Laparoscopy-Assisted Restorative Proctocolectomy with Ileal Pouch-Anal Anastomosis in Middle Colic Artery Ligation Immediately before Specimen Removal.

Introduction: Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgical procedure for ulcerative colitis (UC). Intestinal ischemia may occur if the main blood vessels are ligated at an early stage of this surgery. Considering that the blood flow in the large intestine can be maintained by preserving the middle colic artery, we have used a new IPAA method: ligating the middle colic artery immediately before removal of the specimens ("M-method"). Here, we evaluated the M-method's clinical outcomes. Methods: Between April 2009 and December 2021, 13 patients underwent a laparoscopy-assisted IPAA procedure at our institution. The conventional method was used for 6 patients, and the M-method was used for the other 7 patients. We retrospectively analyzed the cases' clinical notes. Results: The M-method's rate of postoperative complications (Clavien-Dindo classification grade II or more) was significantly lower than that of the conventional method (14.2% vs. 83.3%). The M-method group's postoperative stay period was also significantly shorter (average 16.4 days vs. 55.5). There were significant differences in the albumin value and the ratio of the modified GPS score 1 or 2 on the 7th postoperative day between the M- and conventional methods (average 3.15 vs. 2.5, average 4/7 vs. 6/6). However, it is necessary to consider the small number of cases and the uncontrolled historical comparison. Conclusion: Late ligation of the middle colic artery may be beneficial for patients' post-surgery recovery and can be recommended for IPAAs in UC patients.

The first case of laparoscopic surgery for cecal cancer occurring in a blind loop.

Cancer occurrence in a blind loop is extremely rare. An 86-year-old Japanese woman underwent colonoscopy for tarry stools and weight loss; it revealed a bypass of the transverse colon and small intestine, cecal cancer, and a polyp. She had suffered from acute appendicitis and had undergone two surgeries at age 25: an appendectomy and then a bypass surgery between the transverse colon and the small intestine. We performed a laparoscopy-assisted ileocecal resection for the cancer and polyp in the blind loop with an end-to-side instrumental anastomosis. The pathological examination demonstrated that the cancer was medullary carcinoma (T2, N0, M0, Stage I) and the polyp was tubular adenoma. Two months have passed since the patient's discharge, and she is free of abdominal complaints. Our literature search identified 10 cases of cancer in a blind loop. Laparoscopy-assisted surgery may be possible in patients who have undergone blind-loop surgery.

Prognostic Value of KRAS Exon-specific Mutations in Patients With Colorectal Cancer.

BACKGROUND/AIM: The clinical significance of many RAS-family mutations in colorectal cancer (CRC) remains unclear. The purpose of this study was to investigate the relationship of RAS mutations on an exon basis (i.e., mutations in KRAS exons 2, 3, and 4 and in NRAS) with clinicopathological features and prognosis in CRC. PATIENTS AND METHODS: We performed a retrospective cohort study of the medical records and frozen tissue samples of 268 consecutive patients with stage I-III CRC who underwent curative resection at a single institution between 2014 and 2018. RESULTS: The RAS mutation rate was significantly associated with age and histology. Patients with KRAS exon 2 mutations exhibited shorter recurrence-free survival compared to those with KRAS wild-type, KRAS exon 3 mutations, KRAS exon 4 mutations, and NRAS mutations (73.0% vs. 85.5%, 86.7%, 85.7%; p=0.031). Age and histology were independent risk factors for RAS mutations. RAS mutations were independent prognostic factors with respect to recurrence-free survival in patients with stage I-III CRC. CONCLUSION: In stage I-III CRC patients, KRAS exon 2 mutations had the worst prognosis, whereas KRAS wild type, exon 3 mutations, exon 4 mutations, and NRAS mutations had better prognoses.

A Non-Invasive Deep Photoablation Technique to Inhibit DCIS Progression and Induce Antitumor Immunity.

Ductal carcinoma in situ (DCIS) of the breast is often managed by lumpectomy and radiation or mastectomy, despite its indolent features. Effective non-invasive treatment strategies could reduce the morbidity of DCIS treatment. We have exploited the high heat shock protein 90 (HSP90) activity in premalignant and malignant breast disease to non-invasively detect and selectively ablate tumors using photodynamic therapy (PDT). PDT with the HSP90-targeting photosensitizer, HS201, can not only ablate invasive breast cancers (BCs) while sparing non-tumor tissue, but also induce antitumor immunity. We hypothesized that HS201-PDT would both non-invasively ablate DCIS and prevent progression to invasive BC. We tested in vitro selective uptake and photosensitivity of HS201 in DCIS cell lines compared to the non-selective parental verteporfin, and assessed in vivo antitumor efficacy in mammary fat pad and intraductal implantation models. Selective uptake of HS201 enabled treatment of intraductal lesions while minimizing toxicity to non-tumor tissue. The in vivo activity of HS201-PDT was also tested in female MMTV-neu mice prior to the development of spontaneous invasive BC. Mice aged 5 months were administered HS201, and their mammary glands were exposed to laser light. HS201-PDT delayed the emergence of invasive BC, significantly prolonged disease-free survival (DFS) (p = 0.0328) and tended to improve overall survival compared to the no-treatment control (p = 0.0872). Systemic administration of anti-PD-L1 was combined with HS201-PDT and was tested in a more aggressive spontaneous tumor model, HER2delta16 transgenic mice. A single PDT dose combined with anti-PD-L1 improved DFS compared to the no-treatment control, which was significantly improved with repetitive HS201-PDT given with anti-PD-L1 (p = 0.0319). In conclusion, a non-invasive, skin- and tissue-sparing PDT strategy in combination with anti-PD-L1 antibodies effectively prevented malignant progression of DCIS to invasive BC. This non-invasive treatment strategy of DCIS may be safe and effective, while providing an option to reduce the morbidity of current conventional treatment for patients with DCIS. Clinical testing of HS201 is currently underway.

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers.

BACKGROUND: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined. METHODS: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved. RESULTS: HS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells. CONCLUSIONS: The increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity.

BACKGROUND: Despite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment systemic antitumor immunity, in part by appropriately presenting tumor antigens, but also by modulating the immunosuppressive tumor microenvironment (TME). We previously validated this concept in models of murine carcinoma treated with a novel predominately microcavitating version of high-intensity focused ultrasound (HIFU), mechanical high-intensity focused ultrasound (M-HIFU). Here we elucidated the mechanisms of enhanced antitumor immunity by M-HIFU over conventional thermal high-intensity focused ultrasound (T-HIFU) and investigated the potential of the combinatorial strategy with an immune checkpoint inhibitor, anti-PD-L1 antibody. METHODS: The antitumor efficacy of treatments was investigated in syngeneic murine breast cancer models using triple-negative (E0771) or human ErbB-2 (HER2) expressing (MM3MG-HER2) tumors in C57BL/6 or BALB/c mice, respectively. Induction of systemic antitumor immunity by the treatments was tested using bilateral tumor implantation models. Flow cytometry, immunohistochemistry, and single-cell RNA sequencing were performed to elucidate detailed effects of HIFU treatments or combination treatment on TME, including the activation status of CD8 T cells and polarization of tumor-associated macrophages (TAMs). RESULTS: More potent systemic antitumor immunity and tumor growth suppression were induced by M-HIFU compared with T-HIFU. Molecular characterization of the TME after M-HIFU by single-cell RNA sequencing demonstrated repolarization of TAM to the immunostimulatory M1 subtype compared with TME post-T-HIFU. Concurrent anti-PD-L1 antibody administration or depletion of CD4+ T cells containing a population of regulatory T cells markedly increased T cell-mediated antitumor immunity and tumor growth suppression at distant, untreated tumor sites in M-HIFU treated mice compared with M-HIFU monotherapy. CD8 T and natural killer cells played major roles as effector cells in the combination treatment. CONCLUSIONS: Physical disruption of the TME by M-HIFU repolarizes TAM, enhances T-cell infiltration, and, when combined with anti-PD-L1 antibody, mediates superior systemic antitumor immune responses and distant tumor growth suppression. These findings suggest M-HIFU combined with anti-PD-L1 may be useful in reducing late recurrence or metastasis when applied to primary tumors.

HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study.

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Heterogenous chemosensitivity of a panel of organoid lines derived from small cell neuroendocrine carcinoma of the uterine cervix.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare disease with a poor prognosis. The lack of established disease models has hampered therapy development. We generated a panel of cancer tissue-originated spheroid (CTOS) lines derived from SCNEC of the uterine cervix using a method based upon cell-cell contact throughout the preparation and culturing processes. Using 11 CTOS lines, we assessed the sensitivity of various drugs used in clinical practice. Drug sensitivity assays revealed significant heterogeneous inter-CTOS chemosensitivity. Microarray analyses were then performed to identify sensitivity-related gene signatures. Specific gene sets were identified which likely contribute to the sensitivity to the tested drugs. We identified a line (Cerv54) that was exceptionally sensitive to irinotecan. Cerv54 had increased levels of CES1, which catalyzes the conversion of irinotecan to the active form, SN38, although in Cerv54 cells, SN38 was undetectable, CES1 expression and activity were markedly low compared to the liver, and a CES1 inhibitor had no effect on irinotecan sensitivity. These results suggested a novel irinotecan mode of action in Cerv54. Our CTOS lines may be useful for understanding the variation and mechanism of drug sensitivity, contributing to the understanding and development of chemotherapeutic drugs.

Heat shock protein 90-targeted photodynamic therapy enables treatment of subcutaneous and visceral tumors.

Photodynamic therapy (PDT) ablates malignancies by applying focused near-infrared (nIR) light onto a lesion of interest after systemic administration of a photosensitizer (PS); however, the accumulation of existing PS is not tumor-exclusive. We developed a tumor-localizing strategy for PDT, exploiting the high expression of heat shock protein 90 (Hsp90) in cancer cells to retain high concentrations of PS by tethering a small molecule Hsp90 inhibitor to a PS (verteporfin, VP) to create an Hsp90-targeted PS (HS201). HS201 accumulates to a greater extent than VP in breast cancer cells both in vitro and in vivo, resulting in increased treatment efficacy of HS201-PDT in various human breast cancer xenografts regardless of molecular and clinical subtypes. The therapeutic index achieved with Hsp90-targeted PDT would permit treatment not only of localized tumors, but also more diffusely infiltrating processes such as inflammatory breast cancer.

In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531-42. ©2017 AACR.

Vaccination targeting human HER3 alters the phenotype of infiltrating T cells and responses to immune checkpoint inhibition.

Expression of human epidermal growth factor family member 3 (HER3), a critical heterodimerization partner with EGFR and HER2, promotes more aggressive biology in breast and other epithelial malignancies. As such, inhibiting HER3 could have broad applicability to the treatment of EGFR- and HER2-driven tumors. Although lack of a functional kinase domain limits the use of receptor tyrosine kinase inhibitors, HER3 contains antigenic targets for T cells and antibodies. Using novel human HER3 transgenic mouse models of breast cancer, we demonstrate that immunization with recombinant adenoviral vectors encoding full length human HER3 (Ad-HER3-FL) induces HER3-specific T cells and antibodies, alters the T cell infiltrate in tumors, and influences responses to immune checkpoint inhibitions. Both preventative and therapeutic Ad-HER3-FL immunization delayed tumor growth but were associated with both intratumoral PD-1 expressing CD8+ T cells and regulatory CD4+ T cell infiltrates. Immune checkpoint inhibition with either anti-PD-1 or anti-PD-L1 antibodies increased intratumoral CD8+ T cell infiltration and eliminated tumor following preventive vaccination with Ad-HER3-FL vaccine. The combination of dual PD-1/PD-L1 and CTLA4 blockade slowed the growth of tumor in response to Ad-HER3-FL in the therapeutic model. We conclude that HER3-targeting vaccines activate HER3-specific T cells and induce anti-HER3 specific antibodies, which alters the intratumoral T cell infiltrate and responses to immune checkpoint inhibition.

Clinical significance of mucinous components in rectal cancer after preoperative chemoradiotherapy.

PURPOSE: The clinical implications of mucinous components in rectal tumors, especially with regard to the efficacy of neoadjuvant chemoradiotherapy, remain unclear. METHODS: One hundred and thirty rectal cancer patients who received curative resection after neoadjuvant chemoradiotherapy were retrospectively reviewed. Patients were classified into 3 groups according to the proportion of extracellular mucin: low (<5 %), moderate (5-25 %), and high (>25 %). RESULTS: There were 82 (63.1 %), 26 (20.0 %), and 22 (16.9 %) patients in the low, moderate, and high mucin groups, respectively. Patients with a high mucinous tumor component were significantly more likely to have an advanced tumor stage (p = 0.010) and a shorter disease-free (p = 0.002) and distant recurrence-free survivals (p < 0.001), whereas the mucinous tumor component showed no correlation with local recurrence (p = 0.101). A high mucinous component was also an independent predictive factor for a shorter disease-free survival (p = 0.041, hazard ratio = 2.56) and distant recurrence-free survival (p = 0.001, hazard ratio = 5.74) according to a multivariate analysis. CONCLUSIONS: Because the mucinous components showed little correlation with local recurrence, mucinous cancer should not be a determining factor for chemoradiotherapy. However, the frequent occurrence of metachronous distant metastasis among patients with a high mucin component makes this a possible indicator for more robust postoperative adjuvant treatment and close surveillance of recurrence.

Perioperative Allogeneic Blood Transfusion Is Associated With Surgical Site Infection After Abdominoperineal Resection-a Space for the Implementation of Patient Blood Management Strategies.

Allogeneic blood transfusion (ABT) has been reported as a major risk factor for surgical site infection (SSI) in patients undergoing colorectal surgery. However, the association of ABT with SSI in patients undergoing abdominoperineal resection (APR) and total pelvic exenteration (TPE) still remains to be evaluated. Here, we aim to elucidate this association. The medical records of all patients undergoing APR and TPE at our institution in the period between January 2000 and December 2012 were reviewed. Patients without SSI (no SSI group) were compared with patients who developed SSI (SSI group), in terms of clinicopathologic features, including ABT. In addition, data for 262 patients who underwent transabdominal rectal resection at our institution in the same period were also enrolled, and their data on differential leukocyte counts were evaluated. Multivariate analysis showed that intraoperative transfusion was an independent predictive factor for SSI after APR and TPE (P = 0.004). In addition, the first-operative day lymphocyte count of patients undergoing APR, TPE, and transabdominal rectal resection was significantly higher in nontransfusion patients compared with transfusion ones (P = 0.026). ABT in the perioperative period of APR and TPE may have an important immunomodulatory effect, leading to an increased incidence of SSI. This fact should be carefully considered, and efforts to avoid allogeneic blood exposure while still achieving adequate patient blood management would be very important for patients undergoing APR and TPE as well.

Lysophosphatidylserine stimulates chemotactic migration of colorectal cancer cells through GPR34 and PI3K/Akt pathway.

BACKGROUND: Lysophosphatidylserine (lysoPS) is a type of lysophospholipid mediator, which is involved in allergic conditions and tumor progression. We investigated the physiological function of lysoPS on colorectal cancer (CRC) cell lines, as well as the involved receptor and signaling pathways. MATERIALS AND METHODS: Expression of lysoPS receptors on six cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). The physiological functions of lysoPS were investigated, and experiments using small interfering RNA (siRNA) or inhibitors of the signaling pathways were conducted. RESULTS: Among the three lysoPS receptors, GPR34 was highly expressed on all cell lines. LysoPS stimulated the chemotactic migratory ability. Wortmannin inhibited the migratory ability, as well as the GPR34 knock-down, strongly suggestive of the involvement of this receptor in the PI3K/Akt pathway. CONCLUSION: The involved receptor and pathways in the migratory ability in response to lysoPS was demonstrated, which opens premises for targeting as a new strategy for prevention and treatment of colorectal cancer.

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus.

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Changes in the expression of pituitary gonadotropin subunits during reproductive cycle of multiple spawning female chub mackerel Scomber japonicus.

The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHß, and LHß) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHß, and LHß were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHß mRNA levels remained high during the vitellogenic stages, while GPα and LHß mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHß mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHß mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.