Tomoyuki Moriya

A simple immunohistochemical panel comprising 2 conventional markers, Ki67 and p53, is a powerful tool for predicting patient outcome in luminal-type breast cancer

BackgroundKi67 is widely used in order to distinguish the “A” and “B” subtypes of luminal-type breast cancer. This study aimed to validate the prognostic value of adding p53 to Ki67 for characterizing luminal-type breast cancer.MethodsImmunostaining for Ki67, p53, and the molecular markers HER2, CK5/6, CK14, EGFR, FOXA1, GATA3, and P-cadherin was examined hormone receptor (HR)-positive cancer tissues from 150 patients. The prognostic value of an immunohistochemical panel comprising Ki67 and p53 was compared with that of the single Ki67 labeling index (LI), and uni- and multivariate analyses were performed.ResultsDivision of the patients based on the immunohistochemistry results into favorable- (low Ki67 LI, p53-negative) and unfavorable- (high Ki67 LI and/or p53-positive) phenotype groups yielded distinctly different Kaplan-Meiers curves of both disease-free (P<0.0001) and overall survival (P=0.0007). These differences were much more distinct than those between the corresponding low Ki67 LI vs. high Ki67LI curves. While the prognostic values of the other molecular markers were not significant, combined Ki67-p53 status was an independent prognostic factor by multivariate analysis.ConclusionThese data indicate that an immunohistochemical panel comprising Ki67 and p53 is a practical tool for management of patients with HR-positive breast cancer.

Exogenous interleukin 7 affects gut-associated lymphoid tissue in mice receiving total parenteral nutrition.

In the absence of enteral nutrient delivery, gut-associated lymphoid tissue (GALT) mass and function are reduced. The purpose of this study was to examine whether exogenous interleukin (IL)-7 treatment reverses intravenous (IV)-total parenteral nutrition (TPN)-induced changes in GALT, immunoglobulin (Ig) A levels, and gut barrier function. Eighty-nine mice were randomized to chow, TPN, or TPN + IL-7 (1 μg/kg, administered IV twice a day) and treated for 5 days. The entire small intestine was harvested and lymphocytes were isolated from Peyers patches (PPs), intraepithelial (IE) spaces, and the lamina propria (LP). Small intestinal and bronchoalveolar IgA levels were measured. Proximal and distal small intestinal levels of IgA-stimulating (IL-10) and IgA-inhibiting (IFNγ) cytokines were determined with enzyme-linked immunoabsorbant assay. Moreover, 1 × 1010 live Pseudomonas aeruginosa were delivered by gavage and survival was observed. TPN decreased total cell yields from PPs, IE spaces, and the LP compared with the chow group. IL-7 treatment restored cell numbers. PP CD4+, PP CD8+, IE γδTCR+, and LP CD4+ cell numbers were higher in the TPN + IL-7 group than in the TPN group. Secretory IgA levels were lower in the TPN and TPN + IL-7 than in the chow group. In the distal small intestine, IFNγ levels were similar in the three groups, whereas IL-10 levels were reduced in the TPN and TPN + IL-7 groups relative to the chow group. Survival times were reduced in the TPN compared with the chow group, but IL-7 treatment significantly improved survival. Thus, exogenous IL-7 does not improve secretory IgA levels, nor are there any remarkable effects on levels of gut IgA-mediating cytokines. However, IL-7 treatment during TPN reverses TPN-induced GALT atrophy and improves survival in a gut-derived sepsis model.

Nutritional route affects ERK phosphorylation and cytokine production in hepatic mononuclear cells.

Objective:To clarify the influence of nutritional route on hepatic immunity in a murine model. Summary Background Data:Parenteral nutrition is disadvantageous for preventing infectious complications in critically ill and/or severely injured patients as compared with enteral nutrition. To date, lack of enteral nutrition has been demonstrated to impair mucosal immunity, gut barrier function, and the peritoneal defense system. However, influences of nutritional route on hepatic immunity, another important defense system against infection, have not been well studied. Methods:Male ICR mice were randomized to 3 groups: ad libitum chow (chow), intravenous (IV)-TPN and intragastric (IG)-TPN groups. The TPN groups were given isocaloric and isonitrogenous TPN solutions. After the mice had been fed for 5 days, hepatic mononuclear cells (MNCs) were isolated. Hepatic MNC numbers and functions (cytokine production, intracellular signaling, and LPS receptor expression) were determined. Moreover, 1.0 × 107 Pseudomonas aeruginosa were delivered by intraportal injection. Survival and histology were examined. Results:Hepatic MNC numbers were significantly lower in the IV-TPN group than in the chow and IG-TPN groups, without subpopulation changes. As compared with enterally fed mice, cytokine production (TNF-α, IFN-γ, and IL-10) by hepatic MNCs in response to LPS was impaired in parenterally fed mice in association with blunted phosphorylation of ERK1/2, a MAPK. Hepatic MNCs from IV-TPN mice showed decreased expressions of CD14 and TLR4/MD2, as compared with enterally fed mice. Survival times were reduced in the IV-TPN group as compared with the chow and IG-TPN groups. Conclusion:Preservation of hepatic immunity with enteral feeding is important for prevention of infectious complications in severely injured and/or critically ill patients.

Effects of Adding Butyric Acid to PN on Gut-Associated Lymphoid Tissue and Mucosal Immunoglobulin A Levels

BACKGROUND Parenteral nutrition (PN) causes intestinal mucosal atrophy, gut-associated lymphoid tissue (GALT) atrophy and dysfunction, leading to impaired mucosal immunity and increased susceptibility to infectious complications. Therefore, new PN formulations are needed to maintain mucosal immunity. Short-chain fatty acids have been demonstrated to exert beneficial effects on the intestinal mucosa. We examined the effects of adding butyric acid to PN on GALT lymphocyte numbers, phenotypes, mucosal immunoglobulin A (IgA) levels, and intestinal morphology in mice. METHODS Male Institute of Cancer Research mice (n = 103) were randomized to receive either standard PN (S-PN), butyric acid-supplemented PN (Bu-PN), or ad libitum chow (control) groups. The mice were fed these respective diets for 5 days. In experiment 1, cells were isolated from Peyers patches (PPs) to determine lymphocyte numbers and phenotypes (αβTCR(+), γδTCR(+), CD4(+), CD8(+), B220(+) cells). IgA levels in small intestinal washings were also measured. In experiment 2, IgA levels in respiratory tract (bronchoalveolar and nasal) washings were measured. In experiment 3, small intestinal morphology was evaluated. RESULTS Lymphocyte yields from PPs and small intestinal, bronchoalveolar, and nasal washing IgA levels were all significantly lower in the S-PN group than in the control group. Bu-PN moderately, but significantly, restored PP lymphocyte numbers, as well as intestinal and bronchoalveolar IgA levels, as compared with S-PN. Villous height and crypt depth in the small intestine were significantly decreased in the S-PN group vs the control group, however Bu-PN restored intestinal morphology. CONCLUSIONS A new PN formula containing butyric acid is feasible and would ameliorate PN-induced impairment of mucosal immunity.

Reversal of parenteral nutrition-induced gut mucosal immunity impairment with small amounts of a complex enteral diet.

BACKGROUND Although parenteral nutrition (PN) prevents progressive malnutrition, lack of enteral nutrition (EN) during PN leads to gut associated lymphoid tissue (GALT) atrophy and dysfunction. Administering a small amount of EN with PN reportedly prevents increases in intestinal permeability. However, its effects on GALT remain unclear. We analyzed the minimum amount of EN required to preserve gut immunity during PN. METHODS Male Institute of Cancer Research mice underwent jugular vein catheter insertion and tube gastrostomy. They were randomized into four groups to receive isocaloric and isonitrogenous nutritional support with variable EN to PN ratios (EN 0, EN 33, EN 66, and EN 100). EN was provided with a complex enteral diet. After 5 days of feeding, the mice were killed and whole small intestines were harvested. GALT lymphocytes were isolated and counted. Their phenotypes were analyzed by flow cytometry. IgA levels of small intestinal washings were analyzed with enzyme-linked immunosorbent assay. RESULTS Body weight changes did not differ between any two of the groups. Peyers patch lymphocyte numbers increased in proportion to EN amount, whereas lamina propria lymphocyte numbers were significantly higher in the EN 100 than in the EN 0 group, with no marked increases in the EN 33 and EN 66 groups. Small intestinal IgA levels increased EN amount-dependently and reached a plateau at EN 66. CONCLUSIONS A small amount of EN partially reverses PN-induced GALT changes, suggesting beneficial but limited effects on gut mucosal immunity.

Early Reduction in Standardized Uptake Value After One Cycle of Neoadjuvant Chemotherapy Measured by Sequential FDG PET/CT is an Independent Predictor of Pathological Response of Primary Breast Cancer

To the Editor: F-fluorodeoxyglucose (FDG) levels on positron emission tomography (PET) reflect glucose metabolism in cancer cells (1). Currently, FDG PET combined with computed tomography (FDG PET ⁄ CT) has become employed as a non-invasive method for imaging glucose metabolism in tumors (2,3). The aim of the present study was to evaluate whether early metabolic changes after one cycle of neoadjuvant chemotherapy in FDG uptake evaluated by maximal standardized uptake value (SUVmax) could predict a pathological response of primary breast cancers. Thirty-two tumors in 30 patients having primary invasive breast cancer were investigated. All patients had received a standard neoadjuvant chemotherapy regimen comprising four cycles of epirubicine (90 mg ⁄ m) and cyclophosphamide (600 mg ⁄ m) on a triweekly basis and sequential use of 12 cycles of weekly paclitaxel (80 mg ⁄ m) (25 patients) or four cycles of triweekly docetaxel (60 mg ⁄ m) (five patients). The procedure of FDG PET ⁄ CT has been described (4). Sequential FDG PET ⁄ CT (Biograph LSO Emotion; 3D model, Siemens, Germany) was performed at the baseline (baseline PET ⁄ CT), after one cycle of chemotherapy (PET ⁄ CT2), after four cycles of chemotherapy (PET ⁄ CT3), and prior to surgery (PET ⁄ CT4). Tumors showing a 40% reduction or more in SUVmax on PET ⁄ CT2, when compared with the baseline PET ⁄ CT, were defined as metabolic responders and those showing a change of less than 40% in SUVmax were considered to be metabolic nonresponders (Fig. 1). Baseline characteristics of patients and tumors, determined by conventional modalities, are shown in Table 1. Baseline SUVmax was significantly higher in metabolic responders [10.2 ± 6.4 SD] than nonresponders (6.7 ± 3.1 SD) (p = 0.05). The percentage of tumors with nuclear grade 3 was significantly higher among the metabolic responders (71%) than among the nonresponders (16%) (p = 0.03). There were no significant differences between metabolic responders and nonresponders with regard to patient age, T-stage, nodal status, hormone receptor status, or HER2 status. The average degree of decrease in SUVmax at PET ⁄ CT2 in comparison with the baseline SUVmax was 57.9% (±11.7 SD) in metabolic responders and 10.3% (±15.7 SD) in metabolic nonresponders (p < 0.0001). Clinical response after completion of chemotherapy was measured using ultrasound or CT combined with FDG PET, and evaluated based on RECIST. On the basis of clinical response, five (71%) and two (29%) of the seven tumors with a metabolic response exhibited partial response (PR) and complete response (CR), respectively, while 18 (72%) of the 25 nonresponding tumors had PR. No nonresponding tumors showed CR. Metabolic responders showed a significantly excellent clinical response rate (100%) in terms of PR or CR in comparison with that (72%) for non-responding tumors (p = 0.001) (Table 2a). Histological criteria for assessment of therapeutic response were based on General Rules for Clinical and Pathological Recording of Breast Cancer 2008 (5). Among a total of 32 tumors, six (19%) and two (6%) were found to have grade 3, or pathological complete response (pCR), and to have grade 2b, or near pCR, where only a few residual invasive cancer cells were seen, respectively. Among the seven tumors with a metabolic response, three (43%) and two (29%) showed pCR and near pCR, respectively. Among the 25 tumors without metabolic response, Address corresopondence and reprint requests to: Hitoshi Tsuda, MD, PhD, Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan, or e-mail: hstsuda@ gmail.com.

Lack of preoperative enteral nutrition reduces gut-associated lymphoid cell numbers in colon cancer patients: a possible mechanism underlying increased postoperative infectious complications during parenteral nutrition.

Objective: To examine preoperative dietary influences on gut-associated lymphoid tissue (GALT) cell number in the context of postoperative infectious complications. Background: There is little clinical evidence regarding whether nutritional routes affect GALT size and/or phenotype. The influence of GALT atrophy on clinical outcomes is also unclear. Method: Patients with complete obstruction of the colon due to a tumor were excluded from this study. Study 1. Resected terminal ileum specimens, from 62 patients [preoperative parenteral nutrition (PN): n = 15, preoperative oral feeding (OF): n = 47] who underwent right colectomy during the period from 1997 to 2004 at our department, were immunohistochemically stained for counting numbers of T, IgA-producing, and mature and immature dendritic cells (DCs) in the lamina propria (LP) and intraepithelial space. Study 2. We reviewed 341 patients (PN: n = 99, OF: n = 242) with colon cancer who underwent colectomy during this period for postoperative complications. Results: Study 1. T cell numbers in the LP and intraepithelial space and IgA-producing cell number in the LP were significantly lower in the PN than in the OF group. Mature DC number in the LP was significantly lower in the PN than in the OF group, whereas total DC numbers (both mature and immature DC) were similar in the 2 groups. Study 2. The PN group had significantly higher rates of total infectious complications, surgical site infection, pneumonia, infectious colitis, and central venous catheter infection. Conclusions: Lack of enteral delivery of nutrients reduces numbers of T and IgA-producing cells, as well as mature DCs, in GALT of colon cancer patients, as it does in animal models. A close association between GALT changes and infectious complication morbidity was confirmed.

High levels of DJ‐1 protein and isoelectric point 6.3 isoform in sera of breast cancer patients

In patients with cancer and Parkinsons disease, the DJ‐1 protein may be secreted into the serum during the impaired response of the underlying cell‐protective mechanisms. In order to determine the clinical significance of DJ‐1 protein in the sera of breast cancer patients, we examined blood samples from a breast cancer group (n = 180) and a non‐cancerous control group (n = 300). Higher levels of DJ‐1 were detected in the breast cancer group (mean level, 42.7 ng/mL) than the control group (28.3 ng/mL) by ELISA (P = 0.019). Higher DJ‐1 levels were significantly associated with advanced clinical grade, according to the TNM classification, negative hormone receptor status, and high Ki‐67 labeling index, of biopsied materials; samples showed low DJ‐1 protein expression despite upregulated DJ‐1 mRNA. DJ‐1 isoforms could be detected clearly in 17 blood samples (from 11 breast cancer patients, and 6 non‐cancerous controls) by 2‐D gel electrophoresis and immunoblot analysis. The isoform at the pI of 6.3 showed the highest intensity in all 11 cancer cases. Conversely, in the 6 non‐cancerous cases, isoforms other than the pI 6.3 isoform were highly expressed, and there was a significant difference in the isoform pattern between breast cancer cases and controls (P = 0.00025). These data indicate that high levels of DJ‐1, probably of isoform at pI 6.3, is a candidate serum marker of breast cancer.

Arginine-Enriched Total Parenteral Nutrition Improves Survival in Peritonitis by Normalizing NFκB Activation in Peritoneal Resident and Exudative Leukocytes

Background:Enteral nutrition maintains peritoneal defense more effectively than parenteral nutrition, at least partly by preserving NF&kgr;B activation in peritoneal cells. We hypothesized that arginine (ARG)-enriched parenteral nutrition would normalize NF&kgr;B activation in peritoneal leukocytes, thereby improving the survival of peritonitis models. Methods:A total of 105 ICR mice were randomized to chow (n = 33), IV feeding of a standard (STD) total parenteral nutrition (STD-TPN) solution (ARG 0.3%) (n = 35), or 1% ARG-TPN solution (n = 37), and fed accordingly for 5 days.Experiment 1: Thirty mice were used for intranuclear NF&kgr;B measurement in peritoneal resident cells (PRCs). After incubation with lipopolysaccharide (LPS: 0, 1, 10 &mgr;g/mL) for 30 minutes, intranuclear NF&kgr;B activity was examined by laser scanning cytometry.Experiment 2: Fifty-one mice were injected with 2 mL of 1% glycogen intraperitoneally. Peritoneal exudative cells (PECs) were obtained at 2 or 4 hours after glycogen administration for NF&kgr;B measurement. Cytokine (TNF&agr;, IL-10) levels in peritoneal lavage fluid were also determined by ELISA.Experiment 3: After 5 days of feeding, 24 mice underwent cecal ligation and puncture. Survival was observed up to 5 days. Results:Experiment 1: Intranuclear NF&kgr;B levels in the ARG-TPN and chow groups increased dose-dependently after LPS stimulation, while the level in the STD-TPN group was unchanged.Experiment 2: Intranuclear NF&kgr;B level was significantly higher at 2 hours in the chow than in the STD-TPN group, whereas in the ARG-TPN mice the level was midway between those of the chow and STD-TPN groups. TNF&agr; and IL-10 levels of the chow group were significantly higher than those of STD-TPN mice at 2 hours. TNF&agr; was significantly higher in the ARG-TPN group than in the STD-TPN group, but the IL-10 level showed no recovery.Experiment 3: Survival times were significantly reduced in the STD-TPN as compared with the chow group, though ARG-TPN improved survival. Conclusion:ARG-enriched TPN is a surrogate for enteral feeding which maintains peritoneal defense by preserving NF&kgr;B activation in peritoneal resident and exudative leukocytes.

Nucleostemin expression in invasive breast cancer

BackgroundRecently, the cancer stem cell hypothesis has become widely accepted. Cancer stem cells are thought to possess the ability to undergo self-renewal and differentiation, similar to normal stem cells. Nucleostemin (NS), initially cloned from rat neural stem cells, binds to various proteins, including p53, in the nucleus and is thought to be a key molecule for stemness. NS is expressed in various types of cancers; therefore, its role in cancer pathogenesis is thought to be important. This study was conducted to clarify the clinicopathological and prognostic impact of NS in invasive breast cancers.MethodThe correlation between NS immunoreactivity and clinicopathological parameters was examined in 220 consecutive surgically resected invasive breast cancer tissue samples by using tissue microarrays. The presence of nuclear NS and p53 immunoreactivity in 10% or more of cancer cells was considered as a positive result.ResultsAmong the 220 patients, 154 were hormone-receptor (HR)-positive, 22 HER2-positive/HR-negative, and 44 HR-negative/HER2-negative. One hundred and forty-two tumors (64.5%) showed NS positivity, and this positivity was significantly correlated with estrogen receptor (ER) (P = 0.050), human epidermal growth factor receptor 2 (HER2) (P = 0.021), and p53 (P = 0.031) positivity. The patients with NS-positive tumors showed significantly shorter disease-free survival than those with NS-negative tumors. Furthermore, the patient group with NS- and p53-positive tumors showed significantly poorer prognosis than other patient groups. Multivariate analysis showed that NS status was an independent prognostic indicator.ConclusionsNS may play a significant role in the determination of breast cancer progression in association with p53 alterations. The NS status of patients with luminal and HER2 type breast cancers may be a useful prognostic marker.