Neoplasia risk in patients with Lynch syndrome treated with immune checkpoint blockade

Abstract

Metastatic and localized mismatch repair-deficient (dMMR) tumors are exquisitely sensitive to immune checkpoint blockade (ICB). The ability of ICB to prevent dMMR malignant or pre-malignant neoplasia development in patients with Lynch syndrome (LS) is unknown. Of 172 cancer-affected patients with LS who had received ≥1 ICB cycles, 21 (12%) developed subsequent malignancies after ICB exposure, 91% (29/32) of which were dMMR, with median time to development of 21 months (interquartile range, 6-38). Twenty-four of 61 (39%) ICB-treated patients who subsequently underwent surveillance colonoscopy had premalignant polyps. Within matched pre-ICB and post-ICB follow-up periods, the overall rate of tumor development was unchanged; however, on subgroup analysis, a decreased incidence of post-ICB visceral tumors was observed. These data suggest that ICB treatment of LS-associated tumors does not eliminate risk of new neoplasia development, and LS-specific surveillance strategies should continue. These data have implications for immunopreventative strategies and provide insight into the immunobiology of dMMR tumors.

Extended Data Fig. 1.. Study Cohort of patients with Lynch Syndrome from MSKCC.

Consort diagram of study cohort. Segregated by microsatellite status as designated by MSIsensor. MSKCC: Memorial Sloan Kettering Cancer Center; MSI: Microsatellite unstable; MSS: Microsatellite stable.

Extended Data Fig. 2.. Histological evaluation of patient 7′s sequential tumors demonstrating distinct histological and immunohistochemical features.

(Patient example Fig. 1a, patient 7 Fig. 1b, Extended Data Tables 3/4). a, Tumor 3: Gastric adenocarcinoma. b, Tumor 4: Colorectal adenocarcinoma. c, Tumor 5: Prostate adenocarcinoma. d, Tumor 6: Small bowel adenocarcinoma. The relevant stain, hematoxylin and eosin, was run on each chosen study tissue block once. All antibodies and staining protocols are validated and optimized to current standards.

Extended Data Fig. 3.. Paired analysis of pre- and post-immune checkpoint blockade (ICB) exposed tumors using MSK-IMPACT next-generation sequencing derived data22.

Nonsynonymous somatic mutations from the MSK IMPACT variant call format (VCF) output from each sequenced patient tumor were input into the SnpEff program v5.1d (https://pcingola.github.io/SnpEff/) to predict translated amino acid sequences for each respective mutation. The number of unique mutation associated neoantigens (MANA) was determined by taking the defined peptide sequence and evaluating all potential contiguous 9′mer variations using a sliding reading frame. a, Comparison of total missense mutations in pre- and post-ICB tumors. b, Comparison of mutation associated neoantigens between pre- and post-ICB tumors. All boxplots are composed of median (central line), 25th–75th percentile (box edges), and minimum and maximum values (whiskers). All analyses included 9 distinct pre and 9 distinct post tumors paired for 9 unique patients. p-values are derived from pairwise t-testing based on patient.

Extended Data Fig. 4.. Sensitivity analyses of pre- and post-immune checkpoint blockade (ICB) exposed tumors using MSK-IMPACT next generation sequencing derived data22.

a, d, MSIsensor score, tumor mutational burden (TMB), frameshift mutation rates and number of mutation associated neoantigens (MANA) between pre- and post-ICB exposed tumors when grouped by gastrointestinal cancer only. e-h, MSIsensor score, tumor mutational burden (TMB), frameshift mutation rates and number of mutation associated neoantigens (MANA) between pre- and post-ICB exposed tumors when grouped by urothelial cancers only. All boxplots are composed of median (central line), 25th–75th percentile (box edges), and minimum and maximum values (whiskers). P-values are derived from student’s t-test (unpaired) between pre and post tumors agnostic of patient identity. Analysis a (MSIsensor score) includes 8 unique tumors (6 pre and 2 post) from 7 unique patients, and the other analyses, b-h, include 9 unique tumors (6 pre and 3 post) from 7 unique patients.

Extended Data Fig. 5.. Immunohistochemical analysis of immune cell infiltration of pre-immune checkpoint blockade (ICB) and post-ICB tumors.

a, and b, Intra-patient comparison of paired pre- (a) and post-ICB tumors (b). CD8 tumor infiltrating lymphocytes (CD8 TILS) in the non-ICB exposed urothelial cancer and the post-ICB exposure cutaneous squamous cell carcinoma. HPF: High Power Field. c, Interpatient comparisons of CD8 TILS, programmed cell death 1 protein (PD1) positive TILS and programmed cell death ligand 1 (PDL1) combined positive score (CPS) in tumors arising prior to ICB exposure (N = 8), tumors arising whilst patients were on ICB (N = 5) and tumors arising post completion of ICB (N = 2). Mean and standard deviation (SEM) are represented and compared using ordinary one way ANOVA test corrected for multiple comparisons.*: P ≤ 0.05 and P > 0.01, ns: non significant. Stains with the relevant antibodies, CD8 antibody (clone C8/144B, Catalog # sc-53212, diluted 1:100, Dako), PD-1 (clone NAT105, Catalog # 760–4895, ready to use, Cell Marque), and PD-L1 (cloneE1L3N, Catalog # 13684 S, diluted 1:100, Cell Signaling) were run on each chosen study tissue block once. All antibodies and staining protocols are validated and optimized to current standards.

Extended Data Fig. 6.. Incidence of subsequent primary malignancies (SPM) in the immune checkpoint blockade (ICB) treated and non-ICB (chemotherapy) treated patients with Lynch syndrome.

a, SPM incidence at 5-years matched follow-up in the ICB treated (pre- and post-ICB) and non-ICB treated cohort (pre- and post-chemotherapy). The number of matched patient pre/post ICB or pre/post chemotherapy is reported below the figure. The 5 year incidence of 2nd neoplasm for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 5 year incidence were compared using paired t test. ns: non significant, P > 0.05. b, SPM incidence in patients defined as high risk (HR). HR-patients were defined as patients presenting with multiple non concomitant cancers (> = 2) in a pre-ICB delay matched with post-ICB follow up. 3-year and 5-year incidence calculation was performed excluding the index cancer and concomitant cancers ( < 1 month between diagnoses of cancers). The number of matched patient pre/post ICB or pre/post chemotherapy for 3-year and 5-year incidence of 2nd neoplasm is reported below the figure. The 3-year and 5-year incidence of 2nd neoplasm for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 3-year or 5-year incidence were compared using paired t test. ns: non significant, P > 0.05. c, Swimmers plot demonstration cancer occurrence pre- and post-ICB exposure in the HR patient cohort over time in months for patients with match pre and post ICB follow up. The patient numbers for the ones developing post ICB neoplasms correspond to those reported in Fig. 1 (patients 15, 8, 7 and 4). Other high-risk patients (HR) are numerated from 1 to 12. Median ICB duration and follow up (FU) are reported for patients developing post ICB neoplasms and for the patients not developing post ICB neoplasm. The order of neoplasm occurring during the matched pre and post ICB follow up is reported. No statistical comparison was performed. d, Cumulative post-ICB cancer incidence in the ICB-exposed HR-patient cohort. High risk Patients with a matched follow up pre- and post-ICB of 36 months were selected and all non-index cancers occurring during the window of observation were accounted for. Cumulative incidence is reported as the cumulative sum of non-index cancers occurring during the period of observation. Number of patients and cumulative observation time in months are reported. Fisher’s exact test comparing the cumulative observation time without a neoplasm event to the number of months with an event was used for statistical analysis. ***: P < 0.001.

Figure 1.. Patient case example, clinical timeline, and tumor characteristics of Lynch syndrome patients with subsequent primary malignancies (SPM) after immune checkpoint blockade (ICB).

a, Case example of patient with multiple primary malignancies pre-ICB exposure. (Patient 7 from Figure 1b; Extended Data Table 1) Timeline of cancer diagnoses and sequential local and systemic interventions including combined androgen blockade (CAB), androgen receptor antagonist (ARA) and ICB. Targeted next-generation sequencing (MSK-IMPACT) comparing pre-ICB prostate cancer (PCA) and post-ICB small bowel cancer (SBA) by tumor mutational burden, somatic mutational profile, and copy-number alterations. Single shared somatic mutation, PTPRT at chromosome 20. b, Timeline for SPM development post-ICB exposure (n=21 patients) inclusive of: SBA: Small bowel, CRC: Colorectal, GCA: Gastric, PCA: Prostate, SN: Sebaceous neoplasm, HCC: Hepatocellular, UCC: Urothelial, SCC: Squamous cell, CHO: Cholangiocarcinoma, ECA: Esophageal, BRC: Breast, LYM: Lymphoma, UCE: Uterine, PDA: Pancreatic, VUV: Vulvar. c-f, MSISensor score (c), tumor mutational burden (d), frameshift mutations (e), and comparative analysis of strong binders to HLA Class 1 MHC (f) between pre- and post-ICB tumors. P-values are derived from pairwise t-testing for the paired pre and post tumor from each patient without replicates for each separate analysis. Analyses included nine patients with 9 pre and 9 post tumors except analysis c (MSISensor score) which included 8 patients with 8 pre and 8 post tumors due to an MSI score not available for patient 6. *MSI status on 1 sample with inadequate tumor purity was confirmed via MiMSI

Figure 2.. Incidence of subsequent primary malignancies (SPM) and site of origin in the study cohort and comparator Lynch syndrome (LS) cohorts.

a, Rate of SPM per observation year in the ICB treated cohort (pink) and comparator cohorts inclusive of internal non-immune checkpoint blockade (non-ICB) exposed LS patients (light blue) and external multi-center LS patients undergoing prospective surveillance after the first cancer diagnosis (dark blue). b, Overall SPM incidence at 3-years matched follow-up in the ICB treated (pre- and post-ICB) and non-ICB treated cohort (pre- and post-chemotherapy). The number of matched patient pre/post ICB or pre/post chemotherapy is reported below the figure. The 3 year incidence of 2^nd neoplasm for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 3 year incidence were compared using paired t test. ns: non significant, P>0.05. c, Incidence of cutaneous and visceral neoplasms at 3-years matched follow-up in patients pre- and post-ICB, and pre- and post-chemotherapy. The numbers of matched patient pre/post ICB or pre/post chemotherapy are respectively 65 and 95 patients. The 3 year incidence of 2^nd neoplasm category (skin or visceral) for each clinical condition is reported above the box plot representing the mean and standard deviation (SEM). Pre and post 3 year incidence were compared using repeated measure one way ANOVA test corrected for multiple comparisons comparing 3 year incidences of de novo cutaneous or visceral neoplasms. *: P≤0.05 and P>0.01, ns: non significant, P>0.05. d, Time to first cutaneous or visceral neoplasm post-ICB exposure. The number of patients is reported on the figure, each patient being accounted only once based on first 2^nd neoplasm type (skin or visceral). The median time to first 2^nd neoplasm post ICB is reported. Survival curves were compared using log rank test and the p-value is reported on the figure. The dashed lines represent the upper and lower limit of the confidence interval at 95% for each conditions with matched colors. e, f, Cumulative incidence of cutaneous (e) versus visceral (f) post-ICB SPM. Patients with a matched follow up pre- and post-ICB of 36 months were selected and all non-index cancers occurring during the window of observation were accounted for. Cumulative incidence is reported as the cumulative sum of non-index cancers occurring during the period of observation. Number of patients and cumulative observation time in months is reported. Fisher’s exact test comparing the cumulative observation time without a neoplasm event to the number of months with an event was used for statistical analysis. ****: P<0.0001 ; **: P≤0.01 and >0.001.