| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Division of Hematology and Oncology,* Department of Pathology, Oregon Health and Science University Cancer Institute and Portland Veterans Affairs Medical Center, Portland, Oregon; the Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts; and the Department of Pathology, Indiana University, Indianapolis, Indiana
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Activating mutations of KIT tyrosine kinase are common in gastrointestinal stromal tumors (GISTs)6-8 and mastocytosis/mast cell leukemia.9-11 Rarely, such mutations occur in acute myelogenous leukemia12,13 and sinonasal natural killer/T cell lymphoma.14 The mutations in GISTs are found primarily in exons 11 and 9 of the KIT gene, and the resulting mutant isoforms are sensitive to inhibition by imatinib mesylate (Gleevec, formerly STI571) in vitro.1 Correspondingly, many patients with malignant GIST respond to treatment with this drug.15,16 In contrast, nearly all cases of mastocytosis/mast cell leukemia are related to a mutation in exon 17 of the KIT gene (D816V), which yields an isoform that is resistant to imatinib mesylate.17
To date there have been two reports of KIT mutations in germ cell tumors. Tian and colleagues18 examined 23 cases of seminoma/dysgerminoma and found 2 tumors (1 seminoma [primary site unspecified], 1 ovarian dysgerminoma; 2 of 23 = 8.7%) with an activating mutation in exon 17 (D816H). There were no mutations in 10 non-seminomatous germ cell tumors evaluated. More recently, Przygodzki et al19 found KIT exon 17 mutations in 3 of 8 (37.5%) mediastinal seminomas (K818R, D820V, and N822K). While all three were novel mutations, one of them (K818R) was a conservative change for which the biochemical significance was not established.
To further examine the frequency and spectrum of KIT gene mutations in germ cell tumors, we screened a series of germ cell tumors tumors using the highly sensitive combination of denaturing high performance liquid chromatography (HPLC) and direct sequencing. Selected KIT mutant protein isoforms were profiled biochemically for constitutive KIT kinase activity and sensitivity to imatinib mesylate. In addition, we compared activation of intracellular signaling pathways in seminomas with and without KIT gene mutations, as well as in non-seminomatous germ cell tumors (NSGCT).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Forty-six samples of paraffin-embedded germ cell tumor (32 testicular seminomas and 1 ovarian dysgerminoma, 13 NSGCT [5 also had corresponding frozen tumor]) were obtained from the archives of the Departments of Pathology at Oregon Health and Science University and the Portland VA Medical Center. An additional 8 fresh frozen testicular seminoma samples were from the Department of Pathology of Brigham & Women’s Hospital (Dr. Jonathan Fletcher). Twenty-four samples of paraffin-embedded germ cell tumor NSGCT were from the Department of Pathology, Indiana University (Dr. Oscar Cummings). Fourteen additional samples of fresh-frozen testicular seminoma were obtained from the Germ Cell Tumor Bank of Indiana University (Dr. Cummings). Sections of all paraffin-embedded tumor samples were reviewed by one of the authors (C.L.C.) to verify the diagnosis. Overall, our series included 54 seminomas of testicular origin (46 primary lesions, 8 metastases) and 1 primary ovarian dysgerminoma. The 37 NSGCT included 32 cases with a testicular primary site. In 5 cases of NSGCT the location of the primary site was not available to us. The majority of the NSGCT tumors examined were from recurrent, metastatic lesions. All samples were acquired in accordance with the regulations of the Institutional Review Boards for each institution.
Immunohistochemistry for KIT
All available samples of paraffin-embedded tumor were examined for KIT expression by immunohistochemistry using a rabbit antiserum from Dako (A4502, Dako Corp., Carpinteria, CA) as described previously.20
Genomic DNA Extraction and Analysis
Hematoxylin and eosin (H&E)-stained sections (5 µm) were reviewed under a microscope and areas rich in tumor (>50% cellularity) were marked. Corresponding areas on unstained sections were scraped from the slides with a sterile scalpel blade. In the case of NSGCT, we purposefully selected tumor regions that were rich in high-grade elements (embryonal carcinoma and yolk sac tumor). For lesions with elements of teratoma, we selected regions that were dominated by epithelial-lined cysts and nodules of cartilage rather than pure stroma. In cases of seminoma that had heavy lymphocytic infiltrates (
50% cellularity), 7 µm sections were prepared on non-coated glass slides, deparaffinized, and stained with methyl green. Tumor cells were then microdissected using a Pixcell II Laser Capture Microscope (LCM) (Arcturus Engineering, Mountain View, CA). None of our cases of NSGCT were subjected to LCM. DNA was extracted from scraped tissue or the nuclear pellet of lysed fresh-frozen tumor (detailed below) using the QIAamp DNA Mini Kit and 500 ng was used for PCR (Qiagen, Valencia, CA). Caps bearing tumor cells from laser capture microdissection were incubated overnight in 50 µl of 10 mmol/L Tris-HCL (pH 8.0), 1 mmol/L EDTA, 1% Tween-20, and proteinase K (0.1 mg/ml) at 56°C. The mixture was then heated to 90°C to inactivate the proteinase K and 2 to 5 µl were used for PCR.
PCR was performed on all specimens for KIT exon 17 (High Fidelity System, Roche No. 1732078, Roche Diagnostics, Indianapolis, IN). PCR for KIT exons 9, 11, and 13 was completed in 20 of the seminoma cases that were known to be wild-type at exon 17. The following primer pairs were used:
KIT exon 17 forward 81318 5'-TGTATTCACAGAGACTTGGC-3'
KIT exon 17 reverse 81534 5'-GGATTTACATTATGAAAGTCACAGG-3'
KIT exon 9 forward 5'-ATGCTCTGCTTCTGTACTGCC-3'
KIT exon 9 reverse 5'-AGAGCCTAAACATCCCCTTA-3'
KIT exon 11 forward 5'-CCAGAGTGCTCTAATGACTG-3'
KIT exon 11 reverse 5'-ACCCAAAAAGGTGACATGGA-3'
KIT exon 13 forward 5'-CATCAGTTTGCCAGTTGTGC-3'
KIT exon 13 reverse 5'-ACACGGCTTTACCTCCAATG-3'
DNAs were amplified in 25 to 50 µl PCR reactions of 1 minute at 94°C, 1 minute at 56°C, and 1 minute at 72°C for 45 cycles (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Negative controls (no DNA) were included in every set of amplifications.
DNA from the laser-captured cells usually required nested PCR; 1 µl of the first reaction was used for the nested reaction. The nested PCR reaction was amplified for 25 cycles using the above PCR cycling conditions. Negative controls for the nested PCR reaction included a reaction using 1 µl of negative control (no template) specimen from the first round of amplification as a "template."
Additional exon 17 primers for nested PCR:
KIT exon 17 forward 81289 (before nesting) 5'-GGTTTTCTTTTCTCCTCCAA-3'
KIT exon 17 reverse 81488 (nested) 5'-AGGACGTTTCCTAAAAATCAAAG-3'
Amplicons were analyzed by D-HPLC using a Transgenomic WAVE instrument (Transgenomics, Omaha, NE) as previously described.20 Denaturing temperatures for detection of point mutations were optimized for each primer pair (exon 9, 58°C; exon 11, 57°C; exon 13, 59°C; and exon 17, 56.5°C). Amplicons with an abnormal D-HPLC elution profile at denaturing temperature were subjected to bidirectional sequence analysis on an ABI 377 or 310 sequencer using the ABI Big Dye terminator kit (Applied Biosystems). All mutations were confirmed by repeat HPLC and sequencing analysis of an independently amplified specimen.
KIT Expression Vectors and Cell Transfection
Site-directed mutagenesis of wild-type KIT plasmid cDNA (cloned in pcDNA3.1 [Invitrogen, Carlsbad,CA]) was performed to produce the mutant KIT isoforms (Stratagene QuikChange-XL Site-Directed Mutagenesis Kit). The introduced mutations were confirmed by bi-directional sequencing. Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in F12 (HAM) Nutrient Mixture (Gibco-BRL), + 10% fetal bovine serum. At 70% confluency in T25 flasks, the cells were transiently transfected with wild-type or mutant KIT expression plasmid using Lipofectamine PLUS (Invitrogen Life Technologies) according to the manufacturer’s suggested protocol. The next day, the transfected cells were exposed to concentrations of imatinib mesylate ranging from 0 to 5 µmol/L for 90 minutes (drug generously provided by Dr. Elisabeth Buchdunger, Novartis Pharma, Switzerland). Fresh stock solutions of drug (10 mmol/L) were made before each experiment by dissolving 5 mg STI571 in 1 ml Dulbecco’s phosphate-buffered saline (PBS; Gibco-BRL). When indicated, cells were stimulated with human SCF (final concentration 100 ng/ml) for 10 minutes before their removal from the flask. A 10-minute stimulation with human SCF was found to result in maximal receptor autophosphorylation, consistent with prior results (data not shown).20
Protein Lysates
After the 90-minute incubation period with drug, the transfected CHO cells were then scraped from the flasks and spun (1000 RPM). The cell pellets were lysed with 100 to 150 µl of protein lysis buffer (50 mmol/L Tris, 150 mmol/L sodium chloride, 5 mmol/L EDTA, 1% NP-40, and 0.25% deoxycholate), with addition of the inhibitors aprotinin, leupeptin, pepstatin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, sodium pyrophosphate, and sodium orthovanadate [Sigma, St. Louis, MO]), as previously described.21 Protein concentration of homogenates was determined with Bio-Rad Protein Assay Reagent.
Fresh-frozen tumor specimens were ground in liquid nitrogen with mortar and pestle and solubilized with protein lysis buffer. Lysate was spun at 13,000 RPM for 15 minutes to pellet nuclei. Supernatant was saved for KIT and PI3-kinase immunoprecipitation studies and Western blots.
Immunoprecipitation and Western Blotting
Cellular lysates (250 to 500 µg of protein from CHO cell transfection or 4 mg of fresh-frozen tumor) were pre-cleared with 5 µl (1:100) of normal rabbit IgG agarose conjugate (Santa Cruz Biotech, Santa Cruz, CA) for 1 hour at 4°C. Next, they were immunoprecipitated with 5 µg of anti-KIT antibody agarose conjugate (Santa Cruz Biotech) overnight at 4°C. Separate samples of fresh-frozen tumor lysates were immunoprecipitated with 10 µg of anti-PI3-kinase antibody (N-SH3 domain p85 subunit) (Upstate Biotechnology, Lake Placid, NY). Immunoprecipitates were washed three times with PBS plus inhibitors, resuspended in 20 µl of 4X-Laemlli SDS-PAGE loading buffer, heated at 95°C for 10 minutes, and then separated by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were blocked for one hour with TBS-Tween containing 5% BSA for anti-phosphotyrosine blotting or 5% nonfat milk for anti-KIT blotting. The following primary antibodies were used on the membranes: mouse monoclonal anti-phosphotyrosine antibody (PY20) at 1:1000 dilution (Transduction Laboratories, Lexington, KY) and rabbit anti-KIT polyclonal antibody at 1:500 (Oncogene, Cambridge, MA). Secondary antibodies were peroxidase-conjugated goat anti-mouse antibody at 1:5000 (Biorad, Hercules, CA) and goat-anti-rabbit antibody at 1:10000 (Biorad), respectively. Immunoblots were developed by enhanced chemiluminescence (Amersham Biosciences).
For Western blots of tumor lysates, 25 µg of lysate was combined with 5 µl of 4X-Laemlli SDS-PAGE loading buffer, heated at 95°C for 10 minutes, and then separated by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were blocked for 1 hour with TBS-Tween containing 5% BSA for anti-phospho-blotting or 5% nonfat milk for all other blotting. The following primary antibodies were used on the membranes: anti-phospho-AKT (Ser473) antibody at 1:500 dilution, anti-total AKT 1:500, anti-phospho-MAPK 1:1000, anti-phospho-STAT3 1:666, and anti-STAT3 1:666 (all from Cell Signaling, Beverly, MA); anti-MAPK 1:1000 and anti-PI3-kinase (N-SH3 domain) 1:666 (Upstate Biotechnology, Lake Placid, NY), anti-placental alkaline phosphatase (PLAP) 1:100 (Biomeda Corp, Hayward, CA), and anti-actin 1:500 (Sigma). Secondary antibodies were the same as described above.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The study was performed on 92 germ cells tumors, including 54 pure testicular seminomas, 1 ovarian dysgerminoma, 4 mixed seminoma/NSGCT, and 33 NSGCT. KIT expression was assessed in a subset of the seminomas by immunohistochemistry, which revealed strong surface membrane staining in 82% (31 of 38) of the pure and mixed seminoma tumors. KIT expression in the NSGCT samples was examined in a previous study, which showed patchy cytoplasmic staining in 48% (11 of 23).22 These results are consistent with other reports of KIT expression in testicular germ cell tumors.23-25
For 42 of the 54 cases of seminoma and the one case of ovarian dysgerminoma, the degree of lymphocytic infiltration was <50% and we were able to directly prepare genomic DNA from paraffin slides and/or fresh frozen samples. Amplicons of KIT exon 17 were screened for mutations using D-HPLC (Figure 1)
. Mutations of KIT exon 17 were found in 10 of the 42 cases of testicular seminoma (23.8%), including 3 cases with the D816H mutation originally described by Tian et al (Table 1)
.18
The most common mutation, found in 4 cases, was D816V. Two cases had Y823D, 1 case had Y823C, and 1 case had N822K (Table 1)
. None of these 43 cases required nested PCR for detection of mutations.
|
|
. Overall, fourteen of the pure testicular seminomas (14 of 54, 25.9%) contained a KIT exon 17 point mutation. In five of the seminomas harboring an exon 17 mutation (1 case each of D816V, D816H, Y823C, Y823D, and T801I), DNA extracted from the adjacent normal tissue was wild-type, indicating that the mutations were somatic rather than germline. Twenty pure seminomas that were wild-type for exon 17 were screened for mutations in exons 9, 11, and 13 (all commonly mutated in GISTs); none were found. No KIT mutation was found in the solitary ovarian dysgerminoma. In contrast to our results from pure seminoma/dysgerminoma germ cell tumors, no exon 17 mutations were found in the 33 NSGCT or the 4 mixed seminoma/NSGCT (P = 0.0015, Fisher’s exact test).
KIT Kinase Activity in Transiently Transfected CHO Cells
There are only a few reports of human testicular seminoma cell lines, and these cell lines have not been fully characterized as to their usefulness in modeling seminoma biology in vitro.26-28
In particular, it has not been reported if these cell lines express KIT. Therefore, we used an alternative in vitro eukaryotic system to assess the biochemical consequences of the observed KIT mutations on KIT kinase activity. Plasmid expression vectors encoding each mutation were generated by site-directed mutagenesis of wild-type KIT cDNA. CHO cells were transiently transfected with mutant or wild-type KIT vector. Transfected cells were then exposed to concentrations of imatinib mesylate ranging from 0 to 5 µmol/L for 90 minutes. Cell lysates were then immunoprecipitated with anti-KIT antibodies and analyzed by Western blotting for evidence of KIT kinase activity, as evidenced by phosphorylation of tyrosine residues. As expected, wild-type KIT required ligand stimulation for phosphorylation, while D816V and D816H did not (Figure 2)
.18
The N822K and Y823D mutant isoforms were also constitutively phosphorylated. The T801I mutant isoform was phosphorylated only in the presence of SCF. The kinase activities of mutant isoforms N822K and Y823D were inhibited by imatinib mesylate with an approximate IC50 of 0.5 µmol/L and 0.1 µmol/L, respectively (Figure 2)
. Mutant isoform D816H was much less sensitive to imatinib mesylate, with an IC50 of 5.0 µmol/L. Mutant D816V was resistant to 5.0 µmol/L imatinib mesylate, consistent with previous reports (see Table 1
for summary).17
|
To determine whether KIT kinase is active in seminomas, total KIT protein was immunoprecipitated from 13 fresh-frozen seminoma lysates and analyzed by Western blotting with a phospho-tyrosine-specific monoclonal antibody (PY20). Two of the 13 frozen samples contained the D816V mutation; the remaining 11 were wild-type. While total KIT protein was immunoprecipitated in all of the samples, phosphorylated KIT was detectable only in the specimens containing the D816V mutation (Figure 3A)
. Notably, in two wild-type cases (lanes 6 and 13) in which an equivalent amount of total KIT protein was immunoprecipitated relative to the D816V seminomas, there was no evidence of phosphorylated KIT. Since it has been demonstrated that PI3-kinase is required for SCF-induced cell proliferation and survival in vitro and for male mouse fertility in vivo, we also performed successive immunoblotting of the KIT-immunoprecipitated protein complexes with an antibody to the p85 subunit of PI3-kinase.29-32
PI3-kinase was co-precipitated with KIT in two of the tumors, most strongly in a D816V mutant, but also in a wild-type tumor (Figure 3A
, lanes 5 and 11). Conversely, immunoprecipitation of PI3-kinase protein from the seminoma lysates yielded strong signals for co-precipitated total and phospho-KIT in both of the D816V mutants (Figure 3B)
. In contrast, very little total or phospho-KIT was co-precipitated from the wild-type seminoma samples even though equivalent amounts of p85 subunit of PI3-kinase were immunoprecipitated relative to the D816V seminomas. Thus, evidence of strong KIT activation could be demonstrated only in the seminomas with KIT-activating mutations.
|
. The signal for placental alkaline phosphatase (PLAP) was compared across all lanes to ensure that comparable amounts of seminoma lysate were loaded even in the one case (lane 13) for which LCM had been used for the genotyping analyses. Notably, total KIT was expressed at much higher levels in the two D816V tumors than in any of the KIT wild-type specimens, even though the PLAP staining suggested that these samples contained slightly less seminoma-derived protein than most of the other lanes. In reviewing the KIT IHC staining, we also noted that the mutant KIT seminomas had much stronger KIT staining than the non-mutant cases (data not shown).
|
Five frozen samples of NSGCT were available for comparison of intracellular signaling. Total KIT expression was not detected in any of the NSGCT, as predicted by their paucity of KIT staining on immunohistochemistry (3 of 5 tumors had <5%, patchy KIT staining; the remaining 2 were negative). There was tumor to tumor variation in the expression of total and activated MAPK, AKT, and STAT3, but the overall signal transduction profile of these tumors was not distinguishable from the seminoma tumors (Figure 5)
. It should be noted that normalization of the protein loading on these blots was problematic due to the inherent heterogeneity of the NSGCTs. Actin was added as an additional loading control because PLAP expression is not as uniform in NSGCT as it is in seminoma. The actin staining would suggest some unequal loading across lanes (Figure 5)
, but PLAP staining was quite similar among all of the NSGCT cases. Given that PLAP expression is more characteristic of embryonal carcinoma and yolk sac tumor than teratoma, the findings in Figure 5
may be somewhat biased toward these high-grade NSGCT elements.33
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The activating mutation frequency in our series (24.1%) is higher than that reported by Tian et al, 18 probably due to methodological differences. Seminomas are frequently triploid or near-tetraploid, and therefore a KIT allele with a gain-of-function mutation may be outnumbered by two or more wild-type alleles.42 This ratio of mutant to wild-type alleles approaches the limit of detection by direct sequencing, a problem that may be compounded by concomitant contamination of the tumor by infiltrating lymphocytes. We believe that D-HPLC screening, along with application of laser capture microscopy to selected cases, allowed more point mutations to be identified in our series. It remains possible that some KIT mutations were missed and could perhaps be detected through the use of even more sensitive methodologies (eg, allele-specific PCR).
Przygodzki et al19 recently assayed for KIT mutations in primary mediastinal seminomas using direct sequencing of exon 17 amplicons. Three of eight studied tumors had protein sequence altering mutations: D820V, N822K, and K818R. Asp-820 and Asn-822 mutations have been previously identified as activating in GISTs, mast cell tumors, and in seminomas as presented here, but the biological significance of the conservative change K818R mutation is unknown.39,43,44 The frequency of KIT mutations in our series of seminomas of testicular origin does not appear different from the frequency reported in the smaller series of mediastinal seminomas.
Interestingly, all 33 NSGCT cases in our study were negative for KIT gene mutations. This observation confirms the trend noted by Tian et al, 18 who found no mutations among 10 NSGCT. The extent to which KIT mutations are present in mixed seminoma/NSGCT is less clear. One of the two D816H mutations identified by Tian et al18 was found in a combined dysgerminoma/yolk sac tumor of the ovary. In contrast, we found no KIT mutations in 4 mixed seminoma/NSGCT originating in the testis.
It is possible that stromal cells present in the NSGCT samples may have contributed wild-type DNA that interfered with KIT mutation detection. This is certainly a concern, but there are several reasons why we believe that our results were not significantly influenced in this regard. First, we purposefully selected areas of NSGCT that were rich in high-grade elements (embryonal carcinoma and yolk sac tumor). Second, the areas of teratoma that were analyzed were dominated by epithelial-lined cysts and nodules of cartilage rather than pure stroma. Third, there is recent evidence that at least some of the stroma present in teratomas is tumor-derived rather than a host reaction.45 For these reasons, it is unlikely that the complete absence of detectable KIT gene mutations in all our NSGCT can be explained by contamination from reactive tissue elements.
To explore the role of KIT activation, whether resulting from mutation or ligand stimulation, we examined the phosphorylation status of KIT in seminoma lysates. KIT tyrosyl-phosphorylation, indicative of kinase activation, was readily detected only in seminomas with the D816V mutation. Another measure of KIT activation is the association of KIT with PI3K; we detected co-immunprecipitated PI3K in 1 of 2 cases with D816V and in 1 of 11 wild-type KIT seminomas. On reciprocal immunoprecipitation with a PI3K antibody there was much more phosphorylated and total D816V KIT associated with PI3K than was detected in wild-type KIT seminomas. Thus, were able to find compelling evidence of KIT kinase activation only in the D816V seminomas and not in any of the wild-type KIT cases. These findings indicate that KIT is not hyperactivated in seminomas lacking KIT mutations in the same manner observed for several KIT mutant seminomas. It is likely that KIT can be activated by ligand, or by other mechanisms, in wild-type tumors. However, such mechanisms presumably result in levels of KIT activation that are below the detection threshold for our assays (estimated to be <1% of the activation seen in seminomas with D816 KIT mutation).
There is ample precedent of KIT expression in human cancers in the absence of substantial or pathogenetically significant KIT activation (eg, melanoma, adenoid cystic carcinoma, small cell lung cancer). Indeed, in the case of melanoma, loss of KIT expression correlates directly with increasing clinical stage.1 We believe, based on our results, that in the majority of seminomas KIT expression may be a differentiation marker without playing a central pathogenetic role in the initiation or progression of these tumors. Indeed, not all seminomas even express KIT as assessed by immunohistochemistry.46 In contrast, seminomas with KIT mutations have biochemical evidence of strong KIT activation and it is likely that KIT plays a role in the initiation or maintenance of the seminoma tumor. Further studies are required to confirm this hypothesis.
If oncogenic activation of KIT is responsible for tumorigenesis and/or the maintenance of tumor phenotype in a subset of seminomas, then these tumors might have a distinct pattern of KIT-mediated intracellular signals that differs from wild-type tumors; however, this was not the case. PI3-kinase, AKT, phospho-AKT, MAP-kinase, phospho-MAP-kinase, STAT3, and phospho-STAT3 were all found at similar levels in 11 wild-type and 2 mutant (both D816V) seminoma lysates. This finding suggests that there might be alternate receptor tyrosine kinase oncogenes or redundant growth signaling pathways in germ cell tumors that have yet to be identified. Indeed, our finding of similar intracellular signal patterns in NSGCT lysates supports this hypothesis. Further proteomic- and/or genomic-based studies will be needed to identify alternative mechanisms for signal transduction pathway activation in seminomas lacking a KIT mutation.
What role activated KIT may play in the biology of seminomas remains unclear. As there is abundant evidence that seminomas and NSGCT share a common neoplastic germ cell of origin, it is intriguing that KIT mutations appear to be limited to seminomas.47 Seminomas likely deviate from NSGCT very early in the oncogenesis of germ cell tumors, and acquired KIT gene mutations may favor the seminoma pathway. From studies in mice it is known that signaling from KIT kinase through the PI3K pathway is essential for normal spermatogenesis.29,48 Insofar as seminomas retain morphological and immunophenotypic features that closely resemble primary germ cells, KIT activity may be important in maintaining this germ cell-like character. "Atypical seminomas," which show evidence of dedifferentiation (eg, expression of cytokeratins or CD30), have a significantly lower frequency of KIT expression than "usual seminomas" (57% vs. 95%, respectively).46 These "atypical seminomas" have higher mitotic counts and tend to present at a higher clinical stage.46 Thus, loss of KIT expression in seminomas may correlate with more aggressive clinical behavior.
To the extent that KIT is important in the biology of seminomas, KIT kinase inhibitors may have a role in their treatment, particularly for those seminomas with activating mutations. Imatinib mesylate has proven very effective in the treatment of advanced GI stromal tumors with KIT mutations.15,16 Unlike the case with GISTs, however, most seminomas in our study harbored activating KIT mutations (9 of 13) that are biochemically resistant to imatinib mesylate in vitro. Thus, only 4 of 54 testicular seminomas in our series had an imatinib mesylate-sensitive KIT mutation. Targeting the D816V and D816H mutations found in seminomas will require different kinase inhibitors that can inhibit the specific KIT conformation induced by these mutations.49
In contemplating the treatment of seminomas with a KIT kinase inhibitor, it must be noted that current medical treatment of seminoma results in a cure in the vast majority of patients. Indeed, even patients with advanced metastatic disease do well with platinum-based chemotherapy. A clinical trial sponsored by Cancer and Leukemia Group B (CALGB) is currently underway to examine the efficacy of imatinib mesylate in the treatment of advanced metastatic seminoma that is refractory to chemotherapy. KIT inhibitors might also be tested in a "window of opportunity" neoadjuvant setting in those patients with low volume retroperitoneal disease before their radiotherapy. If active in these settings, future development could include use of KIT inhibitors as an adjuvant therapy for patients with stage I disease, with the goal of decreasing the need for subsequent radiation treatment or chemotherapy and perhaps reducing consequent toxicities, eg, infertility. Rational design and interpretation of such trials will require molecular assessment of KIT activation, at both the protein and nucleic acid levels, in individual patient tumor specimens.
|
Footnotes |
|---|
Supported in part with federal funds from the National Cancer Institute, National Institutes of Health (Contract N01-CO-12400 [to M.C.H.), and a Department of Veterans Affairs Merit Review Grant (to M.C.H.).
Accepted for publication September 26, 2003.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Faderl, A. Pal, W. Bornmann, M. Albitar, D. Maxwell, Q. Van, Z. Peng, D. Harris, Z. Liu, I. Hazan-Halevy, et al. Kit Inhibitor APcK110 Induces Apoptosis and Inhibits Proliferation of Acute Myeloid Leukemia Cells Cancer Res., May 1, 2009; 69(9): 3910 - 3917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Heinrich, H. Joensuu, G. D. Demetri, C. L. Corless, J. Apperley, J. A. Fletcher, D. Soulieres, S. Dirnhofer, A. Harlow, A. Town, et al. Phase II, Open-Label Study Evaluating the Activity of Imatinib in Treating Life-Threatening Malignancies Known to Be Associated with Imatinib-Sensitive Tyrosine Kinases Clin. Cancer Res., May 1, 2008; 14(9): 2717 - 2725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J A Schumacher, K S J Elenitoba-Johnson, and M S Lim Detection of the c-kit D816V mutation in systemic mastocytosis by allele-specific PCR J. Clin. Pathol., January 1, 2008; 61(1): 109 - 114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Ruhe, S. Streit, S. Hart, C.-H. Wong, K. Specht, P. Knyazev, T. Knyazeva, L. S. Tay, H. L. Loo, P. Foo, et al. Genetic Alterations in the Tyrosine Kinase Transcriptome of Human Cancer Cell Lines Cancer Res., December 1, 2007; 67(23): 11368 - 11376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Voisset, S. Lopez, P. Dubreuil, and P. De Sepulveda The tyrosine kinase FES is an essential effector of KITD816V proliferation signal Blood, October 1, 2007; 110(7): 2593 - 2599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Link, G. Kunter, Y. Kasai, Y. Zhao, T. Miner, M. D. McLellan, R. E. Ries, D. Kapur, R. Nagarajan, D. C. Dale, et al. Distinct patterns of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia Blood, September 1, 2007; 110(5): 1648 - 1655. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G. Roberts, A. F. Odell, E. M. Byrnes, R. M. Baleato, R. Griffith, A. B. Lyons, and L. K. Ashman Resistance to c-KIT kinase inhibitors conferred by V654A mutation Mol. Cancer Ther., March 1, 2007; 6(3): 1159 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.J. Galan, M. De Felici, B. Buch, M.C. Rivero, A. Segura, J.L. Royo, N. Cruz, L.M. Real, and A. Ruiz Association of genetic markers within the KIT and KITLG genes with human male infertility Hum. Reprod., December 1, 2006; 21(12): 3185 - 3192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Corless, P. Harrell, M. Lacouture, T. Bainbridge, C. Le, K. Gatter, C. White Jr, S. Granter, and M. C. Heinrich Allele-Specific Polymerase Chain Reaction for the Imatinib-Resistant KIT D816V and D816F Mutations in Mastocytosis and Acute Myelogenous Leukemia J. Mol. Diagn., November 1, 2006; 8(5): 604 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Heinrich, C. L. Corless, C. D. Blanke, G. D. Demetri, H. Joensuu, P. J. Roberts, B. L. Eisenberg, M. von Mehren, C. D.M. Fletcher, K. Sandau, et al. Molecular Correlates of Imatinib Resistance in Gastrointestinal Stromal Tumors J. Clin. Oncol., October 10, 2006; 24(29): 4764 - 4774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Paschka, G. Marcucci, A. S. Ruppert, K. Mrozek, H. Chen, R. A. Kittles, T. Vukosavljevic, D. Perrotti, J. W. Vardiman, A. J. Carroll, et al. Adverse Prognostic Significance of KIT Mutations in Adult Acute Myeloid Leukemia With inv(16) and t(8;21): A Cancer and Leukemia Group B Study J. Clin. Oncol., August 20, 2006; 24(24): 3904 - 3911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gabillot-Carre, Y. Lepelletier, M. Humbert, P. de Sepuvelda, N. B. Hamouda, J. P. Zappulla, R. Liblau, A. Ribadeau-Dumas, F. Machavoine, S. Letard, et al. Rapamycin inhibits growth and survival of D816V-mutated c-kit mast cells Blood, August 1, 2006; 108(3): 1065 - 1072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Rajpert-De Meyts Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects Hum. Reprod. Update, May 1, 2006; 12(3): 303 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Schittenhelm, S. Shiraga, A. Schroeder, A. S. Corbin, D. Griffith, F. Y. Lee, C. Bokemeyer, M. W.N. Deininger, B. J. Druker, and M. C. Heinrich Dasatinib (BMS-354825), a Dual SRC/ABL Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane, and Activation Loop Mutant KIT Isoforms Associated with Human Malignancies Cancer Res., January 1, 2006; 66(1): 473 - 481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. F. Goemans, C. M. Zwaan, A. Harlow, A. H. Loonen, B. E. S. Gibson, K. Hahlen, D. Reinhardt, U. Creutzig, M. C. Heinrich, and G. J. L. Kaspers In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets Blood, November 15, 2005; 106(10): 3532 - 3537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. McIntyre, B. Summersgill, B. Grygalewicz, A. J.M. Gillis, J. Stoop, R. J.H.L.M. van Gurp, N. Dennis, C. Fisher, R. Huddart, C. Cooper, et al. Amplification and Overexpression of the KIT Gene Is Associated with Progression in the Seminoma Subtype of Testicular Germ Cell Tumors of Adolescents and Adults Cancer Res., September 15, 2005; 65(18): 8085 - 8089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Hofer, T. J. Browne, L. He, R. I. Skotheim, R. A. Lothe, and M. A. Rubin Identification of Two Molecular Groups of Seminomas by Using Expression and Tissue Microarrays Clin. Cancer Res., August 15, 2005; 11(16): 5722 - 5729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Corless, A. Schroeder, D. Griffith, A. Town, L. McGreevey, P. Harrell, S. Shiraga, T. Bainbridge, J. Morich, and M. C. Heinrich PDGFRA Mutations in Gastrointestinal Stromal Tumors: Frequency, Spectrum and In Vitro Sensitivity to Imatinib J. Clin. Oncol., August 10, 2005; 23(23): 5357 - 5364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. P. Rubin, C. R. Antonescu, J. P. Scott-Browne, M. L. Comstock, Y. Gu, M. R. Tanas, C. B. Ware, and J. Woodell A Knock-In Mouse Model of Gastrointestinal Stromal Tumor Harboring Kit K641E Cancer Res., August 1, 2005; 65(15): 6631 - 6639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Krause and R. A. Van Etten Tyrosine Kinases as Targets for Cancer Therapy N. Engl. J. Med., July 14, 2005; 353(2): 172 - 187. [Full Text] [PDF]
|
|
|
|
|
|
R. I. Skotheim, G. E. Lind, O. Monni, J. M. Nesland, V. M. Abeler, S. D. Fossa, N. Duale, G. Brunborg, O. Kallioniemi, P. W. Andrews, et al. Differentiation of Human Embryonal Carcinomas In vitro and In vivo Reveals Expression Profiles Relevant to Normal Development Cancer Res., July 1, 2005; 65(13): 5588 - 5598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Corbin, S. Demehri, I. J. Griswold, Y. Wang, C. A. Metcalf III, R. Sundaramoorthi, W. C. Shakespeare, J. Snodgrass, S. Wardwell, D. Dalgarno, et al. In vitro and in vivo activity of ATP-based kinase inhibitors AP23464 and AP23848 against activation-loop mutants of Kit Blood, July 1, 2005; 106(1): 227 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Antonescu, P. Besmer, T. Guo, K. Arkun, G. Hom, B. Koryotowski, M. A. Leversha, P. D. Jeffrey, D. Desantis, S. Singer, et al. Acquired Resistance to Imatinib in Gastrointestinal Stromal Tumor Occurs Through Secondary Gene Mutation Clin. Cancer Res., June 1, 2005; 11(11): 4182 - 4190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yanagihori, N. Oyama, K. Nakamura, and F. Kaneko c-kit Mutations in Patients with Childhood-Onset Mastocytosis and Genotype-Phenotype Correlation J. Mol. Diagn., May 1, 2005; 7(2): 252 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sihto, M. Sarlomo-Rikala, O. Tynninen, M. Tanner, L. C. Andersson, K. Franssila, N. N. Nupponen, and H. Joensuu KIT and Platelet-Derived Growth Factor Receptor Alpha Tyrosine Kinase Gene Mutations and KIT Amplifications in Human Solid Tumors J. Clin. Oncol., January 1, 2005; 23(1): 49 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Uccini, O. Mannarino, H. P. McDowell, U. Pauser, R. Vitali, P. G. Natali, P. Altavista, T. Andreano, S. Coco, R. Boldrini, et al. Clinical and Molecular Evidence for c-kit Receptor as a Therapeutic Target in Neuroblastic Tumors Clin. Cancer Res., January 1, 2005; 11(1): 380 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Corbin, I. J. Griswold, P. La Rosee, K. W. H. Yee, M. C. Heinrich, C. L. Reimer, B. J. Druker, and M. W. N. Deininger Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970 Blood, December 1, 2004; 104(12): 3754 - 3757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Th. Went, S. Dirnhofer, M. Bundi, M. Mirlacher, P. Schraml, S. Mangialaio, S. Dimitrijevic, J. Kononen, A. Lugli, R. Simon, et al. Prevalence of KIT Expression in Human Tumors J. Clin. Oncol., November 15, 2004; 22(22): 4514 - 4522. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
