Introduction of a New Toxicology Consult Service in a Large Tertiary Care Teaching Hospital.

BACKGROUND: Clinical toxicology is not a certified specialty in Israel, consequently there are a limited number of toxicologists and toxicology services available for consultation. OBJECTIVES: To establish a medical toxicology consultation service focusing on bedside consultations, which had not previously been available in Israel. METHODS: This single-center, retrospective chart review of toxicology consults was conducted during the first years after the initiation of a new toxicology service. RESULTS: From September 2017 to December 2021, 1703 toxicology consultations were conducted. The most common exposures and reasons for consultation included psychotropic medications (427, 23%), analgesics and anti-inflammatory medications (353, 19%), household products (312, 17%), substances of abuse (240, 13%), and natural toxins (142, 8%). Bedside medical toxicology consultations were performed in 1036 cases (62%) during daytime and night shifts. The number of consultation requests increased steadily over the study period. CONCLUSIONS: The new toxicology service led to a significant change in the institution's approach to toxicological patients. A bedside toxicology service could help reduce the healthcare burden on national poison centers and can offer readily available, personalized, medical toxicology care.

Patient-derived xenografts for individualized care in advanced sarcoma.

BACKGROUND: Patients with advanced, metastatic sarcoma have a poor prognosis, and the overall benefit from the few standard-of-care therapeutics available is small. The rarity of this tumor, combined with the wide range of subtypes, leads to difficulties in conducting clinical trials. The authors previously reported the outcome of patients with a variety of common solid tumors who received treatment with drug regimens that were first tested in patient-derived xenografts using a proprietary method ("TumorGrafts"). METHODS: Tumors resected from 29 patients with sarcoma were implanted into immunodeficient mice to identify drug targets and drugs for clinical use. The results of drug sensitivity testing in the TumorGrafts were used to personalize cancer treatment. RESULTS: Of 29 implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although 6 patients died before the completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of 16 (81%) of the remaining individuals. No patients progressed during the TumorGraft-predicted therapy. CONCLUSIONS: The current data support the use of the personalized TumorGraft model as an investigational platform for therapeutic decision-making that can guide treatment for rare tumors such as sarcomas. A randomized phase 3 trial versus physician's choice is warranted.

A Retrospective Study Reviewing Interprofessional Advance Care Planning Group Discussions in Pulmonary Rehabilitation: A Proof-of-Concept and Feasibility Study.

BACKGROUND: Advance care planning (ACP) is a process of reflection and discussion wherein a patient, in consultation with a health-care provider, family, and/or loved ones, clarifies values and treatment preferences and establishes goals, including a plan for end-of-life (EOL) care. Advance care planning encompasses appreciating and understanding illness and treatment options, elucidating patient values and beliefs, and identifying a substitute decision maker (SDM) or designating a power of attorney (POA) for personal care. These discussions have proven to be effective in improving patient-family satisfaction, reducing anxiety regarding EOL care in patients and family members, and improving patient-centered care by empowering patients to direct their care at EOL. However, ACP conversations are often difficult to have due to the sensitive nature of such discussions. OBJECTIVE: The aim of this study was to determine whether group facilitation for teaching and discussing ACP enhances participants' understanding of ACP and allows them to feel comfortable and supported when discussing these sensitive issues. METHODS: Patients who were registered in North York General Hospital's (NYGH) pulmonary rehabilitation program from June 2016 until August 2017 were given the opportunity to attend two 1-hour sessions related to ACP. The first session was dedicated to educating patients on ACP, explaining the hierarchy of the SDM and the role of the POA for personal care. The second session, provided a short time later, was devoted to discussions of values, wishes, fears, and trade-offs for future medical and EOL care. These discussions led by the supportive care nurse practitioner and a physician who are members of the NYGH Freeman Palliative Care Team were held in a group-facilitated format. Anonymous feedback forms, including both qualitative and quantitative feedback, were completed by the participants and analyzed. PARTICIPANTS: Analysis of a sample of 30 participants who attended 1 or 2 of the ACP sessions revealed that 21 identified as female and 9 identified as male. The average age of the participants was 76 years. FINDINGS: Participants felt the content was relevant to their needs and were comfortable asking questions with all feedback averages ranging from good to very good. Participants appreciated the opportunity to share their thoughts in an open and interactive format. CONCLUSION: Discussing issues relevant to ACP, including providing information about ACP, sharing fears, wishes, and tradeoffs, were well-received in a group-support environment. Future studies should assess the impact of ACP group discussion on the individual, such as identifying a POA, having discussions regarding wishes and values with the SDM/POA, and examining the clinical impact of such sessions.

Publication outcomes of abstracts presented at 2013 North American Congress of Clinical Toxicology (NACCT) conference.

Introduction: Scientific conferences are useful in disseminating medical research and advancing the medical and scientific fields. An important measure of the success of such conferences is the proportion of research that is published in peer-reviewed journals. The conversion rates for toxicology abstracts to full-text publications at previous North American toxicology meetings were low. No study has assessed the publication rate from the 2013 North American Congress of Clinical Toxicology (NACCT) conference.Methods: We reviewed 316 abstracts presented at the 2013 NACCT Conference. We searched the PubMed, EMBASE, and Medline databases using the authors' names and keywords, through September 2019. We then identified and excluded cases and case reports to reanalyze the data.Results: Thirty-three of 316 abstracts (10.4%) subsequently appeared in 17 different peer-reviewed journals, led by Clinical Toxicology (13 out of 33, 3%). Leading countries of origin for abstract submission were the USA (285), Canada (9), and the UK (8). Excluding case reports, 25 out of 207 abstracts (12.0%) achieved publication in peer-reviewed journals.Conclusions: Fewer than one in eight abstracts reached publication within six years of the 2013 NACCT meeting, even after accounting for and excluding case reports. This rate is lower than in other specialty medical societies.

Growth of Triple Negative and Progesterone Positive Breast Cancer Causes Oxidative Stress and Down-Regulates Neuroprotective Transcription Factor NPAS4 and NPAS4-Regulated Genes in Hippocampal Tissues of TumorGraft Mice-an Aging Connection.

While the refinement of existing and the development of new chemotherapeutic regimens has significantly improved cancer treatment outcomes and patient survival, chemotherapy still causes many persistent side effects. Central nervous system (CNS) toxicity is of particular concern, as cancer patients experience significant deficits in memory, learning, cognition, and decision-making. These chemotherapy-induced cognitive changes are termed chemo brain, and manifest in more than half of cancer survivors. Moreover, recent studies have emerged suggesting that neurocognitive deficits manifest prior to cancer diagnosis and treatment, and thus may be associated with tumor presence, a phenomenon recently termed "tumor brain." To dissect the molecular mechanisms of tumor brain, we used TumorGraftTM models, wherein part of a patient's tumor is grafted into immune-deficient mice. Here, we analyzed molecular changes in the hippocampal tissues of mice carrying triple negative (TNBC) or progesterone receptor positive (PR+BC) xenografts. TNBC growth led to increased oxidative damage, as detected by elevated levels of 4-hydroxy-2-nonenal, a product of lipid peroxidation. Furthermore, the growth of TNBC and PR+BC tumors altered global gene expression in the murine hippocampus and affected multiple pathways implicated in PI3K-Akt and MAPK signaling, as well as other pathways crucial for the proper functioning of hippocampal neurons. TNBC and PR+BC tumor growth also led to a significant decrease in the levels of neuronal transcription factor NPAS4, a regulator that governs the expression of brain-derived neurotrophic factor (BDNF), and several other key brain neurotrophic factors and pro-survival molecules. The decreased expression of ERK1/2, NPAS4, and BDNF are also seen in neurodegenerative conditions and aging, and may constitute an important tumor brain mechanism.

A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk.

Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer-associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.

Growth of malignant extracranial tumors alters microRNAome in the prefrontal cortex of TumorGraft mice.

A wide array of central nervous system complications, neurological deficits, and cognitive impairments occur and persist as a result of systemic cancer and cancer treatments. This condition is known as chemo brain and it affects over half of cancer survivors. Recent studies reported that cognitive impairments manifest before chemotherapy and are much broader than chemo brain alone, thereby adding in tumor brain as a component. The molecular mechanisms of chemo brain are under-investigated, and the mechanisms of tumor brain have not been analyzed at all. The frequency and timing, as well as the long-term persistence, of chemo brain and tumor brain suggest they may be epigenetic in nature. MicroRNAs, small, single-stranded non-coding RNAs, constitute an important part of the cellular epigenome and are potent regulators of gene expression. miRNAs are crucial for brain development and function, and are affected by a variety of different stresses, diseases and conditions. However, nothing is known about the effects of extracranial tumor growth or chemotherapy agents on the brain microRNAome. We used the well-established TumorGraft ™ mouse models of triple negative (TNBC) and progesterone receptor positive (PR+BC) breast cancer, and profiled global microRNAome changes in tumor-bearing mice upon chemotherapy, as compared to untreated tumor-bearing mice and intact mice. Our analysis focused on the prefrontal cortex (PFC), based on its roles in memory, learning, and executive functions, and on published data showing the PFC is a target in chemo brain. This is the first study showing that tumor presence alone significantly impacted the small RNAome of PFC tissues. Both tumor growth and chemotherapy treatment affected the small RNAome and altered levels of miRNAs, piRNAs, tRNAs, tRNA fragments and other molecules involved in post-transcriptional regulation of gene expression. Amongst those, miRNA changes were the most pronounced, involving several miRNA families, such as the miR-200 family and miR-183/96/182 cluster; both were deregulated in tumor-bearing and chemotherapy-treated animals. We saw that miRNA deregulation was associated with altered levels of brain-derived neurotrophic factor (BDNF), which plays an important role in cognition and memory and is one of the known miRNA targets. BDNF downregulation has been associated with an array of neurological conditions and could be one of the mechanisms underlying tumor brain and chemo brain. In the future our study could serve as a roadmap for further analysis of cancer and chemotherapy's neural side effects, and differentially expressed miRNAs should be explored as potential tumor brain and chemo brain biomarkers.

Chemo brain or tumor brain - that is the question: the presence of extracranial tumors profoundly affects molecular processes in the prefrontal cortex of TumorGraft mice.

Cancer chemotherapy causes numerous persistent central nervous system complications. This condition is known as chemo brain. Cognitive impairments occur even before treatment, and hence are referred to as cancer associated cognitive changes, or tumor brain. There is much yet to be learned about the mechanisms of both chemo brain and tumor brain. The frequency and timing of chemo brain and tumor brain occurrence and persistence strongly suggest they may be epigenetic in nature and associated with altered gene expression. Here we used TumorGraftTM models wherein part of a patient's tumor is removed and grafted into immune-deficient mice and conducted global gene expression and DNA methylation analysis. We show that malignant non-central nervous system tumor growth causes profound molecular alterations in the brain. Mice harbouring triple negative or progesterone positive breast cancer TumorGrafts exhibited altered gene expression, decreased levels of DNA methylation, increased levels of DNA hydroxymethylation, and oxidative stress in the prefrontal cortex. Interestingly, chemotherapy did not have any additional synergistic effects on the analyzed processes. The molecular changes observed in this study are known signs of neurodegeneration and brain aging. This study provides an important roadmap for future large-scale analysis of the molecular and cellular mechanisms of tumor brain.

RET Fusion Lung Carcinoma: Response to Therapy and Clinical Features in a Case Series of 14 Patients.

BACKGROUND: RET (rearranged during transfection) fusions have been reported in 1% to 2% of lung adenocarcinoma (LADC) cases. In contrast, KIF5B-RET and CCDC6-RET fusion genes have been identified in 70% to 90% and 10% to 25% of tumors, respectively. The natural history and management of RET-rearranged LADC are still being delineated. MATERIALS AND METHODS: We present a series of 14 patients with RET-rearranged LADC. The response to therapy was assessed by the clinical response and an avatar model in 2 cases. Patients underwent chemotherapy, targeted therapy, and immunotherapy. RESULTS: A total of 14 patients (8 women; 10 never smokers; 4 light smokers; mean age, 57 years) were included. KIF5B-RET and CCDC6-RET variants were diagnosed in 10 and 4 cases, respectively. Eight patients had an early disseminated manifestation, seven with KIF5B-RET rearranged tumor. The features of this subset included bilateral miliary lung metastases, bone metastases, and unusual early visceral abdominal involvement. One such patient demonstrated an early and durable complete response to cabozantinib for 7 months. Another 2 patients treated with cabozantinib experienced a partial response, with rapid significant clinical improvement. Four patients with tumors harboring CCDC6-RET and KIF5B-RET fusions showed pronounced and durable responses to platinum-based chemotherapy that lasted for 8 to 15 months. Two patients' tumors showed programmed cell death ligand 1-positive staining but did not respond to pembrolizumab. The median overall survival was 22.8 months. CONCLUSION: RET-rearranged LADC in our series tended to occur as bilateral disease with early visceral involvement, especially with KIF5B fusion. Treatment with cabozantinib achieved responses, including 1 complete response. However, further studies are required in this group of patients.

Alveolar rhabdomyosarcoma: morphoproteomics and personalized tumor graft testing further define the biology of PAX3-FKHR(FOXO1) subtype and provide targeted therapeutic options.

Alveolar rhabdomyosarcoma (ARMS) represents a block in differentiation of malignant myoblasts. Genomic events implicated in the pathogenesis of ARMS involve PAX3-FKHR (FOXO1) or PAX7-FKHR (FOXO1) translocation with corresponding fusion transcripts and fusion proteins. Commonalities in ARMS include uncontrollable proliferation and failure to differentiate. The genomic-molecular correlates contributing to the etiopathogenesis of ARMS incorporate PAX3-FKHR (FOXO1) fusion protein stimulation of the IGF-1R, c-Met and GSK3-ß pathways. With sequential morphoproteomic profiling on such a case in conjunction with personalized tumor graft testing, we provide an expanded definition of the biology of PAX3-FKHR (FOXO1) ARMS that integrates genomics, proteomics and pharmacogenomics. Moreover, therapies that target the genomic and molecular biology and lead to tumoral regression and/or tumoral growth inhibition in a xenograft model of ARMS are identified. SIGNIFICANCE: This case study could serve as a model for clinical trials using relatively low toxicity agents in both initial and maintenance therapies to induce remission and reduce the risk of recurrent disease in PAX3-FKHR (FOXO1) subtype of ARMS.

MEK inhibitors block growth of lung tumours with mutations in ataxia-telangiectasia mutated.

Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.

Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell phenotypes and promotes aggressive tumor growth.

Stem-like glioma cells reside within a perivascular niche and display hallmark radiation resistance. An understanding of the mechanisms underlying these properties will be vital for the development of effective therapies. Here, we show that the stem cell marker CD44 promotes cancer stem cell phenotypes and radiation resistance. In a mouse model of glioma, Cd44(-/-) and Cd44(+/-) animals showed improved survival compared to controls. The CD44 ligand osteopontin shared a perivascular expression pattern with CD44 and promoted glioma stem cell-like phenotypes. These effects were mediated via the γ-secretase-regulated intracellular domain of CD44, which promoted aggressive glioma growth in vivo and stem cell-like phenotypes via CBP/p300-dependent enhancement of HIF-2α activity. In human glioblastoma multiforme, expression of CD44 correlated with hypoxia-induced gene signatures and poor survival. Altogether, these data suggest that in the glioma perivascular niche, osteopontin promotes stem cell-like properties and radiation resistance in adjacent tumor cells via activation of CD44 signaling.

Integrated next-generation sequencing and avatar mouse models for personalized cancer treatment.

BACKGROUND: Current technology permits an unbiased massive analysis of somatic genetic alterations from tumor DNA as well as the generation of individualized mouse xenografts (Avatar models). This work aimed to evaluate our experience integrating these two strategies to personalize the treatment of patients with cancer. METHODS: We performed whole-exome sequencing analysis of 25 patients with advanced solid tumors to identify putatively actionable tumor-specific genomic alterations. Avatar models were used as an in vivo platform to test proposed treatment strategies. RESULTS: Successful exome sequencing analyses have been obtained for 23 patients. Tumor-specific mutations and copy-number variations were identified. All samples profiled contained relevant genomic alterations. Tumor was implanted to create an Avatar model from 14 patients and 10 succeeded. Occasionally, actionable alterations such as mutations in NF1, PI3KA, and DDR2 failed to provide any benefit when a targeted drug was tested in the Avatar and, accordingly, treatment of the patients with these drugs was not effective. To date, 13 patients have received a personalized treatment and 6 achieved durable partial remissions. Prior testing of candidate treatments in Avatar models correlated with clinical response and helped to select empirical treatments in some patients with no actionable mutations. CONCLUSION: The use of full genomic analysis for cancer care is encouraging but presents important challenges that will need to be solved for broad clinical application. Avatar models are a promising investigational platform for therapeutic decision making. While limitations still exist, this strategy should be further tested.

Astrocyte-specific expression patterns associated with the PDGF-induced glioma microenvironment.

BACKGROUND: The tumor microenvironment contains normal, non-neoplastic cells that may contribute to tumor growth and maintenance. Within PDGF-driven murine gliomas, tumor-associated astrocytes (TAAs) are a large component of the tumor microenvironment. The function of non-neoplastic astrocytes in the glioma microenvironment has not been fully elucidated; moreover, the differences between these astrocytes and normal astrocytes are unknown. We therefore sought to identify genes and pathways that are increased in TAAs relative to normal astrocytes and also to determine whether expression of these genes correlates with glioma behavior. METHODOLOGY/PRINCIPAL FINDINGS: We compared the gene expression profiles of TAAs to normal astrocytes and found the Antigen Presentation Pathway to be significantly increased in TAAs. We then identified a gene signature for glioblastoma (GBM) TAAs and validated the expression of some of those genes within the tumor. We also show that TAAs are derived from the non-tumor, stromal environment, in contrast to the Olig2+ tumor cells that constitute the neoplastic elements in our model. Finally, we validate this GBM TAA signature in patients and show that a TAA-derived gene signature predicts survival specifically in the human proneural subtype of glioma. CONCLUSIONS/SIGNIFICANCE: Our data identifies unique gene expression patterns between populations of TAAs and suggests potential roles for stromal astrocytes within the glioma microenvironment. We show that certain stromal astrocytes in the tumor microenvironment express a GBM-specific gene signature and that the majority of these stromal astrocyte genes can predict survival in the human disease.

Recruited cells can become transformed and overtake PDGF-induced murine gliomas in vivo during tumor progression.

BACKGROUND: Gliomas are thought to form by clonal expansion from a single cell-of-origin, and progression-associated mutations to occur in its progeny cells. Glioma progression is associated with elevated growth factor signaling and loss of function of tumor suppressors Ink4a, Arf and Pten. Yet, gliomas are cellularly heterogeneous; they recruit and trap normal cells during infiltration. METHODOLOGY/PRINCIPAL FINDINGS: We performed lineage tracing in a retrovirally mediated, molecularly and histologically accurate mouse model of hPDGFb-driven gliomagenesis. We were able to distinguish cells in the tumor that were derived from the cell-of-origin from those that were not. Phenotypic, tumorigenic and expression analyses were performed on both populations of these cells. Here we show that during progression of hPDGFb-induced murine gliomas, tumor suppressor loss can expand the recruited cell population not derived from the cell-of-origin within glioma microenvironment to dominate regions of the tumor, with essentially no contribution from the progeny of glioma cell-of-origin. Moreover, the recruited cells can give rise to gliomas upon transplantation and passaging, acquire polysomal expression profiles and genetic aberrations typically present in glioma cells rather than normal progenitors, aid progeny cells in glioma initiation upon transplantation, and become independent of PDGFR signaling. CONCLUSIONS/SIGNIFICANCE: These results indicate that non-cell-of-origin derived cells within glioma environment in the mouse can be corrupted to become bona fide tumor, and deviate from the generally established view of gliomagenesis.

Gli activity correlates with tumor grade in platelet-derived growth factor-induced gliomas.

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor-induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show beta-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity.

Frequent inactivation of the tumor suppressor Kruppel-like factor 6 (KLF6) in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, reflecting incomplete characterization of underlying mechanisms and lack of early detection. Kruppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that is deregulated in multiple cancers through loss of heterozygosity (LOH) and/or inactivating somatic mutation. We analyzed the potential role of the KLF6 tumor suppressor gene in 41 patients who had HCC associated with hepatitis C virus (16 patients), hepatitis B virus (12 patients, one of whom was coinfected with hepatitis C virus), and other etiologies (14 patients) by determining the presence of LOH and mutations. Overall, LOH and/or mutations were present in 20 (49%) of 41 tumors. LOH of the KLF6 gene locus was present in 39% of primary HCCs, and the mutational frequency was 15%. LOH and/or mutations were distributed across all etiologies of HCC evaluated, including patients who did not have cirrhosis. Functionally, wild-type KLF6 decreased cellular proliferation of HepG2 cells, while patient-derived mutants did not. In conclusion, we propose that KLF6 is deregulated by loss and/or mutation in HCC, and its inactivation may contribute to pathogenesis in a significant number of these tumors.

Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer.

BACKGROUND & AIMS: Kruppel-like factor 6 (KLF6) is a ubiquitous zinc finger tumor suppressor that is often mutated in prostate cancer. Our aims were to establish the frequency of KLF6 inactivation in sporadic and inflammatory bowel disease (IBD)-associated colorectal cancers (CRC); to correlate these abnormalities with mutation and/or loss of TP53, APC, and K-RAS; and to characterize the behavior of mutant KLF6 in colon-derived cell lines. METHODS: We analyzed DNA isolated from 50 microdissected CRC cases, including 35 sporadic and 15 IBD-associated tumors. Microsatellite analysis and direct sequencing were used to establish the incidence of microsatellite instability, KLF6 and TP53 allelic imbalance, and KLF6, K-RAS, TP53, and APC mutation. Loss of growth suppressive function of the CRC-derived KLF6 mutants was characterized by in vitro thymidine incorporation assays and Western blotting. RESULTS: KLF6 was inactivated by loss and/or mutation in most sporadic and IBD-related CRCs. The KLF6 locus was deleted in at least 55% of tumors, and mutations were identified in 44%. Rates of KLF6 loss and mutation were similar to those of TP53 and K-RAS in the same samples. KLF6 mutations were present in tumors with either microsatellite or chromosomal instability and were more common, particularly in the IBD-related cancers, in the presence of wild-type APC. Unlike wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21 following transfection into cultured cells. CONCLUSIONS: Deregulation of KLF6 by a combination of allelic imbalance and mutation may play a role in the development of CRC.