Long-term survival and functional outcomes of laparoscopic surgery for clinical stage I ultra-low rectal cancers located within 5 cm of the anal verge: A prospective phase II trial (Ultimate trial).

OBJECTIVE: To clarify the long-term oncological outcomes and postoperative anal, urinary, and sexual functions after laparoscopic surgery for clinical stage I very low rectal carcinoma located near the anal canal. SUMMARY BACKGROUND DATA: Laparoscopic surgery is widely applied for rectal cancer; however, concerns remain, with some studies showing poorer outcomes compared to open surgery. METHODS: This single-arm, phase II trial included patients registered preoperatively from 47 institutions in Japan. The planned sample size was 300. The primary endpoint was the 3-year local recurrence rate. Anal, urinary, and sexual functions were evaluated using a prospective questionnaire. RESULTS: Three-hundred patients were registered between January 2014 and March 2017. Anus-preserving surgery was performed in 278 (93%), including 172 who underwent intersphincteric resection (58%) and 106 (36%) who underwent low anterior resection. The 3-year cumulative local recurrence rate was 6.3%. At 3 years postoperatively, 87% of patients used their own anus, and the median incontinence score improved from 12 at 3 months to 8 at 3 years. Only 5% of patients had severe incontinence (incontinence score of 16 points). Postoperative urinary function evaluation showed that International Prostate Symptom Score and Overactive Bladder Symptom Score decreased 1 week after surgery, but recovered to preoperative level 1 month after surgery. International Consultation on Incontinence Questionnaire-Sort Form remained almost stable after surgery. Sexual function evaluation using the International Index of Erectile Function-5 and International Index of Erectile Function-15 revealed that the patients had deteriorated 3 months after surgery but had recovered only slightly by 6 months. CONCLUSIONS: Laparoscopic surgery achieves feasible long-term oncological outcomes and a high rate of anus preservation with moderate anal function, and an acceptable incontinence score. While urinary function recovered rapidly, sexual function showed poor recovery.

Incidence of low anterior resection syndrome and its association with the quality of life in patients with lower rectal tumors.

PURPOSE: Low anterior resection syndrome (LARS) causes devastating symptoms and impairs the quality of life (QOL). This study investigated the incidence and risk factors of LARS and their association with the QOL in patients with lower rectal tumors. METHODS: Patients who underwent anus-preserving surgery for lower rectal tumors between 2014 and 2019 and who had anal defecation between 2020 and 2021 were surveyed. The LARS score measured severity, and the QOL was evaluated using the Japanese version of the Fecal Incontinence Quality-of-Life Scale (JFIQL). The primary endpoint was the incidence of Major LARS, and the secondary endpoints were risk factors and association with the JFIQL. RESULTS: Of 107 eligible patients, 82 (76.6%) completed the LARS survey. The incidence of Major LARS was 48%. Independent risk factors included neoadjuvant chemoradiotherapy (CRT) and a short interval (< 24 months after surgery; odds ratio, 4.6; 95% confidence interval: 1.1-19, both). The LARS score was moderately correlated with the JFIQL generic score (correlation coefficient: - 0.54). The JFIQL scores were significantly worse in the Minor and Major LARS groups than in the No LARS group. CONCLUSIONS: Major LARS was found in 48% of lower rectal tumors, and independent risk factors include neoadjuvant CRT and a short interval. The QOL was significantly impaired in patients with both Minor and Major LARS.

A Low Mean Closing Load and a Decrease in Load Change at the Tip Increase the Comfort of Scissors.

Introduction During surgery, surgeons intuitively recognize when they are using dull scissors and find them difficult to use. The purpose of this study was to objectively evaluate the physical characteristics of scissors and the comfort reported by surgeons to develop objective quality control standards for scissors used in surgery. Methods Sensory and measurement tests were conducted to evaluate the comfort and physical characteristics of ten pairs of Cooper scissors. As a sensory test, thirty-one volunteer surgeons opened and closed the scissors and selected three that felt comfortable and three that were uncomfortable. The results were scored. For measurement, a load was applied to the handle of the scissors. The load pressure and displacement of the width between each handle when the scissors were closed were measured. Results A strong negative correlation was found between the total comfort score and the mean load value between sensory and measurement tests (r=-0.717, p=0.0195). The correlation between the total score and the change in load at the tip showed a moderate negative correlation (r=-0.687, p=0.0282). Multiple regression analysis showed that the change in load at the tip was an independent factor affecting the total score. Conclusions Surgeons consider scissors with a low mean load required to close the scissors and a small change in load at the tip to be comfortable. The mean load on scissors and the change in load at the tip should be considered in the development of quality control standards for scissors used in surgery.

Risk factors for anastomotic leakage after low anterior resection for obese patients with rectal cancer.

PURPOSE: We aimed to analyze the risk factors for anastomotic leakage (AL) after low anterior resection (LAR) in obese patients (body mass index [BMI] ≥ 25 kg/m2) with rectal cancer. METHODS: Data were collected from four hundred two obese patients who underwent LAR for rectal cancer in 51 institutions. RESULTS: Forty-six (11.4%) patients had clinical AL. The median BMI (27 kg/m2) did not differ between the AL and non-AL groups. In the AL group, comorbid respiratory disease was more common (p = 0.025), and the median tumor size was larger (p = 0.002). The incidence of AL was 11.5% in the open surgery subgroup and 11.4% in the laparoscopic surgery subgroup. Among the patients who underwent open surgery, the AL group showed a male predominance (p = 0.04) in the univariate analysis, but it was not statistically significant in the multivariate analysis. Among the patients who underwent laparoscopic surgery, the AL group included a higher proportion of patients with comorbid respiratory disease (p = 0.003) and larger tumors (p = 0.007). CONCLUSION: Comorbid respiratory disease and tumor size were risk factors for AL in obese patients with rectal cancer. Careful perioperative respiratory management and appropriate selection of surgical procedures are required for obese rectal cancer patients with respiratory diseases.

Low anterior resection syndrome: Incidence and association with quality of life.

Aim: Low anterior resection syndrome (LARS) causes devastating symptoms and impairs quality of life (QOL). Although its incidence and risk factors have been reported, these data are scarce in Japan. This study aimed to elucidate the incidence and risk factors of LARS as well as to evaluate its association with QOL in Japanese patients. Method: Patients with anal defecation at the time of the survey between November 2020 and April 2021 were included, among those who underwent anus-preserving surgery for rectal tumors between 2014 and 2019 in tertiary referral university hospital. The severity of LARS and QOL were evaluated with the LARS score and the Japanese version of the fecal incontinence quality of life scale (JFIQL), respectively. Primary endpoint was the incidence of major LARS. Secondary endpoints were risk factors and association with JFIQL. Results: Of 332 eligible patients, 238 (71.7%) answered the LARS survey completely. The incidence of major LARS was 22% overall, and 48% when limited to lower tumors. Independent risk factors included lower tumors (OR: 7.0, 95% CI: 2.1-23.1, p = 0.001) and surgical procedures with lower anastomoses (OR: 4.6, 95% CI: 1.2-18.5, p = 0.03). The JFIQL generic score correlated moderately with the LARS score (correlation coefficient of -0.65). The JFIQL generic score was also significantly lower in lower tumors. Conclusions: The incidence of major LARS is 22% in Japanese patients, and independent risk factors include lower tumors and surgical procedures with lower anastomoses. More severe LARS is associated with worse QOL which is significantly more impaired in patients with lower tumors.

Impact of Preoperative Stoma Site Marking on Morbidity and Mortality in Patients with Colorectal Perforation: A Nationwide Retrospective Cohort Study.

BACKGROUND: Preoperative stoma site marking reduces the incidence of complications from elective surgery. However, the impact of stoma site marking in emergency patients with colorectal perforation remains unclear. This study aimed to assess the impact of stoma site marking on morbidity and mortality in patients with colorectal perforation who underwent emergency surgery. METHODS: This retrospective cohort study used the Japanese Diagnosis Procedure Combination inpatient database from April 1, 2012, to March 31, 2020. We identified patients who underwent emergency surgery for colorectal perforation. We compared outcomes between those with and without stoma site marking using propensity score matching to adjust for confounding factors. The primary outcome was the overall complication rate, and the secondary outcomes were stoma-related, surgical, and medical complications and 30-day mortality. RESULTS: We identified 21,153 patients (682 with stoma site marking and 20,471 without stoma site marking) and grouped them into 682 pairs using propensity score matching. The overall complication rates were 23.5% and 21.4% in the groups with and without stoma site marking, respectively (p = 0.40). Stoma site marking was not associated with a decrease in stoma-related, surgical, or medical complications. The 30-day mortality did not differ significantly between the groups with and without stoma site marking (7.9% vs. 8.4%, p = 0.843). CONCLUSIONS: Preoperative stoma site marking was not associated with a reduction in morbidity and mortality in patients with colorectal perforation who underwent emergency surgery.

Peripheral low-density granulocytes after colorectal cancer surgery in predicting recurrence.

BACKGROUND: Low-density granulocytes (LDGs) have been shown to be increased in the peripheral blood of patients with inflammatory and malignant diseases. This study evaluated LDGs in patients who underwent radical surgery for colorectal cancer (CRC) and their impact on survival. METHODS: Patients who underwent radical colectomy between 2017 to 2021 were screened for enrolment in the study. Peripheral blood was obtained in the operating room before and after surgery and cells were recovered from the mononuclear layer after density gradient preparations. The ratio of CD66b(+) LDG to CD45(+) leukocytes was determined with flow cytometry, and the association of the ratios with patient outcomes was examined. The main outcome of interest was recurrence-free survival (RFS). RESULTS: Out of 228 patients treated, 176 were enrolled, including 108 colonic and 68 rectal cancers. Overall, 38 patients were stage I, 30 were stage II, 72 were stage 3, and 36 were stage IV. The number of LDGs was markedly increased immediately after surgery and the proportion of LDGs correlated positively with operating time (r = 0.2806, P < 0.001) and intraoperative blood loss (r = 0.1838, P = 0.014). Purified LDGs produced high amounts of neutrophil extracellular traps after short-term culture and efficiently trapped tumour cells in vitro. The proportion of postoperative LDGs was significantly higher in 13 patients who developed recurrence (median 9 (range 1.63-47.0)) per cent versus median 2.93 ((range 0.035-59.45) per cent, P = 0.013). When cut-off values were set at 4.9 per cent, a higher proportion of LDGs was strongly and independently associated with decreased RFS (P = 0.005). In patients with stage III disease, adjuvant chemotherapy significantly improved RFS of patients with high ratios of LDGs, but not low LDGs. CONCLUSION: LDGs are recruited to circulating blood by surgical stress early in the postoperative interval after colectomy for colonic cancer and their postoperative proportion is correlated with recurrence.

The effect of staple height and rectal-wall thickness on anastomotic leakage after laparoscopic low anterior resection.

BACKGROUND: The aim of this study was to evaluate the effect of staple height and rectal wall thickness on the development of an anastomotic leak after laparoscopic low anterior resection performed with the double stapling technique. METHODS: One hundred ninety-nine patients treated from 2013 to 2021 were enrolled. Patients were divided into two groups: those who developed an anastomotic leak (AL (+)) and those who did not (AL (-)). Clinicopathological factors were compared between the groups. RESULTS: Anastomotic leaks were observed in 8/199 patients (4%). A 1.5 mm linear stapler was used for 35/199 patients (17%), 1.8 mm for 89 (45%), and 2 mm for 75 (38%). In the AL (+) group (n = 8), lower staple height (1.5 mm or 1.8 mm) was used more frequently than in the AL (-) group (n = 191). Rectal wall thickness and the rectal wall thickness to staple height ratio was significantly (p < .05) greater in the AL (+) group. However, rectal wall thickness was significantly (p < .05) greater in patients who received neoadjuvant treatment and those with advanced T stage (T3,4) lesions. CONCLUSION: Linear stapler staple height and rectal wall thickness are significantly associated with the development of an anastomotic leak after laparoscopic low anterior resection. Larger staples should be selected in patients with a thicker rectal wall due to neoadjuvant treatment or adjacent advanced rectal tumors.

Idiopathic megacolon complicated by life-threatening giant megacolon and respiratory failure due to diaphragmatic eventration: A case report.

INTRODUCTION AND IMPORTANCE: Giant megacolon requiring emergency surgery is rare. Eventration of the diaphragm associated with giant megacolon is also uncommon. CASE PRESENTATION: We report a 66-year-old male who presented with abdominal distention and progressive dyspnea. After resuscitation following cardiopulmonary arrest, the patient underwent emergent subtotal abdominal colectomy. Eventration of the diaphragm was found postoperatively and his respiratory condition was insufficient to allow liberation. Plication of both diaphragms was performed through left and right thoracotomy via the 8th intercostal space. Postoperatively the patient made a full recovery. CLINICAL DISCUSSIONS: Chronic constipation is a common health condition. A life-threatening condition secondary to chronic constipation is a rarely documented complication. Diaphragmatic eventration that was caused due to chronic megacolon in symptomatic patients requires surgical treatment. CONCLUSIONS: We describe a patient with giant megacolon and diaphragmatic eventration secondary to idiopathic megacolon. The patient underwent subtotal colectomy and diaphragmatic plication and recovered fully.

Metformin combined with local irradiation provokes abscopal effects in a murine rectal cancer model.

Although preoperative chemoradiation therapy can down-stage locally advanced rectal cancer (LARC), it has little effect on distant metastases. Metformin exerts an anti-cancer effect partly through the activation of host immunity. LuM1, a highly lung metastatic subclone of colon 26, was injected subcutaneously (sc) in BALB/c mice and treated with metformin and/or local radiation (RT). Lung metastases and the primary tumors were evaluated and the phenotypes of immune cells in the spleen and lung metastases were examined with flow cytometry and immunohistochemistry. Local RT, but not metformin, partially delayed the growth of sc tumor which was augmented with metformin. Lung metastases were unchanged in metformin or RT alone, but significantly reduced in the combined therapy. The ratios of splenic T cells tended to be low in the RT group, which were increased by the addition of metformin. IFN-γ production of the splenic CD4(+) and CD8(+) T cells was enhanced and CD49b (+) CD335(+) activated NK cells was increased after combined treatment group. Density of NK cells infiltrating in lung metastases was increased after combination treatment. Metformin effectively enhances local and abscopal effects of RT though the activation of cell-mediated immunity and might be clinically useful for LARC.

Programmed cell death ligand 1 expression on monocytes is inversely correlated with tumour response to preoperative chemoradiotherapy for locally advanced rectal cancer.

AIM: The clinical efficacy of chemoradiotherapy (CRT) is largely dependent on host immune status. The aim of this study was to identify possible markers expressed on circulating mononuclear cells to predict tumour response in patients with locally advanced rectal cancer (LARC). METHODS: Peripheral blood samples were obtained from 47 patients diagnosed with LARC before and after CRT. The numbers of lymphocytes and monocyte subsets were analysed using flow cytometry. Based on clinical and pathological findings, patients were classified as high or low responders. RESULTS: Lymphocyte counts were markedly decreased after CRT. Total numbers of lymphocytes (p = 0.030) and CD4(+) T cells (p = 0.041) in post-CRT samples were significantly lower in low responders than in high responders. In contrast, monocyte counts were not reduced and the number of CD14dim (+) CD16(+) nonclassical (patrolling) monocytes were somewhat increased after CRT (p = 0.050). Moreover, the ratios of programmed cell death ligand 1 (PD-L1) (+) cells on patrolling monocytes before and after CRT were significantly higher in low responders than in high responders (p = 0.0046, p = 0.0006). The same trend was observed for classical and intermediate monocytes. The expression of PD-L1 on patrolling monocytes before CRT correlated inversely with the number of T cells and natural killer (NK) cells after CRT. PD-L1(+) ratio in patrolling monocytes was an independent predictor for response to CRT. CONCLUSION: Programmed cell death ligand 1 (PD-L1) expression on patrolling monocytes suppresses cell-mediated immunity in patients receiving CRT which could be related to tumour response, and may be a useful biomarker for decision-making in the management of patients with LARC.

The Regional Specificity of Mucosa-Associated Microbiota in Patients with Distal Colorectal Cancer.

BACKGROUND/AIMS: Recent studies have demonstrated that the populations of several microbes are significantly increased in fecal samples from patients with colorectal cancer (CRC), suggesting their involvement in the development of CRC. The aim of this study was to identify microbes which are increased in distal CRCs and to identify the specific location of microbes increased in mucosal tissue around the tumor. METHODS: Tissue specimens were collected from surgical resections of 28 distal CRCs. Five samples were collected from each specimen (location A: tumor, B: adjacent normal mucosa, C: normal mucosa 1 cm proximal to the tumor, D: normal mucosa 3 cm proximally, and E: normal mucosa 6 cm proximally). The microbiota in the sample were analyzed using 16S rRNA gene amplicon sequencing and the relative abundance (RA) of microbiota compared among the 5 locations. RESULTS: At the genus level, the RA of Fusobacterium and Streptococcus at location A was the highest among the 5 locations, significantly different from that in location E. The dominant species of each genus was Fusobacterium nucleatum and Streptococcus anginosus. The RAs of these species gradually decreased from locations B to E with a statistically significant difference in F. nucleatum. The genus Peptostreptococcus also showed a similar trend, and the RA of Peptostreptococcus stomatis in location A was significantly associated with depth of tumor invasion and tumor size. CONCLUSION: Although the clinical relevance is not clear yet, these results suggest that F. nucleatum, S. anginosus, and P. stomatis can spread to the adjacent normal tissues and may change the surrounding microenvironment to support the progression of CRC.

The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

Metachronous Krukenberg tumor from adenocarcinoma in a Meckel's diverticulum: a case report.

Adenocarcinoma in a Meckel's diverticulum is rare and difficult to diagnose preoperatively. We report the first case of a metachronous Krukenberg tumor from adenocarcinoma in a Meckel's diverticulum. A 45-year-old woman was admitted for recurrent abdominal pain. Computed tomography scan showed a lesion with contrast enhancement, and a Meckel's diverticulum-associated tumor was suspected. Double-ballon enteroscopy revealed intestinal stenosis and biopsy showed adenocarcinoma. Operative findings showed a Meckel's diverticulum with tumor. Histopathological evaluation revealed well-differentiated adenocarcinoma, interrupted by ectopic gastric mucosa, diagnosed as adenocarcinoma in a Meckel's diverticulum. Two years postoperatively, a multi-cystic mass with contrast enhancement was observed in the pelvis on imaging evaluation and oophorectomy performed. Histological examination of the resected ovary showed proliferation of atypical glandular ducts, consistent with metastatic adenocarcinoma. This case demonstrates that adenocarcinoma in a Meckel's diverticulum may result in distant metastases and requires appropriate follow-up.

Metachronous common iliac lymph node metastasis after rectosigmoid colon cancer resection: A case report.

INTRODUCTION AND IMPORTANCE: Metastases to common iliac lymph nodes from cancer of the rectosigmoid are extremely rare. We report a patient with a right common iliac lymph node metastasis after rectosigmoid cancer resection. CASE PRESENTATION: The patient is a 57-year-old woman diagnosed with rectosigmoid cancer (Stage IIIc) who underwent laparoscopic resection followed by 8 courses of adjuvant chemotherapy with capecitabine. Sixteen months after resection, an intra-abdominal mass and a left lung nodule were found on computed tomography scans, which were suspected to be recurrences. Exploratory laparoscopy showed that the abdominal lesion was an enlarged common iliac lymph node, which was completely excised. No other intraabdominal recurrences were found. Subsequently, a left upper lobe lung metastasis was resected thoracoscopically. However, multiple lung metastases developed four months after the lung resection, and systemic therapy was begun. CLINICAL DISCUSSION: A lower incidence of lateral lymph node metastases from cancer in the rectosigmoid has been reported. Direct lymphatic pathways from the sigmoid colon or rectosigmoid to lateral lymph nodes have been suspected, which may be associated with the poor prognosis in this patient. CONCLUSION: A metachronous metastasis to a common iliac lymph node from primary rectosigmoid cancer is reported. Common iliac lymph node metastases from rectosigmoid cancer might have more malignant potential, and should be treated in the same manner as peri-aortic lymph node metastases.

Clinical predictors of gangrenous appendicitis: elevated total bilirubin level and computed tomography scan findings.

AIM: Patients with gangrenous appendicitis usually require emergency surgery. Preoperative diagnosis of gangrenous appendicitis is clinically important but not always straightforward. We undertook this study to identify preoperative predictors of gangrenous appendicitis. METHODS: This was a single-center case-control study. We identified 162 patients who underwent appendectomy between September 2011 and August 2014 after the diagnosis of acute appendicitis was established. We identified laboratory parameters and computed tomography (CT) scan findings predictive of histologically or surgically diagnosed gangrenous appendicitis by univariable and multivariable analyses. RESULTS: Of 146 study patients, gangrenous appendicitis was confirmed in 102. Univariable analysis showed that two laboratory factors (C-reactive protein []and total bilirubin [T-Bil]) and three CT scan findings were significant predictors for gangrenous appendicitis. Multivariable analysis showed that T-Bil and two CT scan findings (appendicolith and fat stranding around the appendix) were independent predictors. The combination of "T-Bil ≥ 1.0 mg/dL or appendicolith" was able to predict gangrenous appendicitis with a sensitivity of 90.5%, positive predictive value of 80.4%, and accuracy of 77.8%. The combination of "T-Bil ≥ 1.0 mg/dL or fat stranding around the appendix" was able to predict gangrenous appendicitis with a sensitivity of 98.9%, positive predictive value of 76.4%, and accuracy of 71.9%. CONCLUSION: These combinations of laboratory and CT scan findings could be valuable as predictors of gangrenous appendicitis.

Intestinal mucosa staple line integrity and anastomotic leak pressure after healing in a porcine model.

PURPOSE: The aim of this study was to evaluate both the intestinal mucosa staple line integrity and anastomotic leak pressure after healing in a porcine survival model. METHODS: We used two suture models using two different size staples (incomplete mucosal closure model: group G [staple height 0.75 mm], complete mucosal closure model: group B [staple height 1.5 mm]) in the porcine ileum. Five staple lines were created in each group made in the ileum for each model, and the staple sites harvested on days 0, 2, and 7. The leak pressure at the staple site was measured at each time point. RESULTS: On day 0, the leak pressure for group G (79.5 mmHg) was significantly lower than that for group B (182.3 mmHg) (p < 0.01). On days 2 and 7, there was no significant difference between groups G and B (171 mmHg and 175.5 mmHg on day 2, 175.5 mmHg and 175.5 mmHg on day 7, p > 0.05). The histological findings in both groups showed similar healing at postoperative days 2 and 7. CONCLUSION: The integrity of the mucosal staple lines was associated with the postoperative leak pressure on day 0. However, there was no association with the leak pressure at two days or more postoperatively in a porcine model.

Flow cytometry detection of cell type-specific expression of programmed death receptor ligand-1 (PD-L1) in colorectal cancer specimens.

AIM: PD-1/PD-L1 blockade therapy is now widely used for the treatment of advanced malignancies. Although PD-L1 is known to be expressed by various host cells as well as tumor cells, the role of PD-L1 on non-malignant cells and its clinical significance is unknown. We evaluated cell type-specific expression of PD-L1 in colorectal cancer (CRC) specimens using multicolor flow cytometry. METHODS: Single cell suspensions were made from 21 surgically resected CRC specimens, and immunostained with various mAbs conjugated with different fluorescent dyes. Tumor cells, stromal cells, and immune cells were identified as CD326(+), CD90(+) and CD45(+) phenotype, respectively. CD11b(+) myeloid cells, CD19(+) B cells and CD4(+) or CD8(+) T cells were also stained in different samples, and their frequencies in the total cell population and the ratio of PD-L1(+) cells to each phenotype were determined. RESULTS: PD-L1 was expressed by all the cell types. The ratio of PD-L1(+) cells to CD326(+) tumor cells was 19.1% ± 14.0%, lower than those for CD90(+) stromal cells (39.6% ± 16.0%) and CD11b(+) myeloid cells (31.9% ± 14.3%). The ratio of PD-L1(+) cells in tumor cells correlated strongly with the ratio in stromal cells, while only weakly with that in myeloid cells. Tumor cells were divided into two populations by CD326 expression levels, and the PD-L1 positive ratios were inversely correlated with the rate of CD326 highly expressing cells as well as mean fluorescein intensity of CD326 in tumor cells, while positively correlated with the frequencies of stromal cells or myeloid cells in CRC. CONCLUSION: PD-L1 is differentially expressed on various cell types in CRC. PD-L1 on tumor cells may be upregulated together with CD326 downregulation in the process of epithelial mesenchymal transition. Quantification of cell type-specific expression of PD-L1 using multicolor flow cytometry may provide useful information for the immunotherapy of solid tumors.