Mi Ryoung Choi

Frameshift mutation of a histone methylation-related gene SETD1B and its regional heterogeneity in gastric and colorectal cancers with high microsatellite instability.

Histone methyltransferase (HMT), which catalyzes a histone methylation, is frequently altered in cancers at mutation and expression levels. The aims of this study were to explore whether SETD1B, SETDB2, and SETD2, SET domain-containing HMT genes, are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that SETD1B, SETDB2, and SETD2 had mononucleotide repeats in coding sequences that might be mutation targets in cancers with microsatellite instability (MSI). We analyzed the mutations in 76 GCs and 93 CRCs and found SETD1B (38.7% of GC and 35.6% of CRC with high MSI [MSI-H]), SETDB2 (11.1% of CRC with MSI-H), and SETD2 frameshift mutations (6.7% of CRC with MSI-H). These mutations were not found in stable MSI/low MSI. In addition, we analyzed intratumoral heterogeneity (ITH) of SETD1B mutation in 6 CRCs and found that 2 CRCs harbored regional ITH of SETD1B. We also analyzed SETD1B expression in GC and CRC by immunohistochemistry. Loss of SETD1B expression was identified in 15% to 55% of the GC and CRC with respect to the MSI status. Of note, the loss of expression was more common in those with SETD1B mutations than those with wild-type SETD1B. We identified alterations of SET domain-containing HMT at various levels (frameshift mutations, genetic ITH, and expression loss), which together might play a role in tumorigenesis of GC and CRC with MSI-H. Our data suggest that mutation analysis in multiple regions is needed for a better evaluation of mutation status in CRC with MSI-H.

Laminin gene LAMB4 is somatically mutated and expressionally altered in gastric and colorectal cancers

Laminins are important in tumor invasion and metastasis as well as in maintenance of normal epithelial cell structures. However, mutation status of laminin chain‐encoding genes remains unknown in cancers. Aim of this study was to explore whether laminin chain genes are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that laminin chain genes LAMA1, LAMA3, LAMB1 and LAMB4 had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the genes in 88 GC and 139 CRC [high MSI (MSI‐H) or stable MSI/low MSI (MSS/MSI‐L)] by single strand conformation polymorphism analysis and DNA sequencing. In the present study, we found LAMB4 (11.8% of GC and 7.6% of CRC with MSI‐H), LAMA3 (2.9% of GC and 2.5 of CRC with MSI‐H), LAMA1 (5.9% of GC with MSI‐H) and LAMB1 frameshift mutations (1.3% of CRC with MSI‐H). These mutations were not found in MSS/MSI‐L (0/114). We also analyzed LAMB4 expression in GC and CRC by immunohistochemistry. Loss of LAMB4 expression was identified in 17–32% of the GC and CRC. Of note, the loss expression was more common in the cancers with LAMB4 mutation or those with MSI‐H. Our data show that frameshift mutations of LAMA1, LAMA3, LAMB1 and LAMB4, and loss of LAMB4 may be features of GC and CRC with MSI‐H.

Frameshift mutations of axon guidance genes ROBO1 and ROBO2 in gastric and colorectal cancers with microsatellite instability

Aims: Several lines of evidence indicate that axon guidance genes are involved not only in neural development but also in cancer development. ROBO1 and ROBO2, crucial regulators of axon guidance, are considered potential tumour suppressor genes. The aim of this study was to explore whether ROBO1 and ROBO2 genes are somatically mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). Methods: In a public database, we observed that both ROBO1 and ROBO2 had mononucleotide repeats in their coding exons that could be mutation targets in cancers with microsatellite instability (MSI). We analysed mutations of these repeats in 77 GC and 88 CRC either with high MSI (MSI-H) or low MSI/microsatellite stability (MSI-L/MSS) by single-strand conformation polymorphism (SSCP) and DNA sequencing. We analysed ROBO1 and ROBO2 expressions in GC and CRC by immunohistochemistry as well. Results: Overall, we found five ROBO1 and five ROBO2 frameshift mutations in the repeats. They were detected exclusively in the cancers with MSI-H (10/70, 14.2%), but not in MSI-L/MSS (0/95, 0%) (p = 0.018). In the immunohistochemistry, loss of ROBO2 expression was identified in 22 (29%) and 17 (19%) of GC and CRC, respectively, while increased expression of ROBO2 was found in 15 (20%) and 22 (25%) of GC and CRC, respectively. There were co-occurrences of mutation and loss of expression in both ROBO1 (4/5, 80% mutated cases, p < 0.001) and ROBO2 (5/5, 100% mutated cases, p < 0.05) genes. Conclusion: This is the first report of ROBO1 and ROBO2 frameshift mutations in GC and CRC. Frameshift mutations of ROBO1 and ROBO2 genes and alteration of ROBO2 expression in GC and CRC suggest that both genes might play roles in the pathogenesis of GC and CRC.

Mutational and expressional analysis of ERBB3 gene in common solid cancers

ERBB3 is a member of EGFR family receptor tyrosine kinases, genetic alterations of which are common and therapeutically targeted in human cancers. Recently, somatic mutations of ERBB3 gene, including recurrent mutation in exon 3 altering Val104, were reported in gastric cancers (GC) and colorectal cancers (CRC), strongly suggesting its role in the development of GC and CRC. To examine whether the recurrent ERBB3 mutations of exon 3 occur in GC and CRC, and other malignancies as well, we analyzed the ERBB3 in 1677 cancer tissues by a single‐strand conformation polymorphism (SSCP) assay. We identified ERBB3 mutations altering the Val104 mutations in GC (0.5%) and CRC (2.2%). However, we did not find the ERBB3 mutations in the other cancers besides GC and CRC. We observed that an increased intensity of phosphorylated ERBB3 (pERBB3) in GC and CRC. Of note, all of the cancers with ERBB3 mutations displayed an increased intensity of pERBB3 immunostaining. Our data indicate that the recurrent ERBB3 mutations altering Val104 occur predominantly in GC and CRC. Also, the data suggest that ERBB3 is altered in GC and CRC by various ways, including somatic mutations and increased expression that might play roles in tumorigenesis.

Frameshift mutations in mammalian target of rapamycin pathway genes and their regional heterogeneity in sporadic colorectal cancers

Mammalian target of rapamycin (mTOR) pathway is known to be involved in cancer pathogenesis. The aim of our study was to find whether mTOR-related genes were mutated and expressionally altered in colorectal cancers (CRCs). Through public database searching, we found that PIK3CB, insulin receptor substrate 1/2 (IRS1), RPS6, EIF4B, RPS6KA5, and PRKAA2 that were known as mTOR-related genes possessed mononucleotide repeats in DNA coding sequences that could be mutated in cancers with microsatellite instability (MSI). We analyzed 124 CRCs by single-strand conformation polymorphism analysis and DNA sequencing and found 7 (8.9%), 8 (10.1%), and 3 (3.8%) of 79 CRCs with high MSI that harbored IRS1, EIF4B, and RPS6KA5 frameshift mutations, respectively. These mutations were not identified in stable MSI/low MSI (0/45). In addition, we analyzed intratumoral heterogeneity (ITH) of PIK3CB, IRS1, RPS6, EIF4B, RPS6KA5, and PRKAA2 frameshift mutations in 16 CRCs and found that IRS1, EIF4B, and RPS6KA5 mutations had regional ITH in 2, 2, and 1 CRCs, respectively. We also analyzed IRS1 expression in the CRCs by immunohistochemistry. Loss of IRS1 expression was identified in 31% of the CRCs. The loss of expression was more common in those with IRS1 mutation than those with wild-type IRS1. Our data indicate mTOR-related genes harbored not only somatic mutations but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of CRC, especially with high MSI. Our data also suggest that mutation analysis in multiregional areas is needed for a precise evaluation of mutation status in CRC with MSI-H.

Inactivating frameshift mutation of AKT1S1, an mTOR inhibitory gene, in colorectal cancers

Dear Editor, Mammalian target of rapamycin (mTOR) integrates growth factorand nutrient-linked signals and regulates cell growth, proliferation, motility, survival, protein synthesis and transcription [1]. The mTORC1 complex is composed of mTOR, Raptor, DEPTOR and AKT1S1 [2]. Alterations of mTOR signaling are associated with hamartoma syndromes, including Peutz–Jeghers syndrome. They are caused by mutations of STK11, PTEN and TSC1/2, which are also mutated in many cancers [2,3]. AKT1S1 is known to interact with AKT and Raptor [4]. AKT1S1 inhibits cell growth and RHEBinduced activation of the mTORC1 pathway, indicating that it is a negative regulator of mTOR signaling. Because negative regulators of mTOR are frequently mutated in cancers, it can be hypothesized that AKT1S1 gene might be mutated in cancers. However, it remains unknown whether AKT1S1 is somatically mutated in cancers. In a public genome database (http://genome.cse. ucsc.edu/), we found that human AKT1S1 had a mononucleotide repeat in the coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of colorectal cancers (CRC) with MSI [5]. To date, however, it is not known whether AKT1S1 gene is mutationally altered in CRC with MSI. In this study, we analyzed a C7 repeat in the AKT1S1 exon 4 by polymerase chain reaction (PCR)-based singlestrand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 89 CRC with high MSI (MSI-H) and 45 CRC with stable MSI (MSS). In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [6]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [7]. On the SSCP, we observed aberrant bands of AKT1S1 gene in three CRCs. DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had raised somatically. DNA sequencing analysis confirmed that the aberrant bands represented a recurrent AKT1S1 somatic mutation, which was a heterozygous frameshift mutation (deletion of one base) in the C7 repeat (c.495ddelC) that would result in a frameshift mutation (p.Thr166ProfsX87) (Figure 1). They were detected in four of the CRC with MSI-H (3/89: 3.4%), but not in those with CRC with MSS. The frameshift mutation detected in the current study would result in a premature stop of amino-acid synthesis in AKT1S1 protein and hence resembles a typical loss-of-function mutation. Because earlier data suggested that AKT1S1 possessed tumor suppressor

Oncogenic ERBB3 mutations altering p.Val104 is rare in acute leukemias and non‐Hodgkin lymphomas

To the Editor: Receptor tyrosine kinases (RTK) that regulate biological processes in cell growth and survival are a major oncogene family. The ERBB family of RTK consists of ERBB1 (EGFR/HER1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) (1). When compared to the other three ERBBs, ERBB3 has a weaker kinase activity (1). Upon binding with ligand, ERBB3 heteromerizes with other RTK, especially ERBB2, and transmits downstream signals (1). Mounting evidence indicates that ERBB genes are altered in human cancers. For example, lung adenocarcinomas harbor EGFR mutations and breast cancers harbor ERBB2 amplification (2, 3). These genetic alterations are therapeutically targeted and currently used in clinic (2, 3), indicating that oncogenic mutations of ERBB genes are common targets of genetic alterations in human cancers. A recent study discovered that ERBB3 somatic mutations were common in colorectal and gastric cancers (~11%), and less common in other solid cancers (4). Of note, most of the ERBB3 mutations were discovered in the extracellular ligand-binding domain (ECD) and were recurrent at specific amino acids. Of these, somatic mutations altering an amino acid residue at 104 (p.Val104Met and p.Val104Leu) were the most common recurrent mutations. These mutations conferred transformation activities in cells in a ligand-independent fashion. Furthermore, anti-ERBB antibodies and small molecule inhibitors blocked the mutant ERBB3-related oncogenic activities. Although ERBB3 signaling and alterations are well known in solid tumors, but they are not known well in hematologic neoplasia (5). Thus, it is interesting to know whether recurrent ERBB3 mutation occurs in neoplasia from hematopoietic tissues as well. For this, we analyzed somatic mutations of ERBB3 gene using genomic DNA from in fresh bone marrow aspirates of 811 hematologic tumors (acute myelogenous leukemias (AML), acute lymphoblastic leukemias (ALL), multiple myelomas, and myelodysplastic syndromes) (Table 1) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed the gene in paraffin-embedded tissues of 63 non-Hodgkin lymphomas (NHL). Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Genomic DNA each from tumor cells and normal cells (remission bone marrow cells in the cases of leukemias) was used in this study. Genomic DNA each from tumor cells and corresponding normal cells was amplified with a primer pair covering the exon 3 of ERBB3 gene that contained coding sequences of amino acid residue Val104. Radioisotope was incorporated into the PCR products for detection by autoradiogram. After the SSCP, DNAs showing mobility shifts were cut out from the dried gel, and re-amplified for 30 cycles using the same primer sets. In colon carcinomas, we were able to find the recurrent mutations p.Val104Met and p.Val104Leu, which were subsequently used as positive controls in SSCP. However, none of the SSCP from the hematologic neoplasia revealed aberrantly migrating bands compared with wild-type bands from the normal tissues (Fig. 1), indicating there was no evidence of ERBB3 mutation altering the Val104 in the neoplasia analyzed. To confirm the SSCP results, we repeated the experiments twice and found that the data were consistent.

Abstract 545: Frameshift mutations of chromosome cohesion-related genes SGOL1 and PDS5B in gastric and colorectal cancers with high microsatellite instability

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cohesin is a protein complex that regulates chromatid cohesion and plays a role in preventing aneuploidy and maintaining chromosomal stability. SGOL1 encodes a cohesin protector, and PDS5B encodes a regulatory cohesion factor. Both SGOL1 and PDS5B are considered putative tumor suppressor genes. The aim of this study was to explore whether SGOL1 and PDS5B genes are mutated and expressionally altered in gastric and colorectal cancers. A genome database indicated that both genes possessed mononucleotide repeats in coding sequences, which could be mutation targets in cancers with microsatellite instability. We analyzed mutations in 91 gastric cancers and 100 colorectal cancers with high microsatellite instability or stable/low microsatellite instability by single-strand conformation polymorphism analysis and DNA sequencing. We also analyzed SGOL1 and PDS5B expression by immunohistochemistry. Overall, we found 21 SGOL1 frameshift mutations in 21 cases and 18 PDS5B frameshift mutations in 16 cases. SGOL1 and PDS5B frameshift mutations were detected in 26.6% and 20.3%, respectively, of high microsatellite instability but not in stable/low microsatellite instability (0/112). By immunohistochemistry, losses of SGOL1 and PDS5B were identified in 19% to 47% of the gastric and colorectal cancers irrespective of microsatellite instability status. The losses were more common in those with frameshift mutations or high microsatellite instability than those without mutations or high microsatellite instability. The data indicate that frameshift mutations of SGOL1 and PDS5B and the loss of their expression may be a feature of gastric and colorectal cancers with high microsatellite instability. In addition, the data suggest that these alterations might contribute to cancer pathogenesis by deregulating cohesin-related functions. Citation Format: Min Sung Kim, Youn Jin Choi, Min Gwak, Hyerim Oh, Mi Ryoung Choi, Sun Yong Hwang, Nam Jin Yoo, Sug Hyung Lee. Frameshift mutations of chromosome cohesion-related genes SGOL1 and PDS5B in gastric and colorectal cancers with high microsatellite instability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2014-545

Frameshift Mutations of CAB39L, an Activator of LKB1 Tumor Suppressor, in Gastric and Colorectal Cancers.

To the Editor: Calcium binding protein 39-like (CAB39L) is a core component of LKB1 tumor suppressor complex and activates LKB1 [1]. Activation of LKB1 plays crucial roles maintaining cell polarity and loss of LKB1 leads to disorganization of cell polarity and facilitates tumor growth. Germline mutations in LKB1 are associated with Peutz-Jeghers syndrome characterized by polyps in the gastrointestinal tract and other neoplasms [2]. Somatic mutations of LKB1 gene were also found in many tumors [3]. Also, CAB39L binds and activates STK24, a STE20 family protein kinase [4]. STK24 not only inhibits cell cycle progression by phosphorylating NDR kinase, but also promotes cell death [5]. These data indicate that that CAB39L is possibly involved in cancer-related pathways and suggest that inactivation of CAB39L might be related to tumorigenesis. However, its implications in cancer development are not unknown. In a public genome database (http://genome.cse.ucsc.edu/), we found that human CAB39L had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) withMSI [6]. To date, however, it is not known whether CAB39L gene is mutationally altered in GC and CRC. In this study, we analyzed an A7 repeat in theCAB39L exon 2 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. For this, we used methacarn-fixed tissues of 34 GC with high MSI (MSI-H), 45 GC with stable MSI (MSS), 89 CRC with MSI-H and 45 CRCwithMSS. For 16 of the 89 CRCwithMSI, we collected four to seven different tumor areas from the same patients and analyzed intratumoral heterogeneity (ITH) of CAB39L mutation. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [7]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [8]. On the SSCP, we observed aberrant bands ofCAB39L gene in two cancers (a GC and a CRC). DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands representedCAB39L somatic mutations, which were a frameshift mutation by duplication of one base (c.10dupA (p.Met4AsnfsX5)) and another frameshift mutation by deletion of one base (c.10delA (p.Met4CysfsX15)) in the A7 repeat. They were detected in a GCwithMSI-H (1/34: 2.9%) and a CRCwithMSI-H (1/89: 1.1 %), but not in those with MSS. The mutational ITH analysis in the 16 CRCs (4–7 fragments per case) with MSI-H identified that the same CRC described above harbored ITH Mi Ryoung Choi and Chang Hyeok An contributed equally to this work.

Frameshift Mutation of an Angiogenesis Factor VEGFB and its Mutational Heterogeneity in Colorectal Cancers

To the Editor: During tumor development and progression, angiogenic switch causes quiescent blood vessels to sprout new vessels that help sustain expanding neoplastic growths [1]. A wellknown prototype of angiogenesis inducers is vascular endothelial growth factor (VEGF) protein family that is comprised of VEGFA, VEGFB, VEGFC and VEGFD, all of which may exhibit angiogenic functions. Angiogenic function of VEGFB has been identified in many studies, albeit less definite than that of VEGFA [2, 3]. With respect to the cancer, alterations of VEGFB gene are largely unknown. Promoter CpG methylation of VEGFB has been reported in ovarian cancers [4]. Cancer development initiates through a clonal expansion of a single cell. The resulting cell population usually becomes heterogeneous after branching sub-clonal expansions, which leads to intra-tumor heterogeneity (ITH). This ITH contributes to acquired tumor aggressiveness and may impede the accurate diagnosis/prognosis and the proper selection of cancer therapies [5]. In a public genome database (http://genome.cse.ucsc.edu/), we found that human VEGFB had a mononucleotide repeat in the coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric cancer, endometrial cancer and colorectal cancers (CRCs) with MSI [6]. Somatic missense mutations of this gene have been identified in CRCs with MSI, whereas frameshift mutations have not [7]. To see whether VEGFB gene harbored frameshift mutations and mutational ITH in CRC, we analyzed the A8 repeat in VEFGB exon 5 by polymerase chain reaction (PCR)-based singlestrand conformation polymorphism (SSCP) assay. For this, we used methacarn-fixed tissues of 79 MSI-high (MSI-H) CRCs and 45 MSI-low (MSI-L) or MSI-stable (MSS) CRCs. For 57 (16 MSI-H and 41 MSI-L/MSS) of the 124 CRCs, we collected four to seven different tumor areas from the same patients and analyzed ITH of VEGFB mutation. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [8, 9]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [8, 9]. On the SSCP, we observed aberrant bands of VEGFB gene in 11 CRCs. DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the M. R. Choi :N. J. Yoo : S. H. Lee (*) Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea e-mail: suhulee@catholic.ac.kr