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Background Nanoliposomal irinotecan (nal-IRI) was recently authorized in Japan for unresectable pancreatic cancer after disease progression following chemotherapy. Physicians now consider certain aspects of nal-IRI safety profile as slightly different from conventional irinotecan. This report aims to explore additional aspects of the nal-IRI safety in Japanese phase 2 study. Methods We analyzed the incidence, time to first onset, and time to resolution for adverse events that require special attention and other selected toxicities in the nal-IRI combination group (n = 46). Results Leukopenia/neutropenia (76.1%/71.7%), diarrhea (58.7%) and hepatic dysfunction (41.3%) were the most commonly reported treatment-emergent adverse events, with a median time to onset of 21.0 days (range: 8, 97), 9.0 days (1, 61) and 22.0 days (2, 325), respectively, and a median time to resolution of 8.0 days (95% confidence intervals: 8, 9), 4.0 days (4, 8) and 40.0 days (9, –), respectively. Eight patients experienced Grade ≥ 3 diarrhea and their symptoms were well controlled by dose modification except one patient who had drug withdrawal. The median time to resolution for Grade ≥ 3 and Grade ≤ 2 diarrhea was 17.5 days (95% confidence intervals: 1, 31) and 4 days (3, 7), respectively. Anorexia occurred in 28/46 patients (60.9%) with a median time to onset of 4.0 days (range: 2, 132) and a median time to resolution of 12.0 days (95% confidence intervals: 6, 26). Conclusions We explored safety profile of nal-IRI combination regimen recognized as effective and tolerable treatment for Japanese unresectable pancreatic cancer patients. Although the treatment-emergent adverse events occurred were controllable, patients with prolonged toxicities should be closely managed.

Introduction In quantitative assays for hepatitis B virus (HBV) DNA, although the amplification reaction signal is detected for low-positive cases, quantification remains challenging. HBV reactivation has been reported in many studies, but only a few have focused on HBV low-positive cases. This study aimed to determine the reactivation rate and risk factors for HBV reactivation in low-positive cases. Methods In this retrospective cohort study, we analyzed 7498 patients who had their HBV DNA measured at Sapporo Medical University Hospital between April 2008 and November 2020. Patient selection criteria were defined as follows: hepatitis B surface antigen was negative; HBV DNA was detectable but not quantifiable at least once. HBV DNA was monitored according to the guidelines for HBV reactivation. Results In total, 49,086 HBV DNA quantitative tests were performed. HBV DNA levels of 2578 tests were detectable but not quantifiable. Eighty patients met the criteria in this study. The median observation period was 497 days, and the 2-year reactivation rate was 15%. Ten patients had low HBV DNA positivity at baseline. Malignant lymphoma was observed in 15 patients; chemotherapy was used to treat other solid tumors in 35 patients, and immunosuppressive therapy was used in 30 patients. Multivariate analysis revealed that HBV DNA detected below the quantification level at baseline was an independent risk factor for HBV reactivation (adjusted hazard ratio 5.82; P = 0.010). Conclusions Patients with low HBV DNA positivity, especially at baseline, are at high risk for HBV reactivation and therefore require closer monitoring.

549 Background: Based on a phase II study conducted in Japanese metastatic pancreatic cancer (NCT02697058) and a global phase III study, nal-IRI obtained market authorization in March 2020 in Japan for "Unresectable pancreatic cancer after disease progression following cancer chemotherapy". After one year of practice, physicians have a sense that some of the safety profile of nal-IRI to be slightly different from conventional irinotecan. The purpose of this report is to explore additional points of the nal-IRI safety in the Japanese study. Methods: This analysis with the safety population of nal-IRI+5-FU/ l-LV group was done for the incidence, time to first onset (TTO), and time to resolution (TTR) for the events associated with 12 identified risks in the Risk Management Plan (RMP)(Table) and other selected GI toxicities. Prophylaxis used were also analyzed. Results: Myelosuppression (82.6%), diarrhoea (58.7%) and hepatic dysfunction (41.3%) were commonly reported of the 12 identified risks. The median (m) TTO of these TEAEs was 16 days (range: 6, 73), 9 days (1, 61), and 22days (2,325) respectively. As for the mTTR, it was 8.0 days (95% CI: 8.00, 9.00), 4.0 days (4.00, 8.00), and 40.0 days (9.00, -) respectively. For the myelosuppression, there were 4 patients who received G-CSF for the treatment of ≥ grade 3 myelosuppression. The 8 patients who experienced diarrhoea leading to dose reduction had no recurrence of ≥grade3 diarrhoea after intervention. For other GI toxicities, anorexia occurred in 28/46 patients (60.9%) with mTTO 4.0 days (range: 2, 132) and mTTR 12.0 days (95% CI: 6.00, 26.00). Summary of TTO for the events associated with identified risks in the RMP. * Intestinal obstruction, enteritis, disseminated intravascular coagulation, interstitial lung disease, ventricular extrasystoles are also included in the RMP but not observed. Discussion: This is the first report of nal-IRI safety profile details such as timing in Japanese patients. It is interesting to note that some TEAEs appear different in practice regarding TTR in comparison with conventional irinotecan. This difference can be due to the unique PK profile of nal-IRI as a result of utilizing liposomal technology but needs to be further elucidated. Conclusions: Although the TEAEs occurred with nal-IRI+5-FU/ l-LV were controllable with proper management in Japanese patients, some with prolonged toxicities should be carefully managed. Clinical trial information: NCT02697058. [Table: see text]

The lymphatic vasculature collects and drains fluid and cells from the periphery through lymph nodes (LNs) for immune monitoring, and then returns lymph to the bloodstream. During immune responses LNs enlarge and remodel, featuring extensive growth of lymphatic sinuses (lymphangiogenesis). This LN lymphangiogenesis also arises in cancer, and is associated with altered lymph drainage through LNs. Studies of mouse solid tumor models identified lymphatic sinus growth throughout tumor-draining LNs (TDLNs), and increased lymph flow through the expanded sinuses. Mice developing B cell lymphomas also feature LN lymphangiogenesis and increased lymph flow, indicating that these changes occur in lymphoma as well as in solid tumors. These LN alterations may be key to promote tumor growth and metastasis to draining LNs and distant organs. Lymphatic sinus growth within the TDLN may suppress anti-tumor-immune responses, and/or the increased lymph drainage could promote metastasis to draining LNs and distant organs. Investigations of human cancers and lymphomas are now identifying TDLN lymphatic sinus growth and increased lymph flow, that correlate with metastasis and poor prognosis. Pathology assessment of TDLN lymphangiogenesis or noninvasive imaging of tumor lymph drainage thus could potentially be useful to assist with diagnosis and treatment decisions. Moreover, the expanded lymphatic sinuses and increased lymph flow could facilitate vaccine or drug delivery, to manipulate TDLN immune functioning or to treat metastases. The insights obtained thus far should encourage further investigation of the mechanisms and consequences of TDLN lymphatic sinus growth and lymph flow alterations in mouse cancer models, and in human cancer patients.

The earlier step of cutaneous wound healing process, re-epithelialization of the wounded skin, is triggered by a variety of growth factors. However, molecular mechanisms through which growth factors trigger skin wound healing are less understood. Here, we demonstrate that hepatocyte growth factor (HGF)/c-Met signaling-induced expression of the small G protein Arf6 mRNA in keratinocytes is essential for the skin wound healing. Arf6 mRNA expression was dramatically induced in keratinocytes at the wounded skin, which was specifically suppressed by the c-Met inhibitor. Wound healing of the skin was significantly delayed in keratinocyte-specific Arf6 conditional knockout mice. Furthermore, Arf6 deletion from keratinocytes remarkably suppressed HGF-stimulated cell migration and peripheral membrane ruffle formation, but did not affect skin morphology and proliferation/differentiation of keratinocytes. These results are consistent with the notion that Arf6 expressed in skin keratinocytes through the HGF/c-Met signaling pathway in response to skin wounding plays an important role in skin wound healing by regulating membrane dynamics-based motogenic cellular function of keratinocytes.

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1–LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1–related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1– or LFA-1–expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.

The ability of tumor cells to metastasize is increasingly viewed as an interaction between the primary tumor and host tissues. Deletion of the p19/Arf or p53 tumor suppressor genes accelerates malignant progression and metastatic spread of 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced squamous cell carcinomas, providing a model system to address mechanisms of metastasis. Here, we show that benign pre-metastatic papillomas from wild-type mice trigger lymphangiogenesis within draining lymph nodes, whereas there is no growth of primary tumor lymphatic vessels. Lymph node lymphangiogenesis is greatly accelerated in papilloma-bearing p19/Arf- or p53-deficient mice, which coincides with the greater propensity of these tumors to progress to carcinomas and to metastasize. The extent of accumulation of B cells within the tumor-draining lymph nodes of wild-type mice predicted the level of lymph node lymphangiogenesis and metastatic potential. Arf or p53 deficiency strongly accelerated lymph node immune cell accumulation, in a manner that was associated with the extent of lymph node lymphatic sinus growth. This immune cell accumulation and lymph node lymphangiogenesis phenotype identifies host anti-tumor responses that could drive metastatic spread of cancers via the lymphatics.

Expression of survivin, a member of the inhibitor-of-apoptosis (IAP) family, is elevated in fetal tissues and in various human cancers originating in the breast, lung, prostate, colon, pancreas, and stomach. Since overexpression of the survivin gene has been linked to poor patient survival in several cancers, survivin may be an important prognostic marker. Mechanisms up-regulating survivin gene expression in cancer are poorly understood. Recently, wild-type p53 was found to repress expression of the survivin gene by binding to the survivin promoter, thereby inhibiting promoter activity. Further, loss of heterozygosity (LOH) at 17p13 distal to the p53 gene is associated with more aggressive behavior of breast cancers. We therefore tested the hypothesis that not only p53 gene mutation but also LOH at 17p13 can up-regulate survivin expression in breast cancer. Survivin mRNA expression was greater in cancers than in uninvolved tissues (p < 0.0001). Mutations of the p53 gene were detected in 5 of 25 tumors; higher survivin gene expression was evident in these. LOH at the D17S938 locus (17p13.1) was found in 10 of 25 tumors, and most of these also showed increased survivin gene expression. Thus expression of survivin may be regulated not only by p53 but additionally by a putative tumor suppressor gene located at 17p13 distal to the p53 gene.

Background Tumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other. Methods The murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels. Results Each peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors. Conclusions Implanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

Angiomodulin (AGM/IGFBP‐rP1), a glycoprotein of about 30 kDa, is overexpressed in tumor vasculature as well as some human cancer cell lines, but it has been suggested to be a tumor suppressor. To elucidate roles of angiomodulin (AGM) in tumor progression, we here examined distribution of AGM in three types of human cancer tissues by immunohistochemistry. The results showed that AGM was overexpressed in the stroma as well as the vasculature surrounding tumor cells in the human cancer tissues. AGM and α‐smooth muscle actin (α‐SMA) as an activated fibroblast marker were often colocalized in cancer‐associated fibroblasts (CAFs). In vitro analysis indicated that transforming growth factor (TGF)‐β1 might be an important inducer of AGM in normal human fibroblasts. AGM strongly stimulated the expression of fibronectin and weakly that of α‐SMA in normal fibroblasts. AGM significantly stimulated the proliferation and migration of fibroblasts. The AGM‐induced expression of fibronectin and α‐SMA was blocked by a TGF‐β signal inhibitor but neither the stimulation of cell growth nor migration. These results imply that AGM activates normal fibroblasts by TGF‐β‐dependent and independent mechanisms. These findings also suggest that AGM and TGF‐β1 cooperatively or complementarily contribute to the stromal activation and connective tissue formation in human cancer tissues, contributing to tumor progression. (Cancer Sci 2012; 103: 691–699)

The small GTPase Arf6 is a member of the Arf (ADP-ribosylation factor) family. Although the function of Arf6 has been heavily studied at the cellular level, its physiological function at the whole animal level is largely unknown. In this study, we examined both the tissue distribution and developmental timing of Arf6 expression in wild type mice to obtain valuable information to speculate on the physiological function of Arf6. Western blot analysis using anti-Arf6 antibody revealed that Arf6 was ubiquitously expressed with its developmental timing differing in a tissue-specific manner. These results were supported by Arf6 mRNA in situ hybridization experiments, which showed that Arf6 was highly expressed in the polarized epithelial cells and embryonic mesenchymal cells of most tissues in a temporally dependent manner. Taken in toto, our results suggest that the expression of Arf6 in mouse tissues is precisely regulated in a development- and tissue-dependent manner.

Despite surgical removal of the primary tumor of breast cancer in patients with apparently localized disease, relapse at local or distant sites may occur because undetectable micrometastases were present at the time of diagnosis. Identification of molecules associated with breast cancer metastasis suggests possible new treatments. PCD1, a gene encoding a new member of the PDZ and LIM domain-containing protein family, recently was identified. We examined the relationships between PCD1 mRNA expression in breast cancers and metastasis.PCD1 mRNA expression in breast cancer tissues was examined using a quantitative reverse transcription polymerase chain reaction.PCD1 mRNA expression was greater in cancers than in noncancerous tissues (p0.0001). In addition, high PCD1 gene expression was more frequent in patients with lymph node metastasis.PCD1 appears to contribute to breast cancer progression and nodal metastasis, thus representing a potential target molecule in breast cancer diagnosis and treatment.

The recently identified aspartate protease gene ALP56 is up-regulated in human malignant tumors, including colorectal cancers, but the relationship remain unclear between ALP56 gene expression and clinicopathological findings, as well as when genetic alterations in ALP56 occur during the colorectal adenoma-carcinoma sequence. We therefore investigated expression of ALP56 mRNA in various human colorectal tissues.We examined 18 colorectal adenomas 22 cancers, and 24 adjacent normal mucosal samples from patients undergoing conventional resection or endoscopic mucosal resection. Expression of ALP56 mRNA was determined by quantitative reverse-transcription polymerase chain reaction.Up-regulation of ALP56 gene transcription was observed in both adenomas and cancers compared to normal mucosa. ALP56 expression in exophytic adenomas was significantly greater than in flat adenomas.ALP56 may contribute to colorectal adenoma formation and to an exophytic growth pattern in these adenomas.

The process of colorectal cancer development involves accumulated genetic alterations affecting APC, K-ras and p53. A recently identified gene, PCD1, was reported to be up-regulated in human malignancies including colorectal cancers, but relationships between PCD1 gene expression and clinicopathological findings, as well as the timing of genetic alteration of PCD1 in colorectal cancer development, are not clear. To determine whether PCD1 contributes to colorectal cancer progression, we investigated the expression of PCD1 mRNA in human colorectal tissues.The expression of PCD1 mRNA was determined by quantitative RT-PCR. The mutation of p53 was detected by a PCR-SSCP method.Up-regulation of PCD1 gene transcription was observed not in adenomas but in cancers compared to normal mucosa (p0.0001). Primary tumors with a mutation of p53 showed significantly greater PCD1 gene expression than tumors without such a mutation (p = 0.0134).The PCD1 gene may play a role in colorectal cancer development from adenomas.

GAPDH, beta-actin and 18S rRNA are widely employed as internal control genes, with the assumption that they are expressed constitutively to similar degrees in different cells and tissues and under different experimental conditions. In this study, we tested this assumption by assessment of the transcription of these three genes in human colonic tissues using a quantitative RT-PCR.GAPDH transcription was significantly greater in both colonic adenomas and cancers than in normal mucosa. In addition, transcription of beta-actin was significantly increased in cancers. The expression of 18S rRNA was essentially constant among these various tissues. Stable expression of 18S rRNA was observed during the growth of colonic cancer cells stimulated with serum, but both GAPDH and beta-actin transcription were up-regulated, coinciding with cell proliferation.These results indicate that 18S rRNA is more reliable than GAPDH and beta-actin as an internal control gene for quantitative comparison of mRNA in colonic cancers.