Efficacy of Recombinant Methioninase on Late-stage Patient Cancer in the Histoculture Drug Response Assay (HDRA) as a Potential Functional Biomarker of Sensitivity to Methionine-restriction Therapy in the Clinic.

Background/Aim: The present study utilized the three-dimensional histoculture drug response assay (HDRA) to determine the efficacy of recombinant methioninase (rMETase) on tumor tissue resected from patients with late-stage cancer, as a functional biomarker of sensitivity to methionine restriction therapy. Patients and Methods: Resected peritoneal-metastatic cancer, including colorectal cancer, pancreatic cancer, ovarian cancer, and pseudomyxoma were placed on Gelform in RPMI 1640 medium for seven days and treated with rMETase from 2.5 U/ml to 20 U/ml. Cell viability was determined using the MTT assay. A total of 48 patients with late-stage cancer underwent testing for rMETase responsiveness using the HDRA. Results: Colorectal cancer and pseudomyxoma had the highest sensitivity to rMETase. Pancreatic and ovarian cancer also responded to rMETase, but to a lesser degree. Conclusion: Patients with tumors with at least 40% sensitivity to rMETase in the HDRA are being considered as candidates for methionine restriction therapy, which includes the use of rMETase in combination with a low-methionine diet.

Isobutyric acid enhances the anti-tumour effect of anti-PD-1 antibody.

The low response rate of immune checkpoint inhibitors (ICIs) is a challenge. The efficacy of ICIs is influenced by the tumour microenvironment, which is controlled by the gut microbiota. In particular, intestinal bacteria and their metabolites, such as short chain fatty acids (SCFAs), are important regulators of cancer immunity; however, our knowledge on the effects of individual SCFAs remains limited. Here, we show that isobutyric acid has the strongest effect among SCFAs on both immune activity and tumour growth. In vitro, cancer cell numbers were suppressed by approximately 75% in humans and mice compared with those in controls. Oral administration of isobutyric acid to carcinoma-bearing mice enhanced the effect of anti-PD-1 immunotherapy, reducing tumour volume by approximately 80% and 60% compared with those in the control group and anti-PD-1 antibody alone group, respectively. Taken together, these findings may support the development of novel cancer therapies that can improve the response rate to ICIs.

Methionine Dependence of Hair Maintenance in C57BL/6 Mice.

BACKGROUND/AIM: Hair-follicle keratinocytes contain high levels of cysteine, which is derived from methionine, rapidly proliferate, and form the hair shaft. The high proliferation rate of hair-follicle keratinocytes resembles that of aggressive cancer cells. In the present study, we determined the effect of a methionine-deficient diet on hair loss (alopecia) in mice with or without homocysteine supplementation. MATERIALS AND METHODS: Mice were fed a normal rodent diet (2020X, ENVIGO) (Group 1); a methionine-choline-deficient diet (TD.90262, ENVIGO) (Group 2); a methionine-choline-deficient diet with a 10 mg/kg/day supply of homocysteine administered by intra-peritoneal (i.p.) injection for 2 weeks (Group 3). In Group 2, mice were fed a methionine-choline-deficient diet for an additional 2 weeks but with 10 mg/kg/day of i.p. l-homocysteine and the mice were observed for two additional weeks. Subsequently, the mice were fed a standard diet that included methionine. Hair loss was monitored by photography. RESULTS: After 14 days, hair loss was observed in Group 2 mice on a methionine-restricted diet but not in Group 3 mice on the methionine-restricted diet which received i.p. homocysteine. In Group 2, at 2 weeks after methionine restriction, hair loss was not rescued by homocysteine supplementation. However, after restoration of methionine in the diet, hair growth resumed. Thus, after 2 weeks of methionine restriction, only methionine restored hair loss, not homocysteine. CONCLUSION: Hair maintenance requires methionine in the diet. Future experiments will determine the effects of methionine restriction on hair-follicle stem cells.

Anti-EGFR antibody monotherapy for colorectal cancer with severe hyperbilirubinemia: A case report.

BACKGROUND: Hyperbilirubinemia with hepatic metastases is a common complication and a poor prognostic factor for colorectal cancer (CRC). Effective drainage is often impossible before initiating systemic chemotherapy, owing to the liver's diffuse metastatic involvement. Moreover, an appropriate chemotherapeutic approach for the treatment of hyperbilirubinemia is currently unavailable. CASE SUMMARY: The patient, a man in his 50s, presented with progressive fatigue and severe jaundice. Computed tomography revealed multiple hepatic masses with thickened walls in the sigmoid colon, which was pathologically confirmed as a well-differentiated adenocarcinoma. No RAS or BRAF mutations were detected. The Eastern Cooperative Oncology Group (ECOG) performance status (PS) score was 2. Biliary drainage was impossible due to the absence of a dilated bile duct, and panitumumab monotherapy was promptly initiated. Subsequently, the bilirubin level decreased and then normalized, and the patient's PS improved to zero ECOG score after four cycles of therapy without significant adverse events. CONCLUSION: Anti-EGFR antibody monotherapy is a safe and effective treatment for RAS wild-type CRC and hepatic metastases with severe hyperbilirubinemia.

Nivolumab receptor occupancy on effector regulatory T cells predicts clinical benefit.

Immune checkpoint inhibitor discovery represents a turning point in cancer treatment. However, the response rates of solid tumors remain ~10%-30%; consequently, prognostic and immune-related adverse event (irAE) predictors are being explored. The programmed cell death protein 1 (PD-1) receptor occupancy (RO) of PD-1 inhibitors depends on the number of peripheral blood lymphocytes and their PD-1 expression levels, suggesting that the RO may be related to efficacy and adverse events. As PD-1 inhibition affects each T-cell subset differently, the RO of each cell population must be characterized. However, relevant data have not been reported, and the prognostic relevance of this parameter is not known. In this study, we aimed to clarify the association between the nivolumab RO in each T-cell population and patient prognosis and reveal the development of irAEs in nivolumab-treated patients. Thirty-two patients were included in the study, and the mean follow-up period was 364 days. The nivolumab RO on effector regulatory T cells (eTregs) was significantly lower in the group that presented clinical benefits, and a significant negative association was observed between PD-1 occupancy on eTregs and all-cause mortality. The results suggest that the nivolumab RO on eTregs may be a prognostic factor in PD-1 inhibitor therapy, implying that the inhibition of PD-1/PD-ligand 1 (PD-L1) signaling on eTregs may attenuate antitumor effects.

Butyricimonas is a key gut microbiome component for predicting postoperative recurrence of esophageal cancer.

BACKGROUND: Recently, intestinal bacteria have attracted attention as factors affecting the prognosis of patients with cancer. However, the intestinal microbiome is composed of several hundred types of bacteria, necessitating the development of an analytical method that can allow the use of this information as a highly accurate biomarker. In this study, we investigated whether the preoperative intestinal bacterial profile in patients with esophageal cancer who underwent surgery after preoperative chemotherapy could be used as a biomarker of postoperative recurrence of esophageal cancer. METHODS: We determined the gut microbiome of the patients using 16S rRNA metagenome sequencing, followed by statistical analysis. Simultaneously, we performed a machine learning analysis using a random forest model with hyperparameter tuning and compared the data obtained. RESULTS: Statistical and machine learning analyses revealed two common bacterial genera, Butyricimonas and Actinomyces, which were abundant in cases with recurrent esophageal cancer. Butyricimonas primarily produces butyrate, whereas Actinomyces are oral bacteria whose function in the gut is unknown. CONCLUSION: Our results indicate that Butyricimonas spp. may be a biomarker of postoperative recurrence of esophageal cancer. Although the extent of the involvement of these bacteria in immune regulation remains unknown, future research should investigate their presence in other pathological conditions. Such research could potentially lead to a better understanding of the immunological impact of these bacteria on patients with cancer and their application as biomarkers.

Precise Non-invasive Imaging Mouse Model of Pancreatic Cancer: Very Narrow Band-width Laser Fluorescence Excitation of Green Fluorescent Protein Provides Ultra-bright Tumor Images With no Skin Autofluorescence.

Background/Aim: Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Materials and Methods: Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Results: Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. Conclusion: A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.

Methionine restriction of glioma does not induce MGMT and greatly improves temozolomide efficacy in an orthotopic nude-mouse model: A potential curable approach to a clinically-incurable disease.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.

Non-invasive Fluorescence Imaging of Breast Cancer Metastasis to the Brain in an Orthotopic Nude-mouse Model With Very-narrow-band-width Laser Excitation of Red Fluorescent Protein Resulting in an Ultra-bright Signal Without Skin Autofluorescence.

BACKGROUND/AIM: Breast-cancer metastasis to the brain is an intractable disease. To discover improved therapy for this disease, we developed a precise non-invasively-imageable orthotopic nude-mouse model, using very-narrow-band-width laser fluorescence excitation. MATERIALS AND METHODS: Female nu/nu nude mice, aged 4-8 weeks, were inoculated through the midline of the skull with triple-negative human MDA-MB-231 breast cancer cells (5×105) expressing red fluorescent protein (RFP). The mice were imaged with the Analytik Jena UVP Biospectrum Advanced at 520 nm excitation with peak emission at 605 nm. RESULTS: Three weeks after injection of MDA-MB-231-RFP cells in the brain, non-invasive fluorescence images of the breast tumor growing on the brain were obtained. The images of the tumor were very bright, with well-defined margins with no detectable skin autofluorescence background. Images obtained at various angles showed that the extent of the tumor margins could be precisely measured. A skin flap over the skull confirmed that the tumor was growing on the surface of the brain which is a frequent occurrence in breast cancer. CONCLUSION: A precise orthotopic model of RFP-expressing breast-cancer metastasis to the brain was developed that could be non-invasively imaged with very-narrow-band-width laser excitation, resulting in an ultra-bright, ultra-low-background signal. The model will be useful in discovering improved therapeutics for this recalcitrant disease.

[11C] Methionine-PET Imaging as a Cancer Biomarker for Methionine Addiction and Sensitivity to Methionine-restriction-based Combination Chemotherapy.

BACKGROUND/AIM: Methionine addiction is a fundamental and universal hallmark of cancer, termed the Hoffman effect. Methionine addiction of cancer is greater than glucose addiction, termed the Warburg effect, as shown by the comparison of PET imaging with [11C]methionine and [18F]fluorodeoxyglucose. The aim of the present study was to determine whether [11C]methionine PET (MET-PET) images could be a biomarker of methionine addiction of cancer and potential response to methionine-restriction-based combination chemotherapy. PATIENTS AND METHODS: In the present study a patient with invasive lobular carcinoma of the breast metastatic to axillary lymph nodes was imaged by both MET-PET and [18F]fluorodeoxyglucose PET (FDG-PET) before and after combination treatment with methionine restriction, comprising a low-methionine diet and methioninase, along with first-line chemotherapy. RESULTS: MET-PET gave a much stronger and precise image of the patient's metastatic axillary lymph nodes than FDG-PET. The patient had a complete response to methionine restriction-based chemotherapy as shown by MET-PET. CONCLUSION: MET-PET imaging is a biomarker of methionine-addicted cancer and potential response to methionine-restriction-based chemotherapy.

Non-classical Monocytes Enhance the Efficacy of Immune Checkpoint Inhibitors on Colon Cancer in a Syngeneic Mouse Model.

BACKGROUND/AIM: The response rate to immune checkpoint inhibitors (ICIs) is approximately 10%-30% and only in a few cancer types. In the present study, we determined whether non-classical monocytes (NCMs) could enhance ICI efficacy in colon cancer using a syngeneic mouse model. MATERIALS AND METHODS: The MC38 C57BL/6 mouse colon cancer model was used. Cells collected from the bone marrow of C57BL/6 mice were cultured, and NCMs were fractionated by cell sorting and administered via the tail veins to the mice implanted with MC38 cells. The anti-mouse PD-L1 antibody was administered three times, and tumor volume and overall survival were observed. RESULTS: More tumors were eradicated and more complete response occurred, after cotreatment with ICIs and NCMs than after treatment with ICIs alone. Moreover, no efficacy was observed when NCMs were administered alone. CONCLUSION: NCMs enhance ICI efficacy. The underlying mechanisms and clinical applications will be studied in the future.

Soluble PD-L1 changes in advanced non-small cell lung cancer patients treated with PD-1 inhibitors: an individual patient data meta-analysis.

Introduction: Currently, first-line immune checkpoint inhibitors (ICIs), including programmed cell death protein-1 (PD-1) inhibitors, are utilized as monotherapy in advanced non-small cell lung cancer (NSCLC) patients with high programmed death ligand-1 (PD-L1) expression (≧50%). Pre-treatment or post-treatment serum soluble PD-L1 (sPD-L1) has been identified as a potential biomarker for assessing ICI efficacy through fixed-point observations. However, existing studies on sPD-L1 changes have produced inconsistent results or have had sample sizes too small to detect clinically meaningful effect sizes. To elucidate the role of sPD-L1, we conducted a collaborative individual patient data meta-analysis of PD-1 inhibitor treatments. Methods: We conducted a thorough search of articles in PubMed via Medline, Embase, Scopus, and Cochrane databases from inception to October 20, 2023. Trials were deemed eligible if they contained individual datasets for advanced NSCLC patients, including data on overall survival (OS)/progression-free survival (PFS), as well as pre- and post-treatment sPD-L1 levels after 3-4 cycles of PD-1 inhibitor treatments. Our analysis focused on patients who completed 3-4 cycles of PD-1 inhibitor treatments. The primary outcome measure was OS/PFS, and we assessed changes in sPD-L1 concentration pre- and post-treatment through ELISA analyses. Results: From our search, we identified a potential seven trials, encompassing 256 patients. Among these, two trials with 26 patients met the criteria for inclusion in our primary analyses. Over a median follow-up period of 10 months, pooled univariate analysis revealed that increases in sPD-L1 levels during PD-1 inhibitor treatment were not associated with OS (HR = 1.25; CI: 0.52-3.02)/PFS (HR = 1.42; CI: 0.61-3.30) when compared to cases with sPD-L1 decreases. Subgroup analyses indicated that the impact of sPD-L1 changes on overall mortality/progression-related mortality remained consistent regardless of gender, age, or the type of treatment (nivolumab or pembrolizumab). Conclusion: Our findings suggest that changes in sPD-L1 levels during PD-1 inhibitor treatment do not significantly influence the prognosis of advanced NSCLC patients, regardless of gender, age, or treatment type. Continuous monitoring of sPD-L1 may not offer significant advantages compared to fixed-point observations.

Corrigendum: Soluble PD-L1 changes in advanced non-small cell lung cancer patients treated with PD-1 inhibitors: an individual patient data meta-analysis.

[This corrects the article DOI: 10.3389/fimmu.2023.1308381.].

Randomized phase I/II study of vascular endothelial growth factor receptor peptide vaccines for patients with hepatocellular carcinoma.

AIM: We evaluated the safety and efficacy of vascular endothelial growth factor receptor (VEGFR)-targeted peptide vaccines for the immunization of patients with unresectable hepatocellular carcinoma (HCC) who had responded to transarterial chemoembolization. METHODS: Twenty-two patients were randomized 1:1 to receive VEGFR-targeted peptides or placebo. The primary end-point was the safety assessment of the immunization. The secondary end-points were evaluation of immunological responses and clinical outcomes. RESULTS: No severe adverse events were induced by the study agents. Among the 12 patients in the vaccine group, a VEGFR1-specific cytotoxic T lymphocyte (CTL) response was induced in eight (66.7%) patients and a VEGFR2-specific CTL response was induced in 10 (83.3%). The median progression-free survival (PFS) and overall survival (OS) rates were 4.8 and 52.0 months, respectively, in the vaccine group, and 2.7 and 21.8 months, respectively, in the placebo group. No statistically significant differences were found between the two groups (PFS p = 0.925, OS p = 0.190). When divided into two groups according to immunoreactivity, the median PFS of patients with and without a strong immune response to VEGFR1 were 7.4 and 2.7 months, and that to VEGFR2 were 10.6 and 2.7 months, respectively; there were significant differences according to the immune response. CONCLUSIONS: Immunotherapy with peptide vaccines targeting VEGFR1 and VEGFR2 was well tolerated with no serious adverse events. It also effectively induced peptide-specific CTLs in patients with unresectable HCC.

Recombinant Methioninase Lowers the Effective Dose of Regorafenib Against Colon-Cancer Cells: A Strategy for Widespread Clinical Use of a Toxic Drug.

Background/Aim: Regorafenib is a multi-kinase inhibitor, targeting vascular endothelial growth factor receptor 2, fibroblast growth factor receptor 1 and other oncogenic kinases. Regorafenib has efficacy in metastatic colon cancer, but has severe dose-limiting toxicities which cause patients to stop taking the drug. The aim of the present study was to determine if recombinant methioninase (rMETase) could lower the effective concentration of regorafenib in vitro against a colorectal-cancer cell line. Materials and Methods: Firstly, we examined the half-maximal inhibitory concentration (IC50) of regorafenib alone and rMETase alone for the HCT-116 human colorectal-cancer cell line. After that, using the IC50 concentration of each drug, we investigated the efficacy of the combination of regorafenib and rMETase. Results: While both methioninase alone (IC50=0.61 U/ml) and regorafenib alone (IC50=2.26 U/ml) inhibited the viability of HCT-116 cells, the combination of the two agents was more than twice as effective as either alone. Addition of rMETase at 0.61 U/ml lowered the IC50 of regorafenib from 2.26 µM to 1.46 µM. Conclusion: rMETase and regorafenib are synergistic, giving rise to the possibility of lowering the effective dose of regorafenib in patients, thereby reducing its severe toxicity, allowing more cancer patients to be treated with regorafenib.

Recombinant-methioninase-producing Escherichia coli Instilled in the Microbiome Inhibits Triple-negative Breast Cancer in an Orthotopic Cell-line Mouse Model.

Background/Aim: Methionine restriction by diet and recombinant methioninase (rMETase) are effective for cancer therapy by themselves or combined with chemotherapy drugs. We previously showed that oral administration of rMETase-producing Escherichia coli JM109 (E. coli JM109-rMETase) can be installed in the mouse microbiome and inhibit colon-cancer growth in a syngeneic mouse model. In the present report, we investigated the efficacy of oral administration of E. coli JM109-rMETase in an orthotopic triple-negative breast cancer (TNBC) cell-line mouse model. Materials and Methods: First, we established orthotopic 4T1 mouse triple-negative breast cancer on an abdominal mammary gland in female athymic nu/nu nude mice aged 4-6 weeks. After tumor growth, 15 mice were divided into three groups of 5. Group 1 was administered phosphate-buffered saline (PBS) orally by gavage twice daily as a control; Group 2 was administered non-recombinant E. coli JM109 competent cells orally by gavage twice daily as a control; Group 3 was administered E. coli JM109-rMETase cells by gavage twice daily for two weeks. Tumor size was measured with calipers twice per week. On day 15, blood methionine level was examined using an HPLC method. Results: Oral administration of E. coli JM109-rMETase inhibited 4T1 TNBC growth significantly compared to the PBS and E. coli JM109 control groups. On day 15, the blood methionine level was significantly lower in the mice administered E. coli JM109-rMETase than in the PBS control. Conclusion: E. coli JM109-rMETase lowered blood methionine levels and inhibited TNBC growth in an orthotopic cell-line mouse model, suggesting future clinical potential against a highly recalcitrant cancer.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors.

Introduction: Programmed cell death ligand 1 (PD-L1) expression in tumor tissues is measured as a predictor of the therapeutic efficacy of immune checkpoint inhibitors (ICIs) in many cancer types. PD-L1 expression is evaluated by immunohistochemical staining using 3,3´-diaminobenzidine (DAB) chronogenesis (IHC-DAB); however, quantitative and reproducibility issues remain. We focused on a highly sensitive quantitative immunohistochemical method using phosphor-integrated dots (PIDs), which are fluorescent nanoparticles, and evaluated PD-L1 expression between the PID method and conventional DAB method. Methods: In total, 155 patients with metastatic or recurrent cancer treated with ICIs were enrolled from four university hospitals. Tumor tissue specimens collected before treatment were subjected to immunohistochemical staining with both the PID and conventional DAB methods to evaluate PD-L1 protein expression. Results: PD-L1 expression assessed using the PID and DAB methods was positively correlated. We quantified PD-L1 expression using the PID method and calculated PD-L1 PID scores. The PID score was significantly higher in the responder group than in the non-responder group. Survival analysis demonstrated that PD-L1 expression evaluated using the IHC-DAB method was not associated with progression-free survival (PFS) or overall survival (OS). Yet, PFS and OS were strikingly prolonged in the high PD-L1 PID score group. Conclusion: Quantification of PD-L1 expression as a PID score was more effective in predicting the treatment efficacy and prognosis of patients with cancer treated with ICIs. The quantitative evaluation of PD-L1 expression using the PID method is a novel strategy for protein detection. It is highly significant that the PID method was able to identify a group of patients with a favorable prognosis who could not be identified by the conventional DAB method.

Monocyte subsets associated with the efficacy of anti­PD­1 antibody monotherapy.

Immune checkpoint inhibitors (ICIs) are among the most notable advances in cancer immunotherapy; however, reliable biomarkers for the efficacy of ICIs are yet to be reported. Programmed death (PD)-ligand 1 (L1)-expressing CD14+ monocytes are associated with shorter overall survival (OS) time in patients with cancer treated with anti-PD-1 antibodies. The present study focused on the classification of monocytes into three subsets: Classical, intermediate and non-classical. A total of 44 patients with different types of cancer treated with anti-PD-1 monotherapy (pembrolizumab or nivolumab) were enrolled in the present study. The percentage of each monocyte subset was investigated, and the percentage of cells expressing PD-L1 or PD-1 within each of the three subsets was further analyzed. Higher pretreatment classical monocyte percentages were correlated with shorter OS (r=-0.32; P=0.032), whereas higher non-classical monocyte percentages were correlated with a favorable OS (r=0.39; P=0.0083). PD-L1-expressing classical monocytes accounted for a higher percentage of the total monocytes than non-classical monocytes with PD-L1 expression. In patients with non-small cell lung cancer (NSCLC), a higher percentage of PD-L1-expressing classical monocytes was correlated with shorter OS (r=-0.60; P=0.012), which is similar to the observation for the whole patient cohort. Comparatively, higher percentages of non-classical monocytes expressing PD-L1 were significantly associated with better OS, especially in patients with NSCLC (r=0.60; P=0.010). Moreover, a higher percentage of non-classical monocytes contributed to prolonged progression-free survival in patients with NSCLC (r=0.50; P=0.042), with similar results for PD-L1-expressing non-classical monocytes. The results suggested that the percentage of monocyte subsets in patients with cancer before anti-PD-1 monotherapy may predict the treatment efficacy and prognosis. Furthermore, more classical monocytes and fewer non-classical monocytes, especially those expressing PD-L1, are involved in shortening OS time, which may indicate the poor efficiency of anti-PD-1 treatment approaches.

Rapid Reduction of CEA and Stable Metastasis in an NRAS-mutant Rectal-Cancer Patient Treated With FOLFIRI and Bevacizumab Combined With Oral Recombinant Methioninase and a Low-Methionine Diet Upon Metastatic Recurrence After FOLFIRI and Bevacizumab Treatment Alone.

BACKGROUND/AIM: The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer. PATIENTS AND METHODS: A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement. RESULTS: After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow. CONCLUSION: Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone.

Case Report: Combined pembrolizumab, 5-fluorouracil, and cisplatin therapy were remarkably effective in p16-positive squamous cell carcinoma of unknown primary.

Background: Cancer of unknown primary (CUP) is a malignant tumor without a known primary lesion with a frequency of 3-5%. It can be divided into favorable and unfavorable prognosis subsets. While recommended treatments are available for the former group, there is no established treatment for the latter. Here, we report the effective treatment of a 32-year-old woman with p16-positive squamous cell CUP with pembrolizumab plus 5-fluorouracil and cisplatin therapy. Case presentation: A 32-year-old woman presented with metastatic lesions in the liver, lung, bone, cervical region, abdominal region, and pelvic lymph nodes. She was diagnosed with p16-positive squamous cell carcinoma of unknown primary origin. The patient received pembrolizumab plus 5-fluorouracil and cisplatin therapy, which markedly reduced the metastasis and improved her Eastern Cooperative Oncology Group performance status after two courses. Conclusion: This case report highlights the potential of pembrolizumab plus 5-fluorouracil and cisplatin therapy for treating CUP with an unfavorable prognosis. p16 positivity is worth examining for squamous cell carcinoma of unknown primary origin, and if present, this therapy should be considered a promising treatment option.