In recent years, immune checkpoint inhibitors (ICIs) have been widely used in malignant solid tumors with remarkable efficacy. However, in colorectal cancer (CRC), ICIs have shown significant therapeutic effects only in patients with highly microsatellite unstable/mismatch repair-deficient metastatic CRC and these patients are only a minority of all CRC patients. In contrast, the majority of patients, those with microsatellite stable (MSS)/mismatch repair-complete (pMMR)-type metastatic CRC, could hardly benefit from ICI monotherapies, and immune combination therapies have become the key to solveing this clinical challenge. This article introduces the common patterns and possible mechanisms of immune-combination therapies for MSS/pMMR-type CRC, the exploration and progress made in the application of immune-combination therapies, as well as the possible predictive markers of efficacy of immune therapies. The prospects and directions of ICIs in the treatment of MSS/pMMR-type CRC are also discussed.
近年来，免疫检查点抑制剂（ICIs）在恶性实体肿瘤中应用广泛且疗效显著。然而，在结直肠癌（CRC）中，ICIs仅对占少数的微卫星高度不稳定/错配修复缺失型转移性CRC表现出显著的治疗效果，而占大多数的微卫星稳定（MSS）/错配修复完整（pMMR）型转移性CRC几乎不能从ICIs单药治疗中获益，免疫联合治疗成为破解这一临床难题的关键。文章介绍了MSS/pMMR型CRC免疫联合治疗的常见模式和可能机制，总结归纳了MSS/pMMR型CRC免疫联合治疗方面所做的探索和取得的进展以及免疫治疗疗效的可能预测标志物，对ICIs在MSS/pMMR型CRC治疗中的应用前景进行了展望。.

UNASSIGNED: The open-label, phase II RATIONALE-209 study evaluated tislelizumab (anti-programmed cell death protein 1 antibody) as a tissue-agnostic monotherapy for microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) tumors.
UNASSIGNED: Adults with previously treated, locally advanced unresectable or metastatic MSI-H/dMMR solid tumors were enrolled. Patients received tislelizumab 200 mg intravenously every 3 weeks. Objective response rate (ORR; primary endpoint), duration of response (DoR), and progression-free survival (PFS) were assessed by independent review committee (Response Evaluation Criteria in Solid Tumors v1.1).
UNASSIGNED: Eighty patients were enrolled and treated; 75 (93.8%) patients had measurable disease at baseline. Most had metastatic disease and received at least one prior therapy for advanced/metastatic disease (n=79; 98.8%). At primary analysis (data cutoff July 8, 2021; median follow-up 15.2 months), overall ORR [46.7%; 95% confidence interval (95% CI), 35.1-58.6; one-sided P<0.0001] and ORR across tumor-specific subgroups [colorectal (n=46): 39.1% (95% CI, 25.1-54.6); gastric/gastroesophageal junction (n=9): 55.6% (95% CI, 21.2-86.3); others (n=20): 60.0% (95% CI, 36.1-80.9)] were significantly greater with tislelizumab vs. a prespecified historical control ORR of 10%; five (6.7%) patients had complete responses. Median DoR, PFS, and overall survival were not reached with long-term follow-up (data cutoff December 5, 2022; median follow-up 28.9 months). Tislelizumab was well tolerated with no unexpected safety signals. Treatment-related adverse events (TRAEs) of grade ≥3 occurred in 53.8% of patients; 7.5% of patients discontinued treatment due to TRAEs.
UNASSIGNED: Tislelizumab demonstrated a significant ORR improvement in patients with previously treated, locally advanced unresectable or metastatic MSI-H/dMMR tumors and was generally well tolerated.

BACKGROUND: Colorectal cancer (CRC) patients with mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) status are conventionally perceived as unresponsive to adjuvant chemotherapy (ACT). The mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA copy number (mtDNA-CN) expression. In light of previous findings indicating that the frequent truncating-mutation of TFAM affects the chemotherapy resistance of MSI CRC cells, this study aimed to explore the potential of mtDNA-CN as a predictive biomarker for ACT efficacy in dMMR CRC patients.
METHODS: Levels of MtDNA-CN were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) in a cohort of 308 CRC patients with dMMR comprising 180 stage II and 128 stage III patients. Clinicopathologic and therapeutic data were collected. The study examined the association between mtDNA-CN levels and prognosis, as well as the impact of ACT benefit on dMMR CRC patients. Subgroup analyses were performed based mainly on tumor stage and mtDNA-CN level. Kaplan-Meier and Cox regression models were used to evaluate the effect of mtDNA-CN on disease-free survival (DFS) and overall survival (OS).
RESULTS: A substantial reduction in mtDNA-CN expression was observed in tumor tissue, and higher mtDNA-CN levels were correlated with improved DFS (73.4% vs 85.7%; P = 0.0055) and OS (82.5% vs 90.3%; P = 0.0366) in dMMR CRC patients. Cox regression analysis identified high mtDNA-CN as an independent protective factor for DFS (hazard ratio [HR] 0.547; 95% confidence interval [CI] 0.321-0.934; P = 0.0270) and OS (HR 0.520; 95% CI 0.272-0.998; P = 0.0492). Notably, for dMMR CRC patients with elevated mtDNA-CN, ACT significantly improved DFS (74.6% vs 93.4%; P = 0.0015) and OS (81.0% vs 96.7%; P = 0.0017), including those with stage II or III disease.
CONCLUSIONS: The mtDNA-CN levels exhibited a correlation with the prognosis of stage II or III CRC patients with dMMR. Elevated mtDNA-CN emerges as a robust prognostic factor, indicating improved ACT outcomes for stages II and III CRC patients with dMMR. These findings suggest the potential utility of mtDNA-CN as a biomarker for guiding personalized ACT treatment in this population.

BACKGROUND: Microsatellite instability (MSI) and mismatch repair (MMR) detection is valuable in assessing prognosis and treatment options. However, the conventional detection methods such as immunohistochemistry (IHC) are limited by not fully consistent results as well as a long turnaround time. TrueMark™ MSI Assay is a novel solution for MSI analysis, but lack of research support in the Chinese colorectal cancer (CRC) patients.
METHODS: 60 dMMR and 60 pMMR CRC samples identified by IHC were collected and their MSI status were detected using TrueMark™ MSI assay with an expanded panel of 13 markers. The overall performance and diagnostic concordance between TrueMark™ MSI test and MMR IHC analysis were assessed and analyzed.
RESULTS: According to the TrueMark™ test, 55 out of the 120 (45.8 %) CRCs were identified as MSI-high (MSI-H) with an instability at ≥ 4/13 markers. Compared with the MMR IHC analysis, an overall percent agreement of 94.2 % and a Kappa of 0.883 were achieved. For the seven inconsistent samples, tumor mutation burden analysis was performed and the results supported the diagnosis by TrueMark™ test. To confirm the robustness of the above findings, a validation was performed in an independent cohort comprising 51 consecutive CRCs. Furthermore, an optimized panel composed of NR-21, NR-24, NR-27, ABI-16, ABI-17 and ABI-20B was developed by multivariate logistic regression model, and showed 100 % agreement with the 13-marker panel for MSI detection in both the derivation and validation sets.
CONCLUSIONS: TrueMark™ MSI provides a fast, reliable and highly automated solution to MSI detection in Chinese CRC patients, and the new 6-marker panel we established shows promise deserving further evaluation.

BACKGROUND: The long-term survival benefit of immune checkpoint inhibitors (ICIs) in neoadjuvant and adjuvant settings is unclear for colorectal cancers (CRC) and gastric cancers (GC) with deficiency of mismatch repair (dMMR) or microsatellite instability-high (MSI-H).
METHODS: This retrospective study enrolled patients with dMMR/MSI-H CRC and GC who received at least one dose of neoadjuvant ICIs (neoadjuvant cohort, NAC) or adjuvant ICIs (adjuvant cohort, AC) at 17 centers in China. Patients with stage IV disease were also eligible if all tumor lesions were radically resectable.
RESULTS: In NAC (n = 124), objective response rates were 75.7% and 55.4%, respectively, in CRC and GC, and pathological complete response rates were 73.4% and 47.7%, respectively. The 3-year disease-free survival (DFS) and overall survival (OS) rates were 96% (95%CI 90-100%) and 100% for CRC (median follow-up [mFU] 29.4 months), respectively, and were 84% (72-96%) and 93% (85-100%) for GC (mFU 33.0 months), respectively. In AC (n = 48), the 3-year DFS and OS rates were 94% (84-100%) and 100% for CRC (mFU 35.5 months), respectively, and were 92% (82-100%) and 96% (88-100%) for GC (mFU 40.4 months), respectively. Among the seven patients with distant relapse, four received dual blockade of PD1 and CTLA4 combined with or without chemo- and targeted drugs, with three partial response and one progressive disease.
CONCLUSIONS: With a relatively long follow-up, this study demonstrated that neoadjuvant and adjuvant ICIs might be both associated with promising DFS and OS in dMMR/MSI-H CRC and GC, which should be confirmed in further randomized clinical trials.

BACKGROUND: Microsatellite instability (MSI) caused by DNA mismatch repair (MMR) deficiency is of great significance in the occurrence, diagnosis and treatment of colorectal cancer (CRC).
OBJECTIVE: This study aimed to analyze the relationship between mismatch repair status and clinical characteristics of CRC.
METHODS: The histopathological results and clinical characteristics of 2029 patients who suffered from CRC and underwent surgery at two centers from 2018 to 2020 were determined. After screening the importance of clinical characteristics through machine learning algorithms, the patients were divided into deficient mismatch repair (dMMR) and proficient mismatch repair (pMMR) groups based on the immunohistochemistry results and the clinical feature data between the two groups were observed by statistical methods.
RESULTS: The dMMR and pMMR groups had significant differences in histologic type, TNM stage, maximum tumor diameter, lymph node metastasis, differentiation grade, gross appearance, and vascular invasion. There were significant differences between the MLH1 groups in age, histologic type, TNM stage, lymph node metastasis, tumor location, and depth of invasion. The MSH2 groups were significantly different in age. The MSH6 groups had significant differences in age, histologic type, and TNM stage. There were significant differences between the PMS2 groups in lymph node metastasis and tumor location. CRC was dominated by MLH1 and PMS2 combined expression loss (41.77%). There was a positive correlation between MLH1 and MSH2 and between MSH6 and PMS2 as well.
CONCLUSIONS: The proportion of mucinous adenocarcinoma, protruding type, and poor differentiation is relatively high in dMMR CRCs, but lymph node metastasis is rare. It is worth noting that the expression of MMR protein has different prognostic significance in different stages of CRC disease.

Objective: To investigate the clinicopathological characteristics and prognostic factors of sporadic mismatch repair deficient (dMMR) colorectal cancer. Methods: A total of 120 cases of sporadic dMMR colorectal cancer from July 2015 to April 2021 were retrospectively collected in Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Patients with Lynch syndrome; synchronous multiple colorectal cancers; preoperative anti-tumor treatments such as chemotherapy and radiotherapy; and those with incomplete follow-up information were excluded based on family history and next-generation sequencing (NGS) test results. Immunohistochemical stains were used to detect the expression of mismatch repair proteins, methylation-specific PCR for methylation testing, and fluorescent PCR for BRAF V600E gene mutation detection. The clinical and pathological data, and gene mutation status were analyzed. Follow-up was done to assess survival and prognosis including progression-free survival and overall survival rate. Results: Sporadic dMMR colorectal cancer occurred more frequently in the right side of the colon, in females, and in the elderly. Morphologically, it was mostly moderately-differentiated, and most patients had low-grade tumor budding. In terms of immunohistochemical expression, MLH1 and PMS2 loss were dominant, and there were age and location-specificities in protein expression. MLH1 methylation was commonly detected in elderly female patients and rare in young male patients; while MLH1 and PMS2 deficiency, and BRAF V600E mutation occurred more often on the right side (P<0.05). The 3-year and 5-year progression-free survival rates were 90.7% and 88.7% respectively, and the 3-year and 5-year overall survival rates were 92.8% and 90.7% respectively. Tumor budding status was an independent risk factor affecting patient recurrence (hazard ratio=3.375, 95% confidence interval: 1.060-10.741, P=0.039), patients with low-grade tumor budding had better prognosis, and those with medium or high-grade tumor budding had poor prognosis. Conclusion: For dMMR colorectal cancer patients, tumor budding status is an independent risk factor for recurrence.
目的： 深入探讨散发性错配修复缺陷（dMMR）结直肠癌的临床病理学特征，并分析影响其预后的关键因素。 方法： 回顾性收集2015年7月至2021年4月在中国医学科学院 北京协和医学院 北京协和医院接受治疗的120例散发性dMMR结直肠癌患者的资料。根据家族史及二代测序检测结果排除林奇综合征患者，排除同时性多发结直肠癌术前进行放化疗等抗肿瘤治疗以及随访资料缺失的患者。采用免疫组织化学方法检测错配修复蛋白的表达情况，利用甲基化特异性PCR方法进行甲基化检测，通过荧光PCR方法对BRAF V600E基因突变进行筛查。主要观察指标包括临床病理资料、基因突变状况、随访数据以及生存与预后分析。预后指标主要关注疾病无进展生存期和总体生存期。 结果： 散发性dMMR结直肠癌更常见于右半结肠、女性和老年患者，形态学以中分化居多，存在低级别肿瘤出芽的患者占多数。免疫组织化学表达方面，以MLH1、PMS2缺失为主，且蛋白表达有年龄及部位的特殊性。MLH1甲基化多见于老年女性患者，而在MLH1和PMS2缺失的年轻男性患者中较为少见。BRAF V600E基因突变在右半结肠的肿瘤中检出频率更高（P<0.05）。此类肿瘤患者的3年和5年无进展生存率分别为90.7%和88.7%，3年和5年总体生存率分别为92.8%和90.7%。肿瘤出芽状态是影响肿瘤复发的独立危险因素（风险比=3.375，95%置信区间：1.060~10.741，P=0.039），其中低级别肿瘤出芽的患者预后较好，而中、高级别肿瘤出芽的患者预后较差。 结论： 在散发性dMMR结直肠癌患者中，肿瘤出芽状态是影响肿瘤复发的独立危险因素。.

Objective: To analyze the differences in clinicopathological features of colon cancers and survival between patients with right- versus left-sided colon cancers. Methods: This was a retrospective cohort study. Information on patients with colon cancer from January 2016 to August 2020 was collected from the prospective registry database at Peking Union Medical College Hospital . Primary tumors located in the cecum, ascending colon, and proximal two-thirds of the transverse colon were defined as right-sided colon cancers (RCCs), whereas primary tumors located in the distal third of the transverse colon, descending colon, or sigmoid colon were defined as left-sided colon cancers (LCCs). Clinicopathological features were compared using the χ2 test or Mann-Whitney U test. Survival was estimated by Kaplan-Meier curves and the log-rank test. Factors that differed significantly between the two groups were identified by multivariate survival analyses performed with the Cox proportional hazards function. One propensity score matching was performed to eliminate the effects of confounding factors. Results: The study cohort comprised 856 patients, with TNM Stage I disease, 391 (45.7%) with Stage II, and 336 (39.3%) with Stage III, including 442 (51.6%) with LCC and 414 (48.4%) with RCC and 129 (15.1%). Defective mismatch repair (dMMR) was identified in 139 patients (16.2%). Compared with RCC, the proportion of men (274/442 [62.0%] vs. 224/414 [54.1%], χ2=5.462, P=0.019), body mass index (24.2 [21.9, 26.6] kg/m2 vs. 23.2 [21.3, 25.5] kg/m2, U=78,789.0, P<0.001), and well/moderately differentiated cancer (412/442 [93.2%] vs. 344/414 [83.1%], χ2=22.266, P<0.001) were higher in the LCC than the RCC group. In contrast, the proportion of dMMR (40/442 [9.0%] vs. 99/414 [23.9%], χ2=34.721, P<0.001) and combined vascular invasion (106/442[24.0%] vs. 125/414[30.2%], χ2=4.186, P=0.041) were lower in the LCC than RCC group. The median follow-up time for all patients was 48 (range 33, 59) months. The log-rank test revealed no significant differences in disease-free survival (DFS) (P=0.668) or overall survival (OS) (P=0.828) between patients with LCC versus RCC. Cox proportional hazards model showed that dMMR was significantly associated with a longer DFS (HR=0.419, 95%CI: 0.204‒0.862, P=0.018), whereas a higher proportion of T3-4 (HR=2.178, 95%CI: 1.089‒4.359, P=0.028), N+ (HR=2.126, 95%CI: 1.443‒3.133, P<0.001), and perineural invasion (HR=1.835, 95%CI: 1.115‒3.020, P=0.017) were associated with poor DFS. Tumor location was not associated with DFS or OS (all P>0.05). Subsequent analysis showed that RCC patients with dMMR had longer DFS than did RCC patients with pMMR (HR=0.338, 95%CI: 0.146‒0.786, P=0.012). However, the difference in OS between the two groups was not statistically significant (HR=0.340, 95%CI:0.103‒1.119, P=0.076). After propensity score matching for independent risk factors for DFS, the log-rank test revealed no significant differences in DFS (P=0.343) or OS (P=0.658) between patients with LCC versus RCC, whereas patient with dMMR had better DFS (P=0.047) and OS (P=0.040) than did patients with pMMR. Conclusions: Tumor location is associated with differences in clinicopathological features; however, this has no impact on survival. dMMR status is significantly associated with longer survival: this association may be stronger in RCC patients.
目的： 分析非转移性结肠癌不同肿瘤位置（左半结肠或右半结肠）患者的临床病理特征及生存的差异，探讨肿瘤位置和错配修复状态（MMR）对生存的影响。 方法： 采用回顾性队列研究的方法。检索北京协和医院结直肠外科结肠癌前瞻性登记数据库2016年1月至2020年8月期间，接受根治性切除、且病理检查证实为非转移性结肠腺癌患者的病例资料和随访信息。将起源于中肠，位于回盲部、升结肠和横结肠近2/3的肿瘤定义为右半结肠癌；而将起源于后肠，位于横结肠远1/3、降结肠和乙状结肠的肿瘤定义为左半结肠癌。使用χ2检验或Mann-Whitney U检验比较两组患者临床病理特征的差异，使用Kaplan-Meier曲线和Log-rank检验进行两组患者的生存分析并比较组间的无病生存率（DFS）和总体生存率（OS）。使用Cox回归分析生存的影响因素，使用倾向性评分匹配以调整混杂后再进行生存分析。 结果： 共纳入856例结肠癌患者，其中肿瘤TNM分期Ⅰ期129例（15.1%），Ⅱ期391例（45.7%），Ⅲ期336例（39.3%）；错配修复缺陷（dMMR）139例（16.2%）。左半结肠癌442例（51.6%，左半结肠癌组），右半结肠癌414例（48.4%，右半结肠癌组）。相比右半结肠癌，左半结肠癌患者的男性比例高［62.0%（274/442）比54.1%（224/414），χ2=5.462，P=0.019］，中位体质指数也高［24.2（21.9，26.6）kg/m2比23.2（21.3，25.5）kg/m2，U=78 789.0，P<0.001］，高、中分化腺癌比例［93.2%（412/442）比83.1%（344/414），χ2=22.266，P<0.001］更高；dMMR状态［9.0%（40/442）比23.9%（99/414），χ2=34.721，P<0.001］和合并脉管侵犯［24.0%（106/442）比30.2%（125/414），χ2=4.186，P=0.041］比例更低。所有患者中位随访时间48（33，59）个月。Log-rank检验结果显示，左半结肠癌组患者与右半结肠癌组患者的DFS（P=0.668）和OS（P=0.828）差异无统计学意义。多因素Cox回归分析发现，dMMR是结肠癌患者DFS的独立保护因素（HR=0.419，95%CI：0.204~0.862，P=0.018）；T3~4（HR=2.178，95%CI：1.089~4.359，P=0.028）、N+（HR=2.126，95%CI：1.443~3.133，P<0.001）和神经侵犯（HR=1.835，95%CI：1.115~3.020，P=0.017）是DFS的独立危险因素。肿瘤位置不是影响非转移性结肠癌患者DFS和OS的独立因素（均P>0.05）。亚组分析发现，在右半结肠癌组患者中，dMMR患者的DFS优于错配修复正常（pMMR）患者（HR=0.338，95%CI：0.146~0.786，P=0.012），但是两组患者的OS差异无统计学意义（HR=0.340，95%CI：0.103~1.119，P=0.076）。对DFS的独立危险因素进行倾向性得分匹配后，Log-rank检验结果显示，两组患者的DFS（P=0.343）和OS（P=0.658）差异无统计学意义，而dMMR患者的DFS（P=0.047）和OS（P=0.040）均优于pMMR患者。 结论： 不同肿瘤位置的非转移性结肠癌患者其临床病理特征存在差异；但患者的生存与肿瘤位置无关，而与MMR有关，dMMR状态与更好的生存有关，在右半结肠癌患者中更为突出。.

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.

BACKGROUND: Patients with DNA mismatch repair-proficient/microsatellite stable (pMMR/MSS) colorectal cancer (CRC), which accounts for 85% of all CRC cases, display a poor respond to immune checkpoint inhibitors (i.e., anti-PD-1 antibodies). pMMR/MSS CRC patients with locally advanced cancers need effective combined therapies.
METHODS: In this pilot study, we administered six preoperative doses of each 2-week cycle of the anti-PD-1 antibody sintilimab (at a fixed dose of 200 mg), oxaliplatin, and 5-FU/CF (mFOLFOX6) combined with five doses of bevacizumab (the number of doses was reduced to prevent surgical delays) to patients with cT4NxM0 colon or upper rectal cancers. And radical surgery was performed approximately 2 weeks after the last dose of neoadjuvant therapy. The primary endpoint was a pathologic complete response (pCR). We also evaluated major pathologic response (MPR, ≤10% residual viable tumor), radiological and pathological regression, safety, and tumor mutation burden (TMB), and tumor microenvironment (TME) characteristics.
RESULTS: By the cutoff date (September 2023), 22 patients with cT4NxM0 pMMR/MSS colon or upper rectal cancers were enrolled and the median follow-up was 24.7 months (IQR: 21.1-26.1). All patients underwent R0 surgical resection without treatment-related surgical delays. pCR occurred in 12 of 22 resected tumors (54.5%) and MPR occurred in 18 of 22 (81.8%) patients. At the cutoff date, all patients were alive, and 21/22 were recurrence-free. Treatment-related adverse events of grade 3 or higher occurred in of 2/22 (9.1%) patients. Among the pCR tumors, two were found to harbor POLE mutations. The degree of pathological regression was significantly greater than that of radiological regression (p = 1.35 × 10-8). The number of CD3+/CD4+ cells in the tumor and stroma in pretreated biopsied tissues was markedly lower in pCR tumors than in non-pCR tumors (p = 0.038 and p = 0.015, respectively).
CONCLUSIONS: Neoadjuvant sintilimab combined with bevacizumab and mFOLFOX6 was associated with few side effects, did not delay surgery, and led to pCR and non-pCR in 54.5% and 81.8% of the cases, respectively. Downregulation of CD3/CD4 expression in the tumor and stroma is related to pCR. However, the molecular mechanisms underlying PD-1 blockade-enhanced targeted chemotherapy require further investigation.