PMC8112237_01
Immunohistochemical staining for detecting MMR, Ki67 and BRAFV600E. All the MMR proteins (MSH6, MSH2, PMS2 and MHL1) is no deletion, namely pMMR. BRAFV600E is positive (Anti-BRAF-V600E immunostaining was considered positive if cytoplasmic staining of tumor cells was observed) and Ki67 is proliferative index of 80% (Three or more high-power (40X) fields were selected to calculate the positive staining rate of Ki67 in tumor cells within these fields, and the average value was taken). H&E sample of MMR proteins, including (A) MSH6(+), (B) MSH2(+), (C) PMS2(+) and (D) MHL1(+), (E) Ki67 and (F) BRAFV600E, respectively under x 100 magnification.
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Immunohistochemistry (IHC) revealed pMMR, BRAFV600E mutation and a Ki67 proliferative index of 80% while the overall pathology report indicated a T3 N2b M0 stage IIIC CRC ( Figure 1 ). Additionally, upon next generation sequencing (NGS) analysis of the circulating tumor DNA (ctDNA), the patient was identified to MSS ( Supplementary Figure 1 ).
Immunohistochemical staining for detecting PD-L1 expression. PD-L1 is positively expressed in patient's surgical specimens (tumor proportion score, TPS=30%; combined positive score, CPS=35; PD-L1 expression was determined using Tumor Proportion Score (TPS), the proportion of viable tumor cells showing partial or complete membrane PD-L1 staining at any intensity. PD-L1 expression was also defined using Combined Positive Score (CPS) by dividing the number of PD-L1-stained cells with the total number of viable tumor cells and multiplying by 100). Staining is performed using a Dako 22C3 assay kit following the manufacturer's instructions. (A) Positive and (B) negative controls as provided by the kit indicated PD-L1-expressing and non-PD-L1-expressing tissues, respectively under x 200 magnification. (C) H&E sample under x100 magnification, and (D) IHC sample under x 200.
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The tumor proportion score (TPS) reached 30%, whereas the combined positive score (CPS) reached 35 ( Figure 2 ). The entire treatment process has been shown in  Supplementary Figure 2 .
PET-CT scans show the patient's therapeutic response following a series of treatments, including second-line and third-line therapeutic regimen based on nivolumab.
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However, positron emission tomography - computed tomography (PET-CT) showed the disease progression as developed retroperitoneal lymph node metastases (29 x 23 mm) ( Figure 3 ). The benefits of this maintenance therapy were confirmed in August 2019 via the PET-CT which had recorded the complete metabolic response ( Figure 3 ).
MRI scans show the chronological response of the patient following a series of treatment. The circle represents the area of the target lesion, and arrows indicate the location of the lesions (lymph nodes of porta hepatis, midsection, and vessels). After treatment with FORFIRI and bevacizumab combined, enlarged tumor was observed in October 2018 (lymph nodes of porta hepatis: 23x11mm, midsection: 24x20mm, vessels: 24x18mm). From the change of immunotherapy to November 2019, MRI showed that the tumor was gradually shrinking (The size of hilar hepatis and epigastric mass is not obvious, and the size of vessel is about 16x15mm.).
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After two months, the magnetic resonance imaging (MRI) scans showed that the disease progressed due to the enlargement (24 x 17 mm) of the lymph node ( Figure 4 ). Due to the partial response (PR) observed in the MRI scan, the tumor collection diameter (19x16 mm) was reduced by 30.1% ( Figure 4 ), subsequently the maintenance therapy was implemented with capecitabine, nivolumab, and bevacizumab.