Understand Tumor Initiation Research Articles

Mapping pediatric brain tumors to their origins in the developing cerebellum.

Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the 3 most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176 645 cells as a reference for an in-depth comparison to 4416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar "lineage of origin."

DIPG-13. NEW IMMUNOCOMPETENT GENETICALLY-ENGINEERED MOUSE MODELS OF PEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMAS REVEAL MECHANISMS OF TUMOR INITIATION AND PROGRESSION

Abstract Pediatric-type diffuse high-grade gliomas (pHGGs) form a group of aggressive brain tumors with very poor prognosis. Recurrent driver mutations in TP53 and histone H3-encoding genes, most commonly H3F3A, are observed in a large proportion of these tumors. H3-K27M substitutions are particularly prevalent in midline gliomas, and H3-G34R mutations occur in supratentorial tumors. To better understand the molecular mechanisms driving pHGG emergence and progression, we set out to develop immunocompetent mouse models recapitulating the oncogenic lesions observed in human tumors. Given that oligodendrocyte progenitor cells (OPCs) may be the cells of origin of some pHGGs, we investigated the effect of mutation induction in the OLIG2-expressing cell lineage, using the Olig2Cre driver. Interestingly, deletion of the P53-encoding gene, Trp53, in Trp53flox/flox;Olig2Cre/+ mice led to the development of diffuse gliomas in nearly all animals. Latency to humane endpoint was relatively long (>1 year), and tumors developed throughout the brain with a mild preference for infratentorial (e.g. brainstem) localization. The gliomas diffusely infiltrated the brain parenchyma, and were heterogeneously immuno-reactive for OPC, astrocytic, and neural progenitor cell (NPC) markers. Based on RNA sequencing, the tumors segregated in two groups: one enriched in expression of OPC genes, and another with a prominent NPC-like signature. Whole exome sequencing revealed frequent Ccnd1 and Kras/Hras copy number gains in advanced tumors. To model additional oncogenic lesions, we generated knock-in mice in which the H3f3aK27M or the H3f3aG34R mutations can be induced in any cell type with Cre recombinase, and extinguished with Flp recombinase. These were combined with conditional Trp53 loss-of-function alleles in various cell lineages, and their effect on tumor initiation and progression was monitored. Collectively, our studies provide new reagents for accurate disease modeling of pHGG in mice, opening opportunities to better understand tumor initiation and progression at high resolution, and for pre-clinical therapeutic testing.

Abstract 5445: Segmentation-free analysis of multiplexed images with unbiased spatial analytics and explainable AI for predicting disease outcomes

Abstract Background: The spatial intratumor heterogeneity (ITH) is widely acknowledged as driving therapeutic response and providing fuel for drug resistance. Currently, patient selection for immunotherapy is driven mostly by PD-1/PD-L1 based IHC tests and mutational analysis. These oversimplified approaches fail to predict the risk of recurrence, therapeutic response and drug resistance with high accuracy. We hypothesize that functional responses of heterogeneous non-random spatial arrangements of tumor, stromal and immune cells in the tumor microenvironment are determined by distinct combinations of their internal states and spatial interactions within neighborhoods. The interactions generate distinct cell-cell communication networks forming functional spatial configurations of cells, termed microdomains, as organizational units of spatial ITH. Deriving the spatial networks within each microdomain with unbiased spatial analytics and the underlying network biology through explainable AI, is key to understanding tumor initiation, tumor progression, and response to therapy. Problem: There has been an explosion of spatial imaging technologies using immunofluorescence and/or mass spectrometry for intact tissues measuring protein expressions, DNA and RNA probes. To extract high-value knowledge from these multiplexed datasets, a key first step is to segment cells accurately. Despite decades of research, this step remains elusive due to imaging artifacts arising from issues with tile stitching, incorrect registration, signal oversaturation, non-uniform illumination, poor or high-background nuclei signal, defocus, varying cell and nuclear shapes, fluorescence emission efficiency variation, overlapping and/or superimposed nuclei and low resolution (e.g., mass cytometry). Those artifacts may lead to incorrect cell phenotypes, incomplete cell phenotype atlases, and missing rare cell, fusion cell or transition cell types. Solution: We present TumorMapr™, a segmentation-free, unbiased spatial analytics and explainable AI platform that extracts information and creates knowledge from patient disease pathology samples imaged with any technology including fluorescence, mass spectrometry etc. Results: We apply TumorMapr on hyperplexed immunofluorescence dataset of colorectal cancer and hyperplexed imaging mass cytometry dataset of triple negative breast cancer to discover functional cell prototypes involving spatial collections of neighboring pixels highly predictive of disease progression and response to therapy, tumor promoting and tumor restraining microdomains and microdomain-specific network biology predictive of disease outcomes. We further compare these results with segmentation-based approaches to showcase the discovery of novel cell prototypes. Citation Format: Filippo Pullara, Brian Falkenstein, Bruce Campbell, Samantha Panakkal, Akif Burak Tosun, Jeffrey Fine, S. Chakra Chennubhotla. Segmentation-free analysis of multiplexed images with unbiased spatial analytics and explainable AI for predicting disease outcomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5445.

Thyroid Follicular Epithelial Cell-Derived Cancer: New Approaches and Treatment Strategies.

Thyroid follicular epithelial cell-derived cancer includes papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, poorly differentiated thyroid cancer, and anaplastic thyroid carcinoma. Although the incidence of thyroid cancer has increased over the past 30 years, there has not been a significant increase in patient mortality. Use of increasingly sensitive detection methods such as high-resolution imaging has enabled earlier detection and better characterization of the thyroid malignancies. In the past several years, researchers have evaluated genetic mutations promoting thyroid carcinogenesis and oncogenesis. The identification of genetic mutations is important in understanding tumor initiation and progression. Additionally, these identified mutations may also serve as potential diagnostic or prognostic indicators and therapeutic molecular targets.

Roles of the BAP1 Tumor Suppressor in Cell Metabolism.

BRCA1-associated protein 1 (BAP1) is emerging as an intensively studied cancer-associated gene. Germline mutations in BAP1 lead to a cancer syndrome, and somatic loss is found in several cancer types. BAP1 encodes a deubiquitinase enzyme, which plays key roles in cell-cycle regulation, cell death, and differentiation. Recent studies have demonstrated that BAP1 is also involved in several aspects of cellular metabolism, including metabolic homeostasis, glucose utilization, control of ferroptosis, and stress response. A better knowledge of the metabolic roles of cancer-associated genes is important to understanding tumor initiation and progression, as well as highlighting potential therapeutic avenues. With this review, we summarize the current knowledge regarding BAP1-mediated regulation of metabolic activities that may support new strategies to treat BAP1-mutated cancers.

Abstract B28: A cross-species enhancer activity analysis approach to identify the Ewing sarcoma cell of origin

Abstract Ewing sarcoma is a malignant bone and soft tissue tumor in children and adolescents. In 85% of all cases the formation of Ewing sarcoma is caused by a chromosomal translocation, leading to the expression of the oncogene EWS-FLI1. This aberrant transcription factor is the main driver of the disease and leads to massive transcriptional dysregulation. Although the genetic mechanism driving Ewing sarcoma is well understood, an animal model adequately mimicking the disease is still lacking. One reason why modeling attempts have remained difficult is the elusive cell of origin in Ewing sarcoma. Several cell types including neural crest progenitor cells and mesenchymal stem cells have been proposed, but none of them has yet been confirmed as cell of origin of Ewing sarcoma. Towards identifying this cell of origin, we are currently following a novel cross-species approach by exploring the use of enhancers, which are specifically active in Ewing sarcoma cells, but were not created de novo by EWS-FLI1. Injection of reporter constructs into zebrafish embryos allows for visualization of cell types with activity of the selected enhancers and provides us with cell-of-origin candidate cell types. To investigate which of these candidate cells are permissive for development of Ewing-like tumors, we will also express EWS-FLI1 in these cells. If successful, this approach will not only reveal the Ewing sarcoma cell of origin but also result in a zebrafish Ewing sarcoma model, which will help to understand tumor initiation and progression and, furthermore, will be a valuable tool to develop novel therapeutic strategies. Citation Format: Sarah Grissenberger, Anna Poetsch, Monika Heinzl, Luisa Morelli, Nathan Sheffield, Niko Popitsch, Heinrich Kovar, Martin Distel. A cross-species enhancer activity analysis approach to identify the Ewing sarcoma cell of origin [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B28.

Emerging Roles of Long Noncoding RNA Regulator of Reprogramming in Cancer Treatment.

Despite numerous advances in cancer treatment, the global prevalence and cancer-related mortality remain high. Understanding tumor initiation and progression mechanisms are critical as it will lead to the development of interventions for improving the prognosis of cancer patients. The roles of long noncoding RNAs (lncRNAs) in cancer have attracted immense research interest. Growing evidence indicates that lncRNA regulator of reprogramming (linc-ROR), a well-studied RNA, regulates the progression of various cancers, such as lung cancer (LC), hepatocellular carcinoma (HCC), breast cancer (BC), colorectal cancer (CRC), pancreatic cancer (PC), papillary thyroid carcinoma (PTC), or esophageal squamous cell carcinoma (ESCC). linc-ROR promotes the proliferation, invasion, migration and chemoresistance of cancer cells. Herein, we reviewed current literature on the modulatory functions and mechanisms of linc-ROR in cancer development. We highlight new linc-ROR-related therapeutic strategies in cancer treatment.

Tracing tumorigenesis in a solid tumor model at single-cell resolution

Characterizing the complex composition of solid tumors is fundamental for understanding tumor initiation, progression and metastasis. While patient-derived samples provide valuable insight, they are heterogeneous on multiple molecular levels, and often originate from advanced tumor stages. Here, we use single-cell transcriptome and epitope profiling together with pathway and lineage analyses to study tumorigenesis from a developmental perspective in a mouse model of salivary gland squamous cell carcinoma. We provide a comprehensive cell atlas and characterize tumor-specific cells. We find that these cells are connected along a reproducible developmental trajectory: initiated in basal cells exhibiting an epithelial-to-mesenchymal transition signature, tumorigenesis proceeds through Wnt-differential cancer stem cell-like subpopulations before differentiating into luminal-like cells. Our work provides unbiased insights into tumor-specific cellular identities in a whole tissue environment, and emphasizes the power of using defined genetic model systems.

Build-a-Mouse: Melanoma Modeling Picks up Speed.

The study by Bok and colleagues in this issue introduces a new paradigm for generating new mouse models for melanoma research. Genetically engineered mouse models (GEMM) have been crucial for understanding tumor initiation and modeling potential therapies, but are time consuming to create. Bok and colleagues generated and validated high-contribution chimeric GEMM models using common melanoma GEMMs as a starting point and added additional CRISPR, Cre-inducible, and Dox-inducible alleles. This rapid method for generating new models has the potential to revolutionize mouse modeling for melanoma.See related article by Bok et al., p. 912.

Mouse Model for Sporadic Mutation of Target Alleles to Understand Tumor Initiation and Progression and Stem Cell Dynamics.

Recent evidence has shown that many different tissues accumulate mutations even though the tissue is phenotypically normal. Therefore, generating mouse models for visualizing the tissue level effects that happen after oncogenic mutation in a single, isolated cell are critical for understanding tumor initiation and the role of competition in stem cell dynamics. Most mouse models have oncogenic mutations at the level of the entire mouse, the entire tissue, or all cells of a specific type in a tissue. However, these mouse models do not mimic the microenvironmental interactions that occur after an isolated cell acquires an oncogenic mutation because of the large number of mutant cells. We developed a mouse model for sporadic and isolated mutation of target alleles to better address the questions of sporadic cancer and stem cell competition. The following chapter describes methods for utilizing this mouse model and a few examples of the novel findings of using such a model.

Paper/PMMA Hybrid 3D Cell Culture Microfluidic Platform for the Study of Cellular Crosstalk.

Studying cellular crosstalk is important for understanding tumor initiation, progression, metastasis, and therapeutic resistance. Moreover, a three-dimensional (3D) cell culture model can provide a more physiologically meaningful culture microenvironment. However, studying cellular crosstalk in a 3D cell culture model involves tedious processing. In this study, a paper/poly(methyl methacrylate) (PMMA) hybrid 3D cell culture microfluidic platform was successfully developed for the study of cellular crosstalk. The platform was a paper substrate with culture microreactors placed on a PMMA substrate with hydrogel-infused channels. Different types of cells were directly seeded and cultured in the microreactors. Aberrant cell proliferation of the affected cells was induced by secretions from transfected cells, and the proliferation ratios were investigated using a colorimetric method. The results showed that the responses of cellular crosstalk were different in different types of cells. Moreover, neutralizing and competitive assays were performed to show the functionality of the platform. Additionally, the triggered signaling pathways of the affected cells were directly analyzed by a subsequent immunoassay. The microfluidic platform provides a simple method for studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model.

Stemness Gene Profiles of Circulating Tumor Cells

Circulating tumor cells (CTCs) are a population of tumor-derived cells that detach from the primary tumor and initiate metastasis. However, the mechanisms of this process are still unknown. This phenomenon renders CTCs as a valuable resource for prognosis and diagnosis of cancer. The involvement of stemness transcription factors and markers, such as NANOG, OCT3/4, CD34, NESTIN, and SOX2, in metastasis initiation has been studied recently because their abnormally elevated expression in cancer cells may be highly important in understanding tumor initiation. This study analyzed the genetic profiles of the above genes in CTCs derived from patients with different types of cancer. Blood samples were randomly collected from 71 cancer patients with various cancer types. CTCs were isolated using enrichment protocols and RNA was extracted. RT-qPCR was performed in triplicate using ACTB as the reference gene. The statistical analysis was performed among the ΔCts of the samples using parametric and non-parametric methods. The molecular analysis revealed that the expression of each gene was different than the others. When each type of cancer was analyzed separately, the gene expression profile was not always the same. It is noteworthy that, in all cases, the gene expression of NESTIN differed from that of transcription factors. According to the above data, gene expression profiles might be used as a potential biomarker or constitute a gene signature.

Development of a 2-chamber culture system for impedimetric monitoring of cell-cell interaction

In cancer research, study of cell-cell interaction is important to understand tumor initiation, progression, metastasis, and therapeutic resistance. Conventionally, transwell system was adopted and cell proliferation was quantified by end-point bio-assays. The operations are labor-intensive and time-consuming while studying of the dynamic cellular responses of cell-cell interaction. Although impedance measurement was suggested to be a promising technique to monitor cellular responses, electrodes cannot be integrated into the transwell for the measurement purpose. In this work, a 2-chamber culture system incorporated with impedance measurement technique was developed to quantitatively study cell-cell interaction. The chamber was composed of 2 sub-chambers separated with a barrier. By this design, two types of cells could be independently cultured and concurrently monitored under common medium supplied. Cell-cell interaction was demonstrated by aberrant cell proliferation induced by the EGF secreted from the transfected cells cultured on another sub-chamber. Real-time and non-invasive monitoring of cell-cell interaction was successfully demonstrated. This work provides a practical solution for monitoring the dynamic cellular responses of cell-cell interaction during the culture course. It is a reliable and convenient platform and facilitate more quantitative assessments in cancer research.

Abstract 1431: An optimized in vitro method to evaluate primary tumor cell invasion using optical imaging technology

Abstract The recent development of human-in-mouse tumor models has provided better representative working models to understand tumor initiation and progression than previous mouse tumor models or cell lines. However, it has not been feasible to use these models for high-throughput functional or therapeutic screening. Our aim was to establish a complementary in vitro culturing system for high-throughput functional studies or therapeutic screening of breast cancer stem cells (BCSCs). We hypothesize that with extracellular matrix and stromal cell support, three-dimensional (3D) cultures can support the survival and invasion of primary human tumors in vitro. We report here a new method to objectively evaluate and quantify invasion of primary breast cancer tumor cells using Transwells and bioluminescence imaging (BLI) technology. We optimized the adhesion and invasion conditions for these primary tumor cells. We also labeled cells using Luc2-EGFP or -tdTomato lentiviral vectors so that the signal of all invaded cells could be quantified using BLI or fluorescence imaging of the entire bottom chamber of the Transwell system. Our results demonstrate that stromal cells are not only necessary for the survival of the primary tumor cells but also facilitate their invasion in vitro. This work establishes a new method for screening candidate genes, miRNAs and compounds that regulate growth, survival, invasion and drug sensitivity of primary tumor cells, including both BCSCs and non-BCSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2011-1431

Abstract 3099: Stress response gadd45a gene as tumor suppressor in MYC expressing myeloid cells is cytokine specific

Abstract MYC, which regulates proliferation, apoptosis/survival and differentiation, is implicated in the etiology of a wide variety of hematologic malignancies. Multiple cooperating molecular pathways of cell survival and apoptosis determine if a cell lives or dies, and understanding how c-MYC interfaces with these pathways to influence the survival of cells is important to understand tumor initiation and progression, and response of tumors to different treatment regimens. Previously, this laboratory has shown that deregulated c-MYC blocks terminal myeloid differentiation and prematurely recruits both the Type I and Type II CD95/Fas apoptotic pathways, promoting an incompletely penetrant apoptotic response. Here we provide data to show that the response of myeloid cells to deregulated MYC expression depends on the status of the Gadd45 family of stress response genes. The gadd45 gene family plays pivotal roles as stress sensors that modulate signaling in response to physiological and environmental stressors, also modulating susceptibility of cells for transformation in vitro and tumor development in vivo. Gadd45 behaves as either tumor suppressor or oncogene depending upon the transforming oncogene and the cell type. To elucidate the role Gadd45a plays in response to the proto-oncogene c-MYC in myeloid cells, bone marrow (BM) cells from wild type (WT) and Gadd45a null mice were retrovirally infected to constitutively express c-MYC. We showed that Gadd45a null BM expressing constitutive c-MYC exhibited less apoptosis than its WT counterpart in expansion media (IL-3, IL-6, SCF), demonstrating that Gadd45a is required for optimal MYC mediated apoptosis. In addition, enhancement of cell cycle progression was observed. Therefore, loss of gadd45a in conjunction with constitutive MYC expression results in enhanced proliferation. Furthermore, in GM-CSF treated cells the MYC-mediated block/delay in differentiation was more extensive in the Gadd45a null cells compared to similarly treated WT cells. Interestingly, the percent of apoptosis was higher in the Gadd54a null cells expressing constitutive MYC as compared to the WT counterpart. This observation was in contrast to the results seen in expansion media, suggesting that the role Gadd45a plays in the presence of deregulated MYC may be cytokine specific. In addition, preliminary data also suggests that Gadd45a null bone marrow expressing deregulated c-myc loose responsiveness to MCSF and GCSF. Data will be presented to explain how gadd45 regulates both the apoptotic response, depending upon the specific cytokine, and differentiation of MYC-expressing myeloid cells. Furthermore, experiments to determine how loss of gadd45a influences MYC-mediated leukemia, including assessing the effect of manipulating the cytokine milieu, are currently underway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3099.

Stress Response Gadd45a Gene as Tumor Suppressor in MYC Expressing Myeloid Cells Is Cytokine Specific.

Abstract 1481Poster Board I-504MYC, which regulates proliferation, apoptosis/survival and differentiation, is implicated in the etiology of a wide variety of hematologic malignancies. Multiple cooperating molecular pathways of cell survival and apoptosis determine if a cell lives or dies, and understanding how c-MYC interfaces with these pathways to influence the survival of cells is important to understand tumor initiation and progression, and response of tumors to different treatment regimens. Previously, this laboratory has shown that deregulated c-MYC blocks terminal myeloid differentiation and prematurely recruits both the Type I and Type II CD95/Fas apoptotic pathways, promoting an incompletely penetrant apoptotic response (Amanullah et al., Oncogene 19:2967-77, 2000; Amanullah et al., Oncogene 21;1600-10, 2002). Here we provide data to show that the response of myeloid cells to deregulated MYC expression depends on the status of the Gadd45 family of stress response genes. The gadd45 gene family plays pivotal roles as stress sensors that modulate signaling in response to physiological and environmental stressors, also modulating susceptibility of cells for transformation in vitro and tumor development in vivo. Gadd45 behaves as either tumor suppressor or oncogene depending upon the transforming oncogene and the cell type (Tront et al., Cancer Research 66:8448-54, 2006; Tront et al., manuscript in press). To elucidate the role Gadd45a plays in response to the proto-oncogene c-MYC in myeloid cells, bone marrow (BM) cells from wild type (WT) and Gadd45a null mice were retrovirally infected to constitutively express c-MYC. We showed that Gadd45a null BM expressing constitutive c-MYC exhibited less apoptosis than its WT counterpart in expansion media (IL-3, IL-6, SCF), demonstrating that Gadd45a is required for optimal MYC mediated apoptosis. In addition, enhancement of cell cycle progression was observed. Therefore, loss of gadd45a in conjunction with constitutive MYC expression results in enhanced proliferation. Furthermore, in GM-CSF treated cells the MYC–mediated block/delay in differentiation was more extensive in the Gadd45a null cells compared to similarly treated WT cells. Interestingly, the percent of apoptosis was higher in the Gadd54a null cells expressing constitutive MYC as compared to the WT counterpart. This observation was in contrast to the results seen in expansion media, suggesting that the role Gadd45a plays in the presence of deregulated MYC may be cytokine specific. Data will be presented to explain how gadd45 regulates both the apoptotic response, depending upon the specific cytokine, and differentiation of MYC-expressing myeloid cells. Furthermore, experiments to determine how loss of gadd45a influences MYC-mediated leukemia, including assessing the effect of manipulating the cytokine milieu, are currently underway. Disclosures:No relevant conflicts of interest to declare.

Genetic predisposition and somatic diversification in tumor development and progression

Studies on human cancer predisposition syndromes have contributed significantly to our understanding on tumor initiation and progression. Work performed on hereditary colon cancer has been particularly fruitful. Much of the molecular background of the various intestinal polyposis syndromes, such as familial adenomatous polyposis (FAP), juvenile polyposis, and Peutz-Jeghers syndrome, has been revealed, pinpointing several key cancer-associated genes. Studies on hereditary nonpolyposis colorectal cancer (HNPCC) have revealed a novel mechanism of tumorigenesis; genomic instability caused by defective DNA mismatch repair (MMR). Understanding the molecular background of these diseases helps us to understand tumor initiation in the affected individuals. Relatively little is known about the details of tumor progression in hereditary and sporadic neoplasia. Certain additional gene mutations can be associated with advancing stages of the disease, but the pace and tempo of the process have remained obscure. A high mutation rate in MMR-deficient tumors has provided a new approach in the analysis of human tumor dynamics. Microsatellite (MS) sequences are frequently mutated in MMR deficient tumors. The high mutation rate allows the use of microsatellite mutations as a tool for analyzing the past patterns of tumor progression. This approach is similar to the use of MS mutations in studying human evolution and migrations. Such tumor studies have revealed progression pathways that differ from the classic adenoma-cancer sequence. The reasons why and how molecular clocks may reveal something new about a well-studied problem are discussed.