Conversion of unresponsiveness to immune checkpoint inhibition by fecal microbiota transplantation in patients with metastatic melanoma: study protocol for a randomized phase Ib/IIa trial.

BACKGROUND: The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune responses. Furthermore, in trials using fecal microbiota transplantation (FMT) to treat melanoma patients unresponsive to immune checkpoint inhibitors (ICI), complete responses (CR), partial responses (PR), and durable stable disease (SD) have been observed. However, the underlying mechanism determining which patients will or will not respond and what the optimal FMT composition is, has not been fully elucidated, and a discrepancy in microbial taxa associated with clinical response has been observed between studies. Furthermore, it is unknown whether a change in the microbiome itself, irrespective of its origin, or FMT from ICI responding donors, is required for reversion of ICI-unresponsiveness. To address this, we will transfer microbiota of either ICI responder or nonresponder metastatic melanoma patients via FMT. METHODS: In this randomized, double-blinded phase Ib/IIa trial, 24 anti-PD1-refractory patients with advanced stage cutaneous melanoma will receive an FMT from either an ICI responding or nonresponding donor, while continuing anti-PD-1 treatment. Donors will be selected from patients with metastatic melanoma treated with anti-PD-1 therapy. Two patients with a good response (≥ 30% decrease according to RECIST 1.1 within the past 24 months) and two patients with progression (≥ 20% increase according to RECIST 1.1 within the past 3 months) will be selected as ICI responding or nonresponding donors, respectively. The primary endpoint is clinical benefit (SD, PR or CR) at 12 weeks, confirmed on a CT scan at 16 weeks. The secondary endpoint is safety, defined as the occurrence of grade ≥ 3 toxicity. Exploratory endpoints are progression-free survival and changes in the gut microbiome, metabolome, and immune cells. DISCUSSION: Transplanting fecal microbiota to restore the patients' perturbed microbiome has proven successful in several indications. However, less is known about the potential role of FMT to improve antitumor immune response. In this trial, we aim to investigate whether administration of FMT can reverse resistance to anti-PD-1 treatment in patients with advanced stage melanoma, and whether the ICI-responsiveness of the feces donor is associated with its effectiveness. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05251389 (registered 22-Feb-2022). Protocol V4.0 (08-02-2022).

Clinical Application and Potential of Fecal Microbiota Transplantation.

Fecal microbiota transplantation (FMT) is a well-established treatment for recurrent Clostridioides difficile infection. FMT has become a more readily available and useful new treatment option as a result of stool banks. The current state of knowledge indicates that dysbiosis of the gut microbiota is implicated in several disorders in addition to C. difficile infection. Randomized controlled studies have shown FMT to be somewhat effective in treating ulcerative colitis, irritable bowel syndrome, and hepatic encephalopathy. In addition, FMT has been beneficial in treating several other conditions, such as the eradication of multidrug-resistant organisms and graft-versus-host disease. We expect that FMT will soon be implemented as a treatment strategy for several new indications, although further studies are needed.

Population pharmacokinetics and pharmacogenetics of once daily tacrolimus formulation in stable liver transplant recipients.

PURPOSE: The once daily formulation of tacrolimus is an important immunosuppressive drug. Interpatient variability in metabolism has been related to genetic variation in CYP3A4 and CYP3A5. However, in liver transplantation, both donor and recipient genotypes may affect pharmacokinetics. The primary objective of this study was to investigate the effect of CYP3A4*22 and CYP3A5*3 of both donor and recipient on once daily tacrolimus pharmacokinetics. The secondary objective was to develop a limited sampling model able to accurately predict exposure. METHODS: Stable liver transplant patients receiving once daily tacrolimus (N = 66) were included. Population pharmacokinetic analysis was performed with patients of whom DNA was available (N = 49), and demographic factors, CYP3A4*22 and CYP3A5*3, were tested as covariates. Moreover, a limited sampling model was developed using data of 66 patients. RESULTS: Pharmacokinetics was best described by a two-compartment model with delayed absorption. CYP3A5*1 carrying recipients engrafted with a CYP3A5*1 carrying liver had an average 1.7-fold higher clearance compared to non-carriers. CYP3A5*1 carrying recipients engrafted with a CYP3A5*1 non-carrying liver or vice versa showed an average 1.3-fold higher clearance compared with non-carriers. CYP3A4*22 was not significantly associated with once daily tacrolimus pharmacokinetics. Using 0, 2, and 3 h postdose as limited sampling model resulted in significantly improved prediction of tacrolimus exposure compared with trough concentration. CONCLUSIONS: Both donor and recipient CYP3A5 genotype significantly influences tacrolimus once daily pharmacokinetics. In contrast, CYP3A4*22 appears not suitable as biomarker. The developed limited sampling model can be used to accurately estimate tacrolimus once daily exposure.

Esophageal and Gastric Cancer Pearl: a nationwide clinical biobanking project in the Netherlands.

Esophageal and gastric cancer is associated with a poor prognosis since many patients develop recurrent disease. Treatment requires specific expertise and a structured multidisciplinary approach. In the Netherlands, this type of expertise is mainly found at the University Medical Centers (UMCs) and a few specialized nonacademic centers. Aim of this study is to implement a national infrastructure for research to gain more insight in the etiology and prognosis of esophageal and gastric cancer and to evaluate and improve the response on (neoadjuvant) treatment. Clinical data are collected in a prospective database, which is linked to the patients' biomaterial. The collection and storage of biomaterial is performed according to standard operating procedures in all participating UMCs as established within the Parelsnoer Institute. The collected biomaterial consists of tumor biopsies, blood samples, samples of malignant and healthy tissue of the resected specimen and biopsies of recurrence. The collected material is stored in the local biobanks and is encoded to respect the privacy of the donors. After approval of the study was obtained from the Institutional Review Board, the first patient was included in October 2014. The target aim is to include 300 patients annually. In conclusion, the eight UMCs of the Netherlands collaborated to establish a nationwide database of clinical information and biomaterial of patients with esophageal and gastric cancer. Due to the national coverage, a high number of patients are expected to be included. This will provide opportunity for future studies to gain more insight in the etiology, treatment and prognosis of esophageal and gastric cancer.

HLA-DRB1*03:01 and HLA-DRB1*04:01 modify the presentation and outcome in autoimmune hepatitis type-1.

The classical human leukocyte antigen (HLA)-DRB1*03:01 and HLA-DRB1*04:01 alleles are established autoimmune hepatitis (AIH) risk alleles. To study the immune-modifying effect of these alleles, we imputed the genotypes from genome-wide association data in 649 Dutch AIH type-1 patients. We therefore compared the international AIH group (IAIHG) diagnostic scores as well as the underlying clinical characteristics between patients positive and negative for these HLA alleles. Seventy-five percent of the AIH patients were HLA-DRB1*03:01/HLA-DRB1*04:01 positive. HLA-DRB1*03:01/HLA-DRB1*04:01-positive patients had a higher median IAIHG score than HLA-DRB1*03:01/HLA-DRB1*04:01-negative patients (P<0.001). We did not observe associations between HLA alleles and alanine transaminase levels (HLA-DRB1*03:01: P=0.2; HLA-DRB1*04:01; P=0.5); however, HLA-DRB1*03:01 was independently associated with higher immunoglobulin G levels (P=0.04). The HLA-DRB1*04:01 allele was independently associated with presentation at older age (P=0.03) and a female predominance (P=0.04). HLA-DRB1*03:01-positive patients received immunosuppressive medication and liver transplantation. In conclusion, the HLA-DRB1*03:01 and HLA-DRB1*04:01 alleles are both independently associated with the aggregate diagnostic IAIHG score in type-1 AIH patients, but are not essential for AIH development. HLA-DRB1*03:01 is the strongest genetic modifier of disease severity in AIH.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

A thirty-year follow-up surveillance study for neoplasia of a dutch ulcerative colitis cohort.

BACKGROUND: Patients with ulcerative colitis have an increased risk of developing colorectal cancer (CRC). The aim of this study is to assess the yield of surveillance colonoscopies in a tertiary referral cohort of ulcerative colitis patients and to identify different risk groups for dysplasia. METHODS: A cohort of 293 patients (148 males, mean age 33.8 years at diagnosis) was built up at our center and started the surveillance program 8-12 years after start of symptoms. They underwent colonoscopies every one to three years. Endpoints were dysplasia or a (sub)total colectomy. RESULTS: After a follow-up period of 10 years, the cumulative incidence of any dysplasia was 23.5%, and of CRC 4.0%. After 15 years these percentages were 33.3% and 6.8%. Patients with pancolitis (n = 178) had a significantly higher cumulative risk of dysplasia than patients with distal disease, HR 1.9 (95%CI 1.1-3.3). Patients who started surveillance at an older age are at increased risk for any dysplasia, HR 1.03 (95%CI 1.01-1.05). CONCLUSIONS: This prospective surveillance study shows a high yield of dysplasia in ulcerative colitis patients. We recommend developing separate surveillance programs for different risk groups. In our opinion patients with distal colitis can follow the general population surveillance program.

MMP-2 and MMP-9 in normal mucosa are independently associated with outcome of colorectal cancer patients.

BACKGROUND: Upregulation of the matrix metalloproteinases MMP-2 and MMP-9 in various cancers has been associated with worse survival of the patients. METHODS: We assessed MMP-2 and MMP-9 levels in normal colorectal mucosa from colorectal cancer patients in relation to the course of the disease. RESULTS: A high protein expression of MMP-2 as well as MMP-9 in normal mucosa was found to be correlated with worse 5-year survival. The combination of both parameters was an even stronger prognostic factor. These protein levels were found not to be related to the corresponding single nucleotide polymorphisms of MMP-2 (-1306C>T) and MMP-9 (-1562C>T). Multivariate analyses indicated that the MMP-2 and MMP-9 levels in normal mucosa are prognostic for survival, independent of TNM classification. CONCLUSION: MMP-2 and MMP-9 levels in normal mucosa are indicative of the course of disease in colorectal cancer patients.

Expression and ligand binding of bombesin receptors in pulmonary and intestinal carcinoids.

INTRODUCTION: Carcinoids are mainly found in the gastrointestinal (65%) and bronchopulmonary tract (25%). These neuroendocrine tumors secrete a wide range of bioactive peptides, including gastrin releasing peptide and neuromedin B, the mammalian analogs of bombesin. The purpose of this study was to investigate the quantity and localization of bombesin receptors in gastrointestinal and pulmonary carcinoids, and to reveal whether bombesin-like peptides (BLP) and their receptors are of any value in distinguishing pulmonary carcinoids from carcinoids of intestinal origin. METHODS: Carcinoid tumors with pulmonary (no.=9) or intestinal (no.=15) localizations were analyzed by immunohistochemistry, autoradiography, and radioimmunoassay, to examine the presence of bombesin receptor subtypes and determine BLP levels in these tumors. RESULTS: All 3 bombesin receptor subtypes (GRPR, NMBR, and BRS-3) were present on pulmonary and intestinal carcinoids by immunohistochemistry. In pulmonary carcinoids, low receptor ligand binding densities together with high and low BLP levels were found. Intestinal carcinoids showed predominantly high receptor ligand binding densities in combination with low BLP levels. CONCLUSIONS: The expression of bombesin receptor subtypes is independent from the carcinoid tumor origin, and is therefore not recommended as a distinction marker, although carcinoids of pulmonary and intestinal origin possess different receptor binding affinities for bombesin and dissimilar BLP levels. The combined presence of bombesin and its receptors might suggest the presence of a paracrine or autocrine growth loop in carcinoids.

Ten-Year Follow-Up of Patients Treated with Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection from a Randomized Controlled Trial and Review of the Literature.

Fecal microbiota transplantation (FMT) has become a well-established treatment for recurrent Clostridioides difficile infection (rCDI). While short-term outcomes and adverse events relating to FMT have been well documented, there still is a paucity of data with regard to long-term safety. In this report, we describe the long-term follow-up of the prospective cohort of the first randomized controlled trial of FMT for rCDI, and review the existing literature. A total of 34 patients were treated with FMT for rCDI. Seven patients were still alive after a follow-up of more than 10 years and three patients were lost to follow-up. None of the 34 patients had experienced a new-onset autoimmune, gastrointestinal, or malignant disorder during follow-up. We did not find any deterioration or amelioration of pre-existing medical conditions. Furthermore, no deaths directly attributable to FMT could be identified. These findings are in accordance with the data in available literature. In conclusion, no long-term adverse events or complications directly attributable to FMT were found in our prospective cohort. Review of the available literature does not point to long-term risks associated with FMT in this elderly population, provided that carefully screened fecal suspensions are being used. No firm conclusion on the long-term safety of FMT in younger patients could be drawn.

Molecular prediction of disease risk and severity in a large Dutch Crohn's disease cohort.

BACKGROUND: Crohn's disease and ulcerative colitis have a complex genetic background. We assessed the risk for both the development and severity of the disease by combining information from genetic variants associated with inflammatory bowel disease (IBD). METHODS: We studied 2804 patients (1684 with Crohn's disease and 1120 with ulcerative colitis) and 1350 controls from seven university hospitals. Details of the phenotype were available for 1600 patients with Crohn's disease and for 800 with ulcerative colitis. Genetic association for disease susceptibility was tested for the nucleotide-binding and oligomerisation domain 2 gene (NOD2), the IBD5 locus, the Drosophila discs large homologue 5 and autophagy-related 16-like 1 genes (DLG5 and ATG16L1) and the interleukin 23 receptor gene (IL23R). Interaction analysis was performed for Crohn's disease using the most associated single nucleotide polymorphism (SNP) for each locus. Odds ratios were calculated in an ordinal regression analysis with the number of risk alleles as an independent variable to analyse disease development and severity. RESULTS: Association with Crohn's disease was confirmed for NOD2, IBD5, DLG5, ATG16L1 and IL23R. Patients with Crohn's disease carry more risk alleles than controls (p = 3.85 x 10(-22)). Individuals carrying an increasing number of risk alleles have an increasing risk for Crohn's disease, consistent with an independent effects multiplicative model (trend analysis p = 4.25 x 10(-23)). Patients with Crohn's disease with a more severe disease course, operations or an age of onset below 40 years have more risk alleles compared to non-stricturing, non-penetrating behaviour (p = 0.0008), no operations (p = 0.02) or age of onset above 40 years (p = 0.028). CONCLUSION: Crohn's disease is a multigenic disorder. An increase in the number of risk alleles is associated with an increased risk for the development of Crohn's disease and with a more severe disease course. Combining information from the known common risk polymorphisms may enable clinicians to predict the course of Crohn's disease.

Clinical significance of stromal apoptosis in colorectal cancer.

BACKGROUND: Epithelial and stromal cells play an important role in the development of colorectal cancer (CRC). We aimed to determine the prognostic significance of both epithelial and stromal cell apoptosis in CRC. METHODS: Total apoptosis was determined by caspase-3 activity measurements in protein homogenates of CRC specimens and adjacent normal mucosa of 211 CRC patients. Epithelial apoptosis was determined by an ELISA specific for a caspase-3-degraded cytokeratin 18 product, the M30 antigen. Stromal apoptosis was determined from the ratio between total and epithelial apoptosis. RESULTS: Epithelial and stromal apoptosis, as well as total apoptosis, were significantly higher in CRC compared with corresponding adjacent normal mucosa. Low total tumour apoptosis (< or = median caspase-3 activity) was associated with a significantly worse disease recurrence (hazard ratio (HR), 95% confidence interval (95% CI): 1.77 (1.05-3.01)), independent of clinocopathological parameters. Epithelial apoptosis was not associated with clinical outcome. In contrast, low stromal apoptosis (< or = median caspase-3/M30) was found to be an independent prognostic factor for overall survival, disease-free survival and disease recurrence, with HRs (95% CI) of 1.66 (1.17-2.35), 1.62 (1.15-2.29) and 1.69 (1.01-2.85), respectively. INTERPRETATION: Stromal apoptosis, in contrast to epithelial apoptosis, is an important factor with respect to survival and disease-recurrence in CRC.

Dysregulation of the transcription factors SOX4, CBFB and SMARCC1 correlates with outcome of colorectal cancer.

The aim of this study was to identify deregulated transcription factors (TFs) in colorectal cancer (CRC) and to evaluate their relation with the recurrence of stage II CRC and overall survival. Microarray-based transcript profiles of 20 normal mucosas and 424 CRC samples were used to identify 51 TFs displaying differential transcript levels between normal mucosa and CRC. For a subset of these we provide in vitro evidence that deregulation of the Wnt signalling pathway can lead to the alterations observed in tissues. Furthermore, in two independent cohorts of microsatellite-stable stage II cancers we found that high SOX4 transcript levels correlated with recurrence (HR 2.7; 95% CI, 1.2-6.0; P=0.01). Analyses of approximately 1000 stage I-III adenocarcinomas, by immunohistochemistry, revealed that patients with tumours displaying high levels of CBFB and SMARCC1 proteins had a significantly better overall survival rate (P=0.0001 and P=0.0275, respectively) than patients with low levels. Multivariate analyses revealed that a high CBFB protein level was an independent predictor of survival. In conclusion, several of the identified TFs seem to be involved in the progression of CRC.

MMP-2 geno-phenotype is prognostic for colorectal cancer survival, whereas MMP-9 is not.

The prognostic significance of single-nucleotide polymorphisms (SNPs) and tumour protein levels of MMP-2 and MMP-9 was evaluated in 215 colorectal cancer patients. Single-nucleotide polymorphism MMP-2(-1306T) and high MMP-2 levels were significantly associated with worse survival. Extreme tumour MMP-9 levels were associated with poor prognosis but SNP MMP-9(-1562C>T) was not. Tumour MMP levels were not determined by their SNP genotypes.

Increased mucosal matrix metalloproteinase-1, -2, -3 and -9 activity in patients with inflammatory bowel disease and the relation with Crohn's disease phenotype.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases are associated with matrix turnover in both physiological and pathological conditions. We postulate an association between aberrant matrix metalloproteinases proteolytic activity and the intestinal tissue destruction, seen in patients with Crohn's disease and/or ulcerative colitis. MATERIALS AND METHODS: Surgically resected inflamed and non-inflamed ileum and colon with/without extensive fibrosis from 122 Crohn's disease, 20 ulcerative colitis and 62 control patients were homogenized. Protein levels of matrix metalloproteinases and tissue inhibitor of metalloproteinases were measured by enzyme-linked immunosorbent assays (ELISA), while matrix metalloproteinases and myeloperoxidase activity were measured by specific activity assays. RESULTS: Expression of total levels of matrix metalloproteinases-1, -2, -3 and -9 relative to tissue inhibitor of metalloproteinases-1 and -2 was increased in inflamed inflammatory bowel disease compared to non-inflamed inflammatory bowel disease and control intestinal mucosa. Also, net matrix metalloproteinases-1, -2, -3 and -9 activity in inflamed inflammatory bowel disease was increased, with similar expression profiles in Crohn's disease and ulcerative colitis. Within inflamed inflammatory bowel disease, a close correlation of matrix metalloproteinases with myeloperoxidase was observed. The expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases was similar in inflamed Crohn's disease tissue with or without extensive fibrosis and not related to fistulizing disease. CONCLUSIONS: We have shown increased net matrix metalloproteinases activity in intestinal inflammatory bowel disease tissue, likely to contribute to the tissue damage and remodelling seen in inflammatory bowel disease.

Liver chimerism after allogeneic blood stem cell transplantation.

UNLABELLED: Blood stem cells can mature into elements of many different lineages. We investigated the presence and nature of donor-derived (chimeric) cells within the liver after allogeneic stem cell transplantation. METHODS: Liver biopsy autopsy specimens were examined from nine female patients who had undergone allogeneic bone marrow (n = 6) or peripheral stem cell (n = 3) transplantation from a male donor. To identify the male origin of cells within the liver, in-situ hybridization for Y-chromosomes was performed in conjunction with CD45 staining to identify leucocytes. RESULTS: Hematopoietic stem cell engraftment was confirmed in all nine recipients. Histologic examination of the liver tissue sections revealed 5.6-fold more Y-chromosome-positive than CD45-positive staining cells (P < .02), indicative of considerable nonleucocytic chimerism. This was particularly observed in patients who had developed graft-versus-host disease. CONCLUSIONS: Donor-derived cells can be found in liver tissue specimens after allogeneic stem cell transplantation. A considerable fraction of chimeric (donor-derived) cells appeared to be of nonlymphohematopoietic origin. This finding supports the theory of blood stem cells developing into liver cells of mesenchymal origin.

How to: Establish and run a stool bank.

BACKGROUND: Since 2013, several stool banks have been developed following publications reporting on clinical success of 'faecal microbiota transplantation' (FMT) for recurrent Clostridium difficile infections (CDI). However, protocols for donor screening, faecal suspension preparation, and transfer of the faecal suspension differ between countries and institutions. Moreover, no European consensus exists regarding the legislative aspects of the faecal suspension product. Internationally standardized recommendations about the above mentioned aspects have not yet been established. OBJECTIVE: In 2015, the Netherlands Donor Feces Bank (NDFB) was founded with the primary aim of providing a standardized product for the treatment of patients with recurrent CDI in the Netherlands. Standard operation procedures for donor recruitment, donor selection, donor screening, and production, storage, and distribution of frozen faecal suspensions for FMT were formulated. RESULTS AND DISCUSSION: Our experience summarized in this review addresses current donor recruitment and screening, preparation of the faecal suspension, transfer of the faecal microbiota suspension, and the experiences and follow-up of the patients treated with donor faeces from the NDFB.

Plasminogen activators and tumor development in the human colon: activity levels in normal mucosa, adenomatous polyps, and adenocarcinomas.

Malignant changes are often accompanied by alterations in activity and composition of the plasminogen activators (PA). To study the relationship between PA expression and the development of colorectal cancer, we determined urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in normal mucosa (n = 80), adenomatous polyps (n = 76), and adenocarcinomas (n = 71) of the colon. Tissues obtained from surgical resection or polypectomy were analyzed for t-PA and u-PA activity in a specific enzymatic assay using plasminogen, a chromogenic substrate, and selective quenching with monospecific antibodies to both activators. The plasminogen activator activities were found to be changed in adenocarcinomas as compared to normal mucosa. The relative contribution of u-PA (expressed as percentage of u-PA) was raised from 6 to 50% for, respectively, normal mucosa and adenocarcinoma. This change could be attributed to a 3-fold decrease in t-PA activity and a 5-fold increase in u-PA activity in the carcinomas. Adenomatous polyps as a group showed percentages of u-PA [20.2 +/- 1.3 (SE)] which were intermediate as well as significantly different (P less than 0.001) from those of normal mucosa and adenocarcinomas. This observation was strengthened by a gradual rise in the relative contribution of u-PA in four resection specimens containing both adenomatous polyps and adenocarcinomas. Zymography showed the presence of minor quantities of PA-PA inhibitor complexes in the tissue extracts studied. The present study shows that the sequence of normal mucosa-adenomatous polyp-adenocarcinoma in the colon is associated with a parallel change in plasminogen activator activity. Thus, change in the regulation of plasminogen activator activity is an early event in the development of colorectal cancer.