Lab Daniel Giglio - Immunotherapy in MSI-high Metastatic Colorectal Cancer

Background

Colorectal cancer (CRC) has an annual incidence in Sweden of 6000 cases of which 4000 are located in the colon. Roughly, 20% of cases present at the metastatic stage (stage IV; mCRC).

Microsatellites are multiple repetitions of short segments of intronic DNA at non-coding genomic locations. Mutations in the mismatch repair genes (MMR; MSH2, MLH1, MSH6, PMS2) cause inactivation and extended number of repetitions of microsatellites. Mutations in the MMR proteins may either be caused by genetic mutations in the patient or appear spontaneously in the tumour. Lynch syndrome (LS) is strongly related to MSI-H/dMMR. These patients have a better prognosis compared with microsatellite stable (MSS) patients. MSI-H/d-MMR is found in 10-15% of CRCs but since MSI-H/d-MMR colorectal tumours less frequently recur, the percentage of mCRCs being MSI-H/dMMR is only 5%.

Chemotherapy has little effect while modern immunotherapy targeting immune checkpoint proteins PD-1 and PD-L1 has shown good clinical efficacy in mCRC. However, not all patients with MSI-H respond to immunotherapy. PD-L1 and PD-L2 are expressed on several cells in organs and inhibit PD-1 on CD8+ cytotoxic T-lymphocytes thereby preventing autoimmunity. Tumour cells also express PD-L1 and PD-L2 and deactivate CD8+ cytotoxic T-lymphocytes. Immunotherapy targets the interaction between PD-1 and PD-L1 and in this way the immune system becomes re-activated and can attack the tumour cells. New promising targets among immune checkpoint proteins have been identified in recent years. This includes lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), B7-homolog 3 (B7-H3), V-domain immunoglobulin-containing suppressor of T cell activation (VISTA), diacylglycerol kinase-α (DGK-α), T cell immunoglobulin and ITIM domain (TIGIT), and B and T lymphocyte attenuator (BTLA).

Reflex testing for MSI-H/dMMR in CRC has not been done until recently. Since 2021 reflex testing for MSI-H/dMMR in CRC is performed in Sweden in stage IIB-IV when adjuvant and neoadjuvant treatment are indicated or when hereditary CRC is suspected and genetic testing is indicated. At present there is limited knowledge on real world data on clinical outcome in patients treated and not treated with modern immunotherapy. With the large-sized almost complete Swedish Colorectal Registry, it is suitable for the assessment of long-term follow up for these patients.

Aims

First, we will describe survival in MSI-H and MSS CRC treated with immunotherapy and co-variates predicting survival in MSI-H and MSS CRC. Second, we will compare the panorama and number of previous cancer diagnoses in patients diagnosed with MSI-H vs. MSS CRC by comparing the Swedish Colorectal Registry with the Swedish Cancer Registry. In this way we may identify patients with suspected LS. Third, we will describe the expression of immune checkpoint proteins and other immune-modulatory proteins in MSI-H/dMMR CRC patients.

Methodology

In the first subproject, we will identify all patients diagnosed 2021-2022 from the Svenska kolorektalcancergregistret (SCRCR), since reflex testing for MSI-H/dMMR started in 2021. Patients will be followed till maximum cut-off date 31 December 2027. Patients with MSS will be used as control group. Two-year and 5-year cancer-specific survival (CSS) and overall survival (OS) will be estimated and compared between MSI-H and MSS. Demographical data, clinical data and pathological data will be retrieved from the SCRCR (see variables below). Data on the administration of oncological therapy will be retrieved from software for administration of oncological therapy (Cytobase and Cytodose). Cause of death will be retrieved from the National Cause of Death Registry. Co-variates retrieved from SCRCR, Cytobase and Cytodose will be correlated to CSS and OS.

In the second subproject, we will assess the number of previous cancers in patients diagnosed with MSI-H CRC vs. patients with MSS CRC. In Sweden, patients with suspected LS are referred for genetic testing at the different departments of clinical genetics in Sweden. However, we hypothesize that there are many patients in Sweden that go undetected with LS. We will therefore assess and compare the number of previous diagnoses of cancers in MSI-H CRC and MSS CRC by studying the Swedish Cancer Registry. In the case we identify patients with suspected LS that previously have not been genetically tested for LS, we will contact their physicians for contact with the patient and for genetical counseling. We will also assess the number of patients with LS referred for genetical testing based on our results.

In the third sub-project, we will assess the expression of immune-regulatory proteins (e.g., PD-1/PD-L1, LAG-3, TIM-3, TIGIT and toll-like receptors; TLRs) in MSI-H/dMMR vs. MSS CRC patients to find sub-groups of patients benefitting from PD-1/PD-L1 targeted therapy or potentially benefitting from new targets among checkpoint proteins and TLRs. Pathological analysis and data will be collected for 100 patients with MSI-H and 100 representative patients with MSS identified in subproject 1 who have or have not undergone treatment targeting PD-1/PD-L1. Paraffin-embedded tumour specimens collected from operation of the primary tumour/metastases or from biopsies will be collected from departments of pathology in Sweden. Tumours will be classified according to histology and stage, treatment and clinical outcome (CSS and OS). Tumour specimens will be assessed for tumour infiltrating lymphocytes (TILs) with conventional eosin-hematoxylin staining and we will also stain subsets of T-lymphocytes for specific immune markers (CD3+, CD4+, CD8+ and CD45RO+) and graded. Moreover, sections will be stained for immune-regulatory proteins to assess the degree of patients that potentially would benefit of immune checkpoint inhibitors currently used and currently assessed in trials. Omics technology including spatial transcriptomics, spatial proteomics, in situ sequencing and FISH will be used on paraffin embedded tissue specimens and on fresh-frozen tissue specimens at core facilities (https://www.scilifelab.se/units/spatial-omics/).  In a subsets of MSI-H CRC patients responding to immunotherapy vs. not responding to immunotherapy spatial omics will be performed on sections of paraffin-embedded specimens. The distribution and concentration of TILs, subsets of lymphocytes, immune checkpoint protein and TLR expressions and spatial biomarkers in the tumour and tumour microenvironment will be tested against CSS and OS. In this way it will be possible to identify patients that will benefit from traditional immunotherapy targeting PD-1/PD-L1 and patients that potentially will benefit from future immune checkpoint inhibitors.

Significance

Immunotherapy in CRC has only recently been introduced. At present little is known of the clinical efficacy of immunotherapy from large real data sets on mCRC. With better understanding of the effects of immunotherapy, the studies will help us to understand and optimize treatments for patients with MSI-H/dMMR and improve OS. Moreover, we will identify patients that may be eligible for new immunotherapies in the future. 

Collaborators

Daniel Giglio, Associate Professor, Principal Investigator, Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Academy.

Kamuran Inci, MD, PhD student, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy.

Bengt Nilsson, MD, PhD, Associate Professor, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy.

Stefan Lindskog, MD, PhD, Associate Professor, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg.

Lars Ny, MD, PhD, Professor, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy.

Fredrik Persson, MD, PhD, Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital.