21 consecutive days, followed by 7 days of rest. We treated 3-6 patients at a particular dose level until the MTD was determined. Results: Twenty patients were treated. The MTD was determined to be 100 mg/m2, as 3 of 6 patients developed dose-limiting toxicities, grade 3 leukopenia, neutropenia, photophobia, fatigue and diarrhea. The RD for the phase II study was thus determined to be 90 mg/m2. The response rate was 41.7%. Conclusions: Combination therapy with CPT-11 and capecitabine was well tolerated with a promising response rate for MBC that had been treated previously with anthracyclines and taxanes. A multi-center phase II study is warranted to evaluate the efficacy and safety of this combination therapy with pharmacokinetic assessment.

Glycogen‐rich clear cell carcinoma of the breast showing carcinomatous lymphangiosis and extremely aggressive clinical behavior

To the Editor: The World Health Organization (WHO) classifies mammary glycogen-rich clear cell carcinoma (GRCC) as an exceptionally rare variant among special types of invasive carcinoma and defines this tumor as >90% of neoplastic cells having abundant clear cytoplasm containing glycogen. Although previous reports suggest that GRCC is more aggressive than invasive carcinoma of no special type, some investigators have recently asserted that the prognosis is no different once GRCC and conventional mammary carcinomas are matched by tumor size, grade, and lymph-node status. Herein, we describe, to our knowledge, the first case of a GRCC with extraordinary vessel invasion (lymphangitic carcinomatosis) in the breast, resulting in a very rapid and aggressive clinical course. The patient, a 62-year-old postmenopausal Japanese woman, presented with a palpable mass in the upper outer quadrant of the right breast. She had no medical or familial history of breast disease. Ultrasonography revealed a well-marginated, hypoechoic right breast tumor showing heterogeneous internal echoes. Systemic CT and bone scintigraphy detected no other suspicious lesions. We performed ultrasound-guided, core needle biopsy of the breast lesion after obtaining informed consent, and the histological diagnosis was invasive carcinoma. The cut surface of the mastectomy specimen contained a lobulated, solid tumor, with grey-whitish and brownish-red areas, measuring 34 × 32 mm. Histologically, this tumor was composed of expansive solid and/or infiltrating trabecular growths of carcinoma cells with geographical coagulation necrosis and hemorrhage (Fig. 1). Carcinoma cells were polygonal with abundant, clear, fine-granular cytoplasm (Fig. 1a) and rich glycogen granules were confirmed by the periodic acid–Schiff method employing diastase (Fig. 1b,c). The nuclei showing a granular chromatin pattern had irregular shapes, frequently with distinct nucleoli (Fig. 1a). Sixty-five mitotic figures were counted in 10 high-power fields. Marked lymphatic permeation (i.e. carcinomatous lymphangiosis) as well as vascular infiltration were detected (Fig. 2a,b). In-situ components composed of similar carcinoma cells with comedo-like necroses were observed near invasive cancer nests (Fig. 1d). Surgical margins of the right breast were negative for cancer. Metastases were identified in 12 of 23 excised right axillary lymph nodes (Fig. 2c). Immunohistochemically, 0% of carcinoma cells were reactive for estrogen or progesterone receptors (Allred’s total scores: 0 and 0, respectively). The HER2 score was 0, and the Ki67 (MIB-1) labelling index was 70.1%. Carcinoma cells were positive for cytokeratin (CK) AE1/AE3, CK7 and GATA3, focally positive for CK CAM5.2, mammaglobin, adipophilin and p63 and negative for CK5/6, CK14, CK20, gross cystic disease fluid protein 15 (GCDFP15), HMB45, Melan-A/ MART-1, smooth muscle actin, muscle specific actin (HHF35), calponin, h-caldesmon, renal cell carcinoma marker (RCC Ma), PAX8, carbonic anhydrase IX, CD10 and androgen receptor. The lining endothelial cells, visualized by D2-40 and/or CD31 staining, were evident in vessels with tumor involvement (Fig. 2b). Myoepithelial markers showed the presence of myoepithelia along the periphery of in-situ carcinoma cell nests. A month after surgery, three nodules were detected by palpation in the right chest wall and were diagnosed as local recurrence with carcinoma verification using fine needle aspiration cytology. On subsequent CT scans, multiple lung, liver, bone (Th5 and L1) and lymph node (right supraclavicular and internal thoracic) metastases were identified. The patient received paclitaxel (90 mg/m) and bevacizumab (10 mg/kg) every 4 weeks for seven cycles. She has remained alive with marked therapeutic effects for 8 months, to date, since surgery. The possibility of metastatic clear cell carcinoma from another site, especially clear cell renal cell carcinoma, should be ruled out. Imaging and clinical history confirmed that our patient had no lesions preoperatively in other organs, and an in-situ component accompanying the invasive breast cancer was demonstrated histologically. These are regarded as the two most important features for diagnosing primary clear cell mammary carcinoma. In addition, GATA3 and mammaglobin immuno-expressions support a diagnosis of primary GRCC of the breast, while negativity for RCC Ma, PAX8, carbonic anhydrase IX and CD10 and positivity for CK7 can rule out clear cell renal cell carcinoma. Differentiation from other mammary neoplasms composed of clear cells is also necessary. Clear cell ‘sugar’ tumor, one member of the family of neoplasms with perivascular epithelioid cell differentiation (so-called PEComas), shows solid proliferation of epithelioid tumor cells with glycogen-rich clear cytoplasm. This tumor, originally described in the lung, is now being recognized at extra-pulmonary sites, including the breast. Immunohistochemically, the PEComa family is characterized by strong reactivity with the HMB45 antibody, variable expressions of muscle markers and negativity for cytokeratins, unlike our present GRCC. Pathology International 2015; 65: 674–676 doi:10.1111/pin.12321 bs_bs_banner

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The efficacy of sodium azulene sulfonate L-glutamine for managing chemotherapy-induced oral mucositis in cancer patients: a prospective comparative study

BackgroundThe efficacy of sodium azulene sulfonate L-glutamine (GA) in treating oral mucositis caused by the administration of anticancer agents has not been previously elucidated. Therefore, this prospective comparative study was conducted to evaluate the efficacy of GA in treating oral mucositis caused by chemotherapy regimens involving fluorinated pyrimidine anticancer drugs.MethodsThe subjects of this study were patients with oral mucositis of grade 2 or higher while on outpatient chemotherapy regimens involving fluorinated pyrimidine anticancer drugs for colorectal or breast cancer. The subjects were randomly divided into a group that received GA (the GA group) or a group that did not receive GA (the control group) by using the closed-envelope method. GA was administered three times a day every day from the first day of the regimen until the final day. The primary endpoint was the development of oral mucositis of grade 2 or higher. The secondary endpoint was the severity of oral pain, which was judged using an 11-stage numerical rating scale (NRS) ranging from 0 to 10.ResultsThe proportion of patients with oral mucositis of grade 2 or higher was 32.4% in the GA group and 57.6% in the control group. The GA group had a significantly lower frequency of occurrence. The changes in the NRS scores before and after the trial began were − 2.9 ± 0.6 in the GA group and − 1.2 ± 0.5 in the control group. The NRS score decreased more significantly in the GA group than in the control group (P = 0.046). One patient stopped GA treatment voluntarily due to nausea; other than nausea, no GA-related side effects were observed.ConclusionsGA protects against oral mucositis and reduces the severity of prevailing oral mucositis symptoms. Our findings indicate that GA is a highly safe and convenient drug.

Effect of olanzapine for breast cancer patients resistant to triplet antiemetic therapy with nausea due to anthracycline-containing adjuvant chemotherapy

OBJECTIVE Triplet antiemetic therapy with neurokinin 1 receptor blocker, 5-hydroxytryptamine receptor blocker and steroids is commonly used in patients who are highly emetic after chemotherapy. However, an alternative antiemetic therapy for patients who are resistant to triplet antiemetic therapy is not established. Olanzapine is recommended in the guidelines as an optional antiemetic drug. However, the effectiveness of adding olanzapine to triplet antiemetic therapy is unknown. In this study, the effectiveness and safety of adding olanzapine to triplet antiemetic therapy with aprepitant, palonosetron and dexamethasone as highly emetic anthracycline-containing adjuvant chemotherapy for primary breast cancer patients were prospectively investigated. METHODS Forty-five patients with breast cancer who experienced >Grade 1 nausea or any vomiting after the first cycle of chemotherapy using both epirubicin and cyclophosphamide were included. Low-dose olanzapine (2.5 mg/day) was administered orally from the first day of chemotherapy for 4 days, and the number of episodes of vomiting, scale of nausea, dietary intake and somnolence were compared with the symptoms after the first cycle. RESULTS As the primary endpoint, the nausea grade was significantly improved by adding olanzapine (P < 0.05). As the secondary endpoints, mean nausea scale (3.2→1.9, Day 1; 3→1.3-1, Days 2-6) and dietary intake (33.6→53.8%, Day 1; 42.0→60.7-78.1%, Days 2-6) were improved by adding olanzapine. Only four patients withdrew due to somnolence and/or dizziness. CONCLUSIONS This study demonstrated the effectiveness and tolerability of adding low-dose olanzapine for patients with insufficient nausea relief with triplet antiemetic therapy consisting of palonosetron, steroid and aprepitant.