Pathologists predict behavior of GIST

Armed Forces Institute of Pathology researchers have focused on GIST
Second of two parts. The first part can be found in the May newsletter.

By Markku Miettinen, M.D., and Jerzy Lasota

The opinions and assertions in this article are the views of the authors and are not to be construed as reflecting the views of the Departments of the Army or Defense. The authors work for the American Registry of Pathology, contracted to work in the Armed Forces Institute of Pathology, offering consultation to government and civilian institutions worldwide. The authors wish to acknowledge the support of Department Defense and American Registry of Pathology in writing this article, as well as the encouragement of the Life Raft Group and its executive director, Norman Scherzer.

PREDICTION of TUMOR BEHAVIOR

The largest clinicopathologic series indicate that GISTs have a spectrum from small benign, typically incidentally diagnosed nodules to malignant tumors (sarcomas) at all sites of occurrence. Perhaps 20 to 30 percent of all GISTs are malignant; the percentage in published series have varied as, for example, series from cancer hospitals have understandably included more malignant tumors.

Mitotic activity and tumor size are the most important gross and microscopic features in the assessment of malignancy. Typically, tumors smaller than 5 cm. with less than 5 mitoses per 50 high magnification fields have been considered benign, although intestinal tumors with such parameters have some unpredictability and a low metastatic rate. Tumors with mitotic activity over 5 per 50 high magnification fields often behave in a malignant manner with potential for metastasis. Tumors greater than 5 cm. with low mitotic activity are somewhat unpredictable. However, they seem to be much more favorable in the stomach than in the intestines. The mutation type may be a prognostic factor, but information is still limited.

MOLECULAR PATHOLOGY OF GISTs

The structure of KIT and PDGFRA genes and their encoded proteins are reviewed here briefly as a background for understanding of KIT and PDGFRA mutations, which are believed to play important role in GIST pathogenesis.

Normal KIT gene and protein: KIT and PDGFRA genes are located on chromosome 4q12. KIT and PDGFRA display extensive structural homology and are members of the type III tyrosine kinase receptor family. Both proteins consist of extracellular, transmembrane, juxtamembrane, and a twopart intracellular tyrosine kinase domain. Activation of TK type III receptors by their ligands leads to downstream phosphorylation of substrate proteins and subsequently activates networks of signal transduction pathways, which regulate important cell functions. In the GI tract, KIT is expressed in interstitial cells of Cajal.

Pathologic activation of TK receptors correlates with enhanced proliferation and development of cancer; its inhibition is considered an important therapeutic approach in oncology.

KIT mutations: Somatic (noninheritable) activating (gain-offunction) mutations in the KIT gene are believed to be a major driving force in the pathogenesis of sporadic non-familial GISTs, and structurally similar, constitutional (inheritable) mutations have been found in patients with familial GISTs. In general, these mutations are believed to confer KIT an independence of the ligand binding signal. Most of the KIT mutations have been heterozygous in nature, consistent with the concept of a dominant oncogene activated by monoallelic mutation. Of some of the specific mutation types, the transforming or autophosphorylating nature has been demonstrated experimentally in vitro. Germ-line KIT mutations similar to those identified in sporadic GISTs have been reported in human familial GIST syndrome characterized by hyperpigmentation and/or urticaria pigmentosa, ICC hyperplasia, and GISTs. Introduction of KIT exon 11 activating mutation into mouse genome reproduced features of human familial GIST syndrome in a mouse model and confirmed the critical role of KIT alteration in GIST development

Four different regions of KIT have been found to be involved. They are, in decreasing order of frequency: exon 11 (juxtamembrane domain), exon 9 (extracellular domain), exon 13 and exon 17 (tyrosine kinase domain).

Exon 11: The most common region involved by KIT mutations in GIST is exon 11, the juxtamembrane domain. This portion, just inside the cell membrane, is a helical domain of KIT, which seems to functionally represent an inhibitory region that regulates the KIT autophosphorylation in response to growth factor signal by KITs ligand, stem cell factor.

Exon 11 mutations were the first KIT mutations described in GISTs and are by far the most common KIT mutations in GIST. The three mutation types involving exon 11 are, in decreasing order of frequency: 1) in frame deletions, usually in the proximal part of the exon; 2) missense mutations, and 3) insertions in the distal part of the exon.

A majority of exon 11 mutations have been in frame deletions of one to several base pairs, in some cases apparently leading also to point mutations occurring in either end of the deletion. Typically these mutations involve the sequences between codons Gln550 and Glu561. In frame deletions in the distal part of exon 11 are rare, however, their functional significance appears to be similar to that of the typical mutational “hotspot.” For example, deletion of Asp579 was shown to activate KIT protein. Exon 11 deletions have been found in GISTs of different organ origins, and some studies have suggested their more common occurrence in large GISTs, and adverse prognostic significance. However, others have shown KIT exon 11 deletions in diminutive incidental tumors known to be clinically indolent. Therefore, prognostic significance of these KIT mutations is currently unclear.

Missense mutations in exon 11 have been reported less frequently than deletions. These mutations involved almost exclusively four codons: Trp557, Val559, Val560 and Leu576. Because of their rarity, their clinical significance is somewhat unclear. However, it was suggested that presence of point mutations in KIT exon 11 might correlate with better overall survival of GISTs patients. Also insertions leading to almost perfect duplication (internal tandem duplication [ITD]) of one to several codons in the distal part of exon 11 appear to be rare in GISTs. These types of KIT-juxtamembrane mutations were previously reported in canine mastocytoma and shown to be associated with constitutive phosphorylation of the KIT protein. Tumors defined by ITDs showed a strong association with gastric location and follow-up data indicated, in gastric cases, less malignant course of the disease than in tumors defined by deletions.

Exon 9: This exon encodes the distal part of the extracellular domain, and seems to be the second most commonly involved region after exon 11. All mutations have been structurally identical duplications of six nucleotides GCC TAT, encoding Ala502- Tyr503. The frequency of this mutation varies from 3 to 18 percent, but this mutation has a strong predilection to intestinal tumors, and therefore its frequency in GIST series will depend on the number of intestinal vs. gastric tumors included. Also, most tumors with the exon 9 mutation have been clinically malignant, suggesting that this type of KIT mutation identifies a clinically aggressive subset of GIST.

Exon 13: Identical missense mutations, resulting in substitution of Glu for Lys642 in exon 13 encoding the tyrosine kinase I, ATP-binding domain of KIT, have been reported with a very low (1-2 percent) frequency. Homozygous exon 13 mutations have been found to lead into constitutive KIT tyrosine phosphorylation. Based on the published series, this mutation seems to be associated with malignant tumor behavior. This mutation was shown to be sensitive to imatinib mesylate (Gleevec/Glivec, formerly STI571), which was able to abolish the phosphorylated status of KIT in a cell line.

Exon 17: This exon encodes part of the catalytic, phosphotransferase domain of KIT, tyrosine kinase II domain. KIT activating replacement mutations causing substitution of Asp816 have been previously reported in mastocytoma, seminoma, and sinonasal natural killer/T-cell lymphoma but a large initial series of GISTs was negative for exon 17 mutations. Subsequently, isolated cases of GISTs with mutations involving this exon have been reported. The number of cases is too small for clinicopathologic correlation.

PDGFRA mutation: Recently, platelet-derived growth factor receptor α (PDGFRA) was shown to be pathologically activated in GISTs. Gain-offunction PDGFRA mutations affecting the activation loop (exon 18 [tyrosine kinase domain]) and juxtamembrane domain (exon 12) were found in 14 of 40 (35 percent) KIT-mutation-negative GISTs. It was suggested that mutational activation of KIT or PDGFRA are mutually exclusive and represent two different alternative ocogenic events leading to similar biological consequences.

Exon 18: Gain-of-function mutations in exon 18 appear to be the most common PDGFRA mutations in GISTs. A great majority of these mutations represent simple T to A missense mutation at the codon 842 leading to substitution of the Val for Asp842. However, in-frame deletions and deletions with point mutations clustering between codons 841-847 were also reported. Germ-line PDGFRA mutations leading to substitution of Tyr for Asp846 has been reported in familial GIST syndromes. Tumors with point mutations affecting codon 842 are smaller and show lower mitotic activity compare to one with deletions. Also PDGFRA exon 18 mutations have a clear predilection to epthelioid morphology and gastric over intestinal location of tumor.

Exon 12: PDGFRA exon 12 mutations are rare, some five to six times less frequent than exon 18 mutations. A majority represent either point mutation leading to substitutions of Asp for Val561 or in-frame deletions with or without coexisting point mutations clustering between codons 566 and 571. Because of their rarity, their clinical significance is somewhat unclear.

Recently, Gleevec was successfully introduced in the treatment of clinically advanced, metastatic GISTs. However, based on “in vitro” studies, it has been suggested that KIT and PDGFRA activated by mutation in the tyrosine kinase domain might not respond well to Gleevec-based TKinhibition. Recent clinical studies on the response of metastatic GISTs to the Gleevec-based treatment revealed limited therapeutic effect in PDGFRA exon18 mutant tumors, however, GISTs with PDGFRA exon 12 mutations maintained a response similar to GISTs with KIT exon 11 mutations.

CONCLUSION

Several aspects of GISTs are under intense investigation. These include the correlation between natural history and mutations, treatment prospects, proper patient selection and mechanisms for possible primary and secondary resistance.

REFERENCES

M. Miettinen, J. Lasota: Gastrointestinal stromal tumors (GISTs): definition, occurrence, pathology, differential diagnosis and molecular genetics. Pol J Pathol 2003; 54:3-24.
S. Hirota, K. Isozaki, Y. Moriyama, et al: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998; 279:577-580.
M.C. Heinrich, B.P. Rubin, B.J. Longley, J.A. Fletcher: Biology and genetic aspects of gastrointestinal stromal tumors. Hum Pathol 2002; 33:484-495.
M.C. Heinrich, C.L. Corless, A. Duensing, et al: PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299:708-710.

About the authors: Pathologist Markku Miettinen, 51, is the chairman and distinguished scientist of the Department of Soft Tissue Pathology of the AFIP where he’s worked since 1996. His current research focus is GIST pathology. He worked eight years as a pathologist in Thomas Jefferson University Hospital in Philadelphia, Penn., and 10 years at the University of Helsinki, Finland, his native country. Jerzy Lasota, 47, has been a pathologist at the Department of Soft Tissue Pathology since 1996. He currently specializes in molecular pathology of GIST, and is responsible for the department’s laboratory. He did seven years of cancer research in Kimmel Cancer Center/Department of Pathology and the Fels Research Institute in Thomas Jefferson and Temple Universities in Philadelphia, and seven years in the Medical Academy of Lodz, Poland, his native country. Together the authors have published more than 30 scientific articles on gastrointestinal stromal tumors.

Address for correspondence: Markku Miettinen, M.D. Department of Soft Tissue Pathology Armed Forces Institute of Pathology 14th Street and Alaska Avenue, N.W. Washington, DC 20306-6000, USA