PMC9791944_01
(A) CT scan performed before surgery on primary tumor. (B) CT at day 42, after the first cycle of immunotherapy. (C) CT after six cycles of immunotherapy (first evidence of partial response according to RECIST 1.1 criteria).
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Moreover, colonic perforation was suspected due to perilesional fluid collection ( Figure 1A ). A CT scan after the first cycle showed an initial reduction of all target lesions ( Figure 1B ). A massive shrinkage and the best response were achieved after six cycles, being maintained thereafter up to the completion of 9 cycles over a 12-month period, without any toxicity or delay ( Figures 1C ,  2B ). Figures 2I-K ) and appeared organized in agglomerates resembling tertiary lymphoid structures ( Supplementary Figure S1 ). Flow cytometry analysis of total leukocytes infiltrating the fresh lesion revealed that the percentages of T- and B cells were higher than in the reference group ( Figures 2L,
Characterization of primary tumor and liver lesion. (A) Re-staging scan before treatment start: large liver metastases in the right lobe associated with a wide abscess in S4. (B) Re-staging CT scan after nine cycles of immunotherapy and surgical resection of residual disease: outcomes of hepatic surgery without any evidence of residual disease. (C) Primary tumor from colonic resection: poorly differentiated adenocarcinoma (hematoxylin and eosin stain at x200 magnification on light microscopy). (D) Liver lesion after nine cycles of immunotherapy and surgical resection: fibrous scar tissue and flogosis, without any evidence of residual neoplastic disease (hematoxylin and eosin stain at x200 magnification on light microscopy). (E, F) Representative seven-color mIHC images of immune cell infiltrates in PT (E) and LL (F); staining antibodies of "panel 1": CD3 (magenta), CD68 (yellow), CD20 (white), CD56 (green), neutrophil elastase (red), pan-cytokeratin (cyan), and nuclei (blue). Original magnification, x20. (G, H) Representative seven-color mIHC images of myeloid composition of PT (G) and LL (H); staining antibodies of "panel 2": CD68 (magenta), CD11c (red), CD11b (green), CD163 (yellow), neutrophil elastase (white), pan-cytokeratin (cyan), and nuclei (blue). Original magnification, x20. (I, J) Pie charts of cumulative mIHC data for PT (I) and LL (J). Data reported for each cell subset are the mean values of 10 fields from the same section. The total number of cells analyzed in PT (I) and LL (J) is 24,272 and 35,141, respectively. T cells, macrophages, neutrophils, B cells, natural killer (NK) cells, and tumor cells are identified as CD3+, CD68+, neutrophil elastase+, CD20+, CD56+, and pan-cytokeratin+ cells, respectively; "other cells" refer to cells negative for all these markers. (K) mIHC cell densities (cells/mm2) of immune cells infiltrating PT and LL. Mean values and SD derived from the analysis of the same 10 fields considered in (I, J) pie charts. (L, M) Percentages of T cells (CD3+) (L) and B cells (CD20+) (M) infiltrating fresh liver lesions, as assessed by flow cytometry. Patient data (case; red square) are reported together with those from the reference group (Ref. group; black dots). Data are gated on total leukocytes (CD45+). Mean and SD of the reference group are reported.
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TNM classification with macroscopic residual of disease ( Figure 2C ). The carcinoembrionyc antigen (CEA) level was determined at the diagnosis and subsequently at CT scan evaluations; it remained within normal limits during all the history of the disease. A re-staging CT scan confirmed large liver metastases, with diameter up to 12.2 cm, associated to hilar and abdominal lymphadenopathy, and a 7-cm abscess associated with cancer in S4 ( Figure 2A ). A massive shrinkage and the best response were achieved after six cycles, being maintained thereafter up to the completion of 9 cycles over a 12-month period, without any toxicity or delay ( Figures 1C ,  2B ). Liver lesion (LL) histology evidenced a pCR with fibrous tissue, necrosis and lympho-histiocytic inflammation, and no residual tumor cells ( Figure 2D ). Fluorescent multiplex IHC (mIHC) was performed to characterize lymphocyte population and immune checkpoint expression. mIHC analysis was carried out on of formalin-fixed paraffin-embedded tissue samples from primary tumor (PT) and secondary LL, whose cell composition is described in  Figures 2E-J . According to histology, no cytokeratin+ tumor cells were detected in LL ( Figures 2F, H, J ), whereas infiltrating immune cells were increased at least five-folds compared to PT ( Figures 2I-K ). Figures 2I-K ) and appeared organized in agglomerates resembling tertiary lymphoid structures ( Supplementary Figure S1 ). Flow cytometry analysis of total leukocytes infiltrating the fresh lesion revealed that the percentages of T- and B cells were higher than in the reference group ( Figures 2L,
T-cell functional state in primary tumor, liver lesion, and PBMCs. (A, B) Representative seven-color mIHC images of T-cell infiltrates of PT (A) and LL (B); staining antibodies of "panel 3": CD3 (pink), CD8 (magenta), CD4 (red), granzyme B (yellow), FoxP3 (green), pan-cytokeratin (cyan), and nuclei (blue). Original magnification, x20. (C) mIHC cell densities of CD4+, CD8+, and granzyme B+ T cells in 10 fields of PT and LL samples. Mean values and SD are reported for each cell population. (D) Percentages of CD4+ and CD8+ T cells infiltrating fresh liver lesions from the patient (Case; red square) and from the reference group (Ref. group; black dots), as assessed by flow cytometry. Data are gated on CD3+ cells. Connected date derived from the same lesion. (E) Percentages of IL-7R-expressing circulating T cells before therapy start (t0) and during treatment (t1 = disease re-staging after four cycles of immunotherapy, t2 = disease re-staging after six cycles of immunotherapy, t3 = disease re-staging after eight cycles of immunotherapy, and t4 = before surgical resection of the liver residual disease), as assessed by flow cytometry. Data are gated on CD4+ and CD8+ T cells before the start of immunotherapy. (F) mIHC cell densities of T-regulatory cells (CD3+/CD4+/FoxP3+) detected in PT and LL samples. Mean values and SD are reported. Data derived from the same 10 fields previously considered (C). (G) Percentages of T-regulatory cells (CD25bright/CD127low) infiltrating fresh liver lesions from the patient (Case; red square) and from the reference group (Ref. group; black dots), as assessed by flow cytometry. Data are gated on CD4+ T cells.
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M ,  S2 ), which consisted of liver metastases (n=7) collected from additional CRC patients treated with standard chemotherapy ( Supplementary Table 1 ). Moreover, peripheral blood T and B cells increased after treatment start ( Supplementary Figure S3 ). T cells in PT (CD8+/CD4+ ratio = 3.2) that was partially maintained in LL (CD8+/CD4+ ratio = 2) ( Figures 3A-C ). These data were confirmed in the fresh sample (CD8+/CD4+ ratio = 1.4) and completely differed from the reference group (CD8+/CD4+ ratio = 0.38;  Figure 3D ). T cells stained positive for granzyme B, irrespective of the subset phenotype ( Figures 3A, C ). This activation status persisted in LL ( Figures 3B, C ), where most of infiltrating T cells showed an effector-memory phenotype with a minority being terminally differentiated ( Supplementary Figure S4 ). T cells disclosed an expression peak of the IL-7 receptor (IL-7Ralpha) just after treatment start ( Figure 3E ), suggesting the induction of a memory pool subsequently observed in the LL. T-regulatory cells were already detected in PT and increased 20-folds in LL ( Figure 3F ). They were higher than in the reference group ( Figure 3G ).
Immune checkpoint expression in primary tumor, liver lesion, and PBMCs. (A, B) Representative seven-color mIHC images of immune checkpoint expression in PT (A) and in LL (B); staining antibodies of "panel 4": CD3 (magenta), PD-1 (white), PD-L1 (green), TIM-3 (yellow), LAG-3 (red), pan-cytokeratin (cyan), and nuclei (blue). Original magnification, x20. (C) Percentages of PD-1-, TIM-3-, and LAG-3-expressing T cells on total CD3+ cells infiltrating the PT and the LL, as assessed by mIHC. Mean values and SD are reported for each cell population. Data derived from analysis of 10 fields of each sample. (D) Percentages of PD1+ cells infiltrating fresh liver lesions from the patient (Case; red square) and from the reference group (Ref. group; black dots), as assessed by flow cytometry. Data are gated on CD4+ and CD8+ T cells. (E) mIHC analysis of PD-1 expression intensity on PD-1+ T cells (CD3+) and other PD-1+ cells (PD-1+/CD3-) infiltrating the PT and the LL. Mean values and SD are reported for each cell population. Data derived from analysis of the same 10 fields previously considered (C). Intensity values are expressed in counts. (F) Flow cytometry data of PD-1 expression intensity (Geomean) on PD-1+ cells infiltrating fresh liver lesions from the patient (Case; red square) and the reference group (Ref. group; black dots). Data are gated on CD4+ and CD8+ T cells. (G) Percentages of PD-1-expressing circulation T cells before therapy start (t0) and during treatment (t1 = disease re-staging after four cycles of immunotherapy, t2 = disease re-staging after six cycles of immunotherapy, t3 = disease re-staging after eight cycles of immunotherapy, and t4 = before surgical resection of the liver residual disease), as assessed by flow cytometry. Data are gated CD+ and CD8+ cells. (H) mIHC cell densities of PD-L1-expressing cells in PT and LL. Mean values and SD are reported for each cell population. Data derived from analysis of the same 10 fields previously considered (C).
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T cells stained positive for granzyme B, irrespective of the subset phenotype ( Figures 3A, C ). This activation status persisted in LL ( Figures 3B, C ), where most of infiltrating T cells showed an effector-memory phenotype with a minority being terminally differentiated ( Supplementary Figure S4 ). C ). These percentages remained essentially unchanged in LL after treatment ( Figures 4B, C ). Cytometry data of LL additionally demonstrated that the percentage of PD-1+ cells within either CD4+ or CD8+ populations was much lower than in the reference group ( Figure 4D ). PT and LL, intensity of PD-1 expression in T cells was low ( Supplementary Figure S5  and  Figure 4E ) and differed clearly from the reference group ( Figure 4F ). PD-1 expression was appreciable in peripheral blood T cells before treatment and decreased after immunotherapy start ( Figure 4G ). PD-L1 in PT ( Figures 4A, H ). Putative PD-L1+ macrophages were organized in rare agglomerates in the PT, whereas they were scattered and highly increased in LL ( Supplementary Figure S6  and  Figures 4B, H ).