Abnormalities in structure and expression of the human retinoblastoma gene in SCLC.

Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in 90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.

Transgenic expression of human thymidylate synthase accelerates the development of hyperplasia and tumors in the endocrine pancreas.

Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and repair and elevated levels of TS have been identified as an important prognostic biomarker for colorectal cancer and several other common human malignancies. In addition, TS gene expression has been linked with cell-cycle regulation and cell proliferation through the ability of retinoblastoma protein to repress the transcriptional activation of E2F target genes such as TS. Therefore, overproduction of TS could participate in the progression to a neoplastic phenotype. Consistent with this model, a recent study has suggested that ectopic TS expression can induce a transformed phenotype in mammalian cells. To investigate the role of deregulated TS activity in tumor development, we generated transgenic mice that express high levels of catalytically active human TS (hTS) exclusively in the pancreas and low levels of hTS in multiple other tissues. Analyses of pancreatic tissue in TS transgenic mice revealed abnormalities within the endocrine pancreas, ranging from pancreatic islet hyperplasia to the detection of islet cell tumors. Overexpression of hTS in murine islets provides a new model to study genetic alterations associated with the progression from normal cells to hyperplasia to islet cell tumors, and suggests that this mouse model may be useful for regulating TS activity in vivo for development of cancer prevention and new therapies.

Sustained expression of Mect1-Maml2 is essential for tumor cell growth in salivary gland cancers carrying the t(11;19) translocation.

Mucoepidermoid (MEC) salivary gland tumors arise from a t(11;19) rearrangement which generates a fusion oncogene, Mect1-Maml2, that functions to activate CREB-responsive target genes. To determine if sustained expression of Mect1-Maml2 is required for tumor cell growth, we first showed that ectopic expression of Mect1-Maml2 in rat epithelial RK3E cells is tumorigenic in vivo in nude mice and that excised xenografts continue to express the fusion oncogene. We then generated a hairpin RNAi vector that selectively suppressed the fusion peptide and showed that ectopic expression in either parotid or pulmonary MEC tumor cell lines containing the t(11;19) rearrangement resulted in at least 90% colony growth inhibition. In contrast, single nucleotide changes within this RNAi sequence abolished the ability to suppress Mect1-Maml2 protein and abolished all growth inhibition of these MEC tumor lines. In addition, the RNAi-specific vector had no effect on colony growth of non-MEC tumors including a lung tumor or two other salivary gland cell lines that do not express Mect1-Maml2. We also generated a mutant Mect1-Maml2 expression plasmid that carried silent nucleotide changes within the RNAi target sequence and observed that co-transfection of this mutant, but not wild-type Mect1-Maml2, could partially rescue RNAi growth inhibition in the MEC tumor line. The recent detection of acquired fusion oncogenes in epithelial solid tumors has suggested new possibilities for the diagnosis and therapy of these cancers. Our data show that the 'gain-of-function' activity from aberrant Mect1-Maml2 expression is a candidate therapeutic target for this group of malignant salivary gland tumors.

Human creatine kinase-B complementary DNA. Nucleotide sequence, gene expression in lung cancer, and chromosomal assignment to two distinct loci.

Using a small cell lung cancer (SCLC) cDNA library, we obtained clones for the creatine kinase-B (CK-B) gene and determined the nucleotide sequence for the protein coding and 3' untranslated region (3' UT). The human translated protein spans 381 residues and the amino acid homology with rabbit CK-B is greater than 98%. We have demonstrated that a nucleic acid probe encompassing the protein coding region will also hybridize to CK-M sequences while a probe derived from the 3' UT region is CK-B specific. When a B-isoenzyme specific sequence is hybridized to Eco RI cut genomic DNA, two independent restriction fragment polymorphisms are detected. We have subsequently localized these two CK-B homologous sequences to chromosomes 14q32 and 16. Finally, we show that increased levels of CK-B seen in SCLC are not accompanied by gene amplification or rearrangement, but reflect a greatly enhanced level of CK-B specific mRNA that is not seen in non-SCLC lines thus far examined.

Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein.

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

The N-terminal domain of c-Myc associates with alpha-tubulin and microtubules in vivo and in vitro.

The polymerization of alpha- and beta-tubulin into microtubules results in a complex network of microfibrils that have important structural and functional roles in all eukaryotic cells. In addition, microtubules can interact with a diverse family of polypeptides which are believed to directly promote the assembly of microtubules and to modulate their functional activity. We have demonstrated that the c-Myc oncoprotein interacts in vivo and in vitro with alpha-tubulin and with polymerized microtubules and have defined the binding site to the N-terminal region within the transactivation domain of c-Myc. In addition, we have shown that c-Myc colocalizes with microtubules and remains tightly bound to the microtubule network after detergent extraction of intact cells. These findings suggest a potential role for Myc-tubulin interaction in vivo.

Disruption of Myc-tubulin interaction by hyperphosphorylation of c-Myc during mitosis or by constitutive hyperphosphorylation of mutant c-Myc in Burkitt's lymphoma.

Somatic mutations at Thr-58 of c-Myc have been detected in Burkitt's lymphoma (BL) tumors and have been shown to affect the transforming potential of the Myc oncoprotein. In addition, the N-terminal domain of c-Myc has been shown to interact with microtubules in vivo, and the binding of c-Myc to alpha-tubulin was localized to amino acids 48 to 135 within the c-Myc protein. We demonstrate that c-Myc proteins harboring a naturally occurring mutation at Thr-58 from BL cell lines have increased stability and are constitutively hyperphosphorylated, which disrupts the in vivo interaction of c-Myc with alpha-tubulin. In addition, we show that wild-type c-Myc-alpha-tubulin interactions are also disrupted during a transient mitosis-specific hyperphosphorylation of c-Myc, which resembles the constitutive hyperphosphorylation pattern of Thr-58 in BL cells.

Immunohistochemical analysis of the p16INK4 cyclin-dependent kinase inhibitor in malignant mesothelioma.

BACKGROUND: The identification in 1994 of the CDKN2 gene as a target for mutations in a wide range of human cancers, including malignant mesothelioma, has been controversial because subsequent studies have detected a lower frequency of CDKN2 gene mutations in primary tumors than in cultured cell lines. These reports raised the hypothesis that another gene, distinct from CDKN2, might be the target of the chromosome 9p21 deletions frequently observed in these tumors. PURPOSE: To address whether inactivation of CDKN2 function is an essential event in the etiology of malignant mesothelioma, we examined p16INK4 protein expression in primary thoracic mesotheliomas, in nonmalignant pleural tissues, and in independent mesothelioma cell lines. We also studied the growth rate of tumor cell lines following stable transfection of CDKN2 gene. METHODS: Retinoblastoma (Rb) and p16INK4 protein expression was determined by immunohistochemical analysis from archival paraffin specimens of 12 primary thoracic mesotheliomas and a nonmalignant pleural biopsy specimen. In addition, protein immunoblot analysis for Rb and p16INK4 expression was conducted on 15 independent mesothelioma cell lines, and the ability of a transfected CDKN2 gene to suppress the growth of the mesothelioma cell lines H2373 and H2461 in vitro was examined. RESULTS: We demonstrated abnormal p16INK4 expression in 12 of 12 primary mesothelioma specimens and in 15 of 15 mesothelioma cell lines. All tumor specimens and the tumor cell lines showed expression of wild-type Rb protein. In addition, we have confirmed the ability of a transfected CDKN2 gene to suppress growth of two independent mesothelioma cell lines. CONCLUSIONS: Immunohistochemical analysis of the p16INK4 gene product is feasible in archival biopsy samples. With this analysis, CDKN2 gene inactivation can be determined in tumors that are contaminated with nonmalignant cells. Furthermore, since loss of p16INK4 protein expression can result from both genetic (gene mutations) and epigenetic (abnormal DNA hypermethylation) mechanisms, as we and others have shown recently, examination of protein expression is a highly sensitive method for analyzing the CDKN2 status in large numbers of tumor samples. IMPLICATIONS: This study suggests that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas. Defining the role of the p16INK4:Rb tumor suppressor pathway and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.

Protein expression and functional analysis of the FHIT gene in human tumor cells.

BACKGROUND: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. METHODS: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal anti-pFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. RESULTS: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. CONCLUSIONS: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the "classic" tumor suppressor paradigm.

Alternative splicing of the RBP1 gene clusters in an internal exon that encodes potential phosphorylation sites.

We have isolated cDNA and genomic clones for the human retinoblastoma binding protein 1 (RBP1) gene, and have identified alternative splicing of RBP1 clustered within a 207-nucleotide internal exon. Three of the predicted RPB1 peptides share amino-terminal and carboxy-terminal domains, while a fourth species encodes a distinct carboxy-terminal domain. Functional analysis of these peptides demonstrated that they are capable of precipitating retinoblastoma (RB) protein in vitro from K562 cell lysates, but cannot bind to mutant RB protein. However, each of the RBP1 peptides differed within an internal exon that contains potential casein kinase II and p34cdc2 phosphorylation sites. Immunoblot analysis using polyclonal alpha-RBP1 antiserum revealed that the RBP1 protein is expressed in a wide range of cell lines of differing histologic type and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis predominantly as a 200-kDa protein. Immunohistochemical analysis using the alpha-RBP1 antiserum demonstrated a distinct nuclear staining pattern that was eliminated when the antiserum was preabsorbed with RBP1 peptide. The RBP1 gene encodes a widely expressed 200-kDa nuclear protein and undergoes alternative splicing that predicts a family of RB-binding peptides.

Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding.

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with alpha-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).

Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB.

Cell cycle dependent phosphorylation of the RB tumor suppressor protein is mediated by a family of G1 cyclin dependent kinases (cdks) and cyclins including the activated cdk4:cyclin D complex. The identification of a cdk4 inhibitor, p16INK4, as a target for mutations in cultured tumor lines and primary tumors suggested that RB activity may be affected in these cells. We have examined 88 lung cancer lines for p16INK4 protein expression and have observed a striking inverse correlation between the presence of p16INK4 and wildtype RB. We demonstrated that only 6/55 (11%) of small cell lung cancer (SCLC) samples had absent p16INK4 protein, and all 6 belonged to the rare subset of SCLC with wildtype RB expression. Conversely of 48 SCLC samples with absent or mutant RB, all showed detectable levels of p16INK4 protein. In contrast, we observed that 23/33 (70%) of non-SCLC samples had loss of p16INK4. Twenty-two of 26 non-SCLC lines with wildtype RB had absent p16INK4 while 6 of 7 non-SCLC lines with absent or mutant RB had detectable p16INK4. The inverse correlation of RB and p16INK4 expression and the absence of p16INK4 inactivation in RB (-/-) SCLC lines (0/48) confirms a common p16INK4/RB growth suppressor pathway in human cancers and provides evidence that p16INK4, and not an adjacent gene on chromosome 9p, is a specific target for mutational events.

CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2'deoxycytidine.

Absent expression of the cyclin dependent kinase-inhibitor, p16INK4, is observed in a wide range of primary human cancers. Although homozygous deletions and point mutations have been reported in a subset of these tumors, the molecular basis for absent p16INK4 in other samples is unknown. We have examined 33 tumor cell lines and have shown that hypermethylation of a G:C-rich region within exon 1 of the CDKN2 gene was present in 100% of samples with wildtype RB expression and no detectable CDKN2 mutations. Treatment for at least 4 hours with the demethylating agent 5-aza 2'deoxycytidine, but not 5-azacytidine or 6-azacytidine, induces the prolonged expression of p16INK4 protein in each of these samples following a discrete 24-48 hour lag period. Consistent with the hypothesis that hypermethylation of the CDKN2 gene is a tumor-specific mechanism for gene inactivation, we observed hypomethylation at the exon 1 site exclusively in tumor lines that expressed p16INK4 or that had sustained inactivating point mutations within the CDKN2 open reading frame. These findings demonstrate a link between DNA methylation and the p16INK4:RB tumor suppressor pathway.

Protein expression of the RB-related gene family and SV40 large T antigen in mesothelioma and lung cancer.

Mutational inactivation of the RB-related gene RBL2/p130 has been reported as a common and important prognostic factor in human lung cancer. To examine the role of the RB-related gene family in lung cancer we analysed the protein expression of the RB gene in cell lines obtained from 83 patients with small cell lung cancer (SCLC) and 114 patients with non-SCLC that included 21 novel lung tumor samples. While we detected five new SCLC with mutant RB expression (RB inactivation in 75/83; 90.4%), we did not detect any RB mutations in the new non-SCLC cell lines (RB inactivation in 13/114 non-SCLC and mesothelioma; 11.4%). In addition, we detected expression of a full-length RBL1/p107 and RBL2/p130 species in every sample tested (RBL1 or RBL2 inactivation in 0/69) and confirmed that both RB-related gene products retain functional binding activity to the E1A viral oncoprotein. Since expression of SV40 Large T antigen (Tag) has been reported in a subset of human lung tumors where it may inactivate RBL1 and RBL2, we also examined mesothelioma and non-mesothelioma lung tumors for Tag expression. Although we detected a faint 85 kDa protein species using specific anti-Tag antibodies, this signal migrated slightly faster than Tag extracted from Cos7 cells and did not exhibit binding activity to the RB or RBL1 proteins. Finally, we subjected 11 lung cancer cell lines to nucleotide sequencing and did not detect mutations within the C-terminal RBL2 exons 19-22 as recently reported. While the RB/p16 tumor suppressor pathway is targeted for mutations in 100% of lung cancers, mutational inactivation of the related RBL1 and RBL2 genes is a rare event.

RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer.

Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb(+/-) p53(+/-) mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.

Partial inactivation of the RB product in a family with incomplete penetrance of familial retinoblastoma and benign retinal tumors.

While familial retinoblastoma has served as the paradigm for the two-hit theory of tumorigenesis and for the concept of the tumor suppressor gene, the etiology of incomplete penetrance of familial retinoblastoma is poorly understood. To address the molecular basis for this phenotype we have studied the functional properties of a mutant Rb gene identified in a kindred with incomplete penetrance of familial retinoblastoma and evidence for regressed retinal lesions (retinomas). In contrast to all previously isolated RB mutant proteins, we demonstrated that the mutant product from this kindred retained the wildtype properties of nuclear localization, the ability to undergo hyperphosphorylation in vivo, and the capacity to suppress growth of RB(-) cells. Protein binding ('pocket') activity, however, was defective defining a new class of RB mutant with partial inactivation. The presence of this unique RB mutant in the germline of obligate carriers with incomplete penetrance and regressed retinal lesions suggests a molecular basis for this phenotype and supports the hypothesis that a minimum 'RB threshold' level of protein binding activity is required to suppress tumorigenesis.

The p16 status of tumor cell lines identifies small molecule inhibitors specific for cyclin-dependent kinase 4.

Loss of p16 functional activity leading to disruption of the p16/cyclin-dependent kinase (CDK) 4:cyclin D/retinoblastoma pathway is the most common event in human tumorigenesis, suggesting that compounds with CDK4 kinase inhibitory activity may be useful to regulate cancer cell growth. To identify such inhibitors, the 60 cancer cell lines of the National Cancer Institute drug screen panel were examined for p16 alterations (biallelic deletion, intragenic mutations, or absent p16 protein), and the growth-inhibitory activity of more than 50,000 compounds against these 60 cell lines was compared with their p16 status. One compound, 3-amino thioacridone (3-ATA; NSC 680434), whose growth-inhibitory activity correlated with the p16 status of the cell lines had an IC50 of 3.1 microM in a CDK4 kinase assay. In addition, four compounds structurally related to 3-ATA inhibited CDK4 kinase with IC50s ranging from 0.2-2.0 microM. All five of these compounds were less potent inhibitors of cell division cycle 2 and CDK2 kinases, with IC50s 30- to 500-fold higher than that for CDK4. ATP competition experiments demonstrated a noncompetitive mode of inhibition for 3-ATA (K(i) = 5.5 microM) and a linear mixed mode for benzothiadiazine (NSC 645787; K(i) = 0.73 microM). We have successfully demonstrated a novel approach to identify specific CDK4 kinase inhibitors that may selectively induce growth inhibition of p16-altered tumors.

A 'core ATPase', Hsp70-like structure is conserved in human, rat, and C. elegans STCH proteins.

We have identified the rat and Caenorhabditis elegans homologues of a 'core ATPase'-encoding Hsp70-like gene, designated Stch. We observed that the human, rat, and C. elegans Stch genes have conserved a stop codon immediately distal to the sequence encoding the Hsp70 ATPase domain. This results in the functional equivalent of an N-terminal, proteolytically cleaved fragment of Hsc70/BiP. Each homologue contains a hydrophobic signal sequence, demonstrates striking identity within the Hsp70 ATPase domain, and retains a similar C-terminal sequence (STCH specific cluster III) that is unique among Hsp70 proteins and which truncates the peptide binding domain. In addition, we have identified an internal 35-aa region that is homologous to the minimal sequence of the Hip chaperone co-factor that is required for direct binding to the ATPase domain of Hsp70. Adjacent to this region, the rat and human STCH protein sequences diverge within a short internal 'insertion' sequence that interrupts the ATPase subdomain between the phosphate-2 and adenosine ATP-binding sites. We have also demonstrated that both human and rat Stch are constitutively produced and are induced by the calcium ionophore A23187, but not by heat shock. The recognition that the truncated 'core ATPase' structure of the STCH molecule is conserved in human, rat, and C. elegans tissues suggests an important role for this unique member of the membrane-bound Hsp70 family.

A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch.

We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.

Clinical significance of the FV:Q506 mutation in unselected oncology patients.

PURPOSE: A common germline mutation in the factor V gene (FV:Q506) has been associated with hypercoagulability in families with heritable predisposition to thrombosis. We examined the prevalence and clinical significance of the FV:Q506 mutation in cancer patients. PATIENTS AND METHODS: We performed a retrospective cohort study by examining 353 consecutive, unselected patients in a general hematology/oncology clinic. We ascertained risk factors, obtained the clinical clotting history, and determined the heterozygous or homozygous presence of the FV:Q506 allele for each patient. RESULTS: We detected a germline mutation in 5.4% (19 of 353) of patients, of whom 18 were heterozygous and 1 was homozygous for the FV:Q506 mutant allele. In 17 of 18 heterozygous patients, there was no history of venous thrombosis or catheter-associated thrombosis. These asymptomatic patients included 13 patients who had been diagnosed with cancer or leukemia for a mean of 66.2 months (median 69) and had received a variety of local and systemic treatments. In contrast, 1 of 18 heterozygous and 1 of 1 homozygous patients had developed deep vein thrombosis that was associated, respectively, with either recurrent thrombotic events or a strong family history for pulmonary embolus. CONCLUSIONS: Routine screening for the FV:Q506 mutation in cancer patients without a personal or family history for venous thrombosis is not helpful in guiding management. In contrast, an episode of venous thrombosis in a patient with a mutant germline FV:Q506 allele was associated with recurrent thrombotic events. These findings suggest that patients heterozygous for the FV:Q506 allele may require an independent "susceptibility" element to manifest a venous hypercoagulable state. In addition, only 2 of 25 clinic patients with a venous clot carried the FV:Q506 allele suggesting this genetic defect plays a minor role in the hypercoagulable state of cancer.