Francesca Miselli

Surgery of Residual Disease Following Molecular-targeted Therapy With Imatinib Mesylate in Advanced/Metastatic GIST

Objective:To explore the role of surgery of residual disease following a period of therapy with imatinib mesylate in advanced gastrointestinal stromal tumors (GIST). Methods:From January 2001 to June 2005, 159 patients with advanced/metastatic GIST were treated with imatinib mesylate at a single institution. As of June 2002, 38 patients were selected for surgery following a variable period of imatinib therapy. Twenty-seven patients were operated on while they were in response, 8 in progression, 3 for localized disease. Clinical, pathologic, and molecular features were assessed and are reported. Results:Postsurgery PFS was 96% at 12 months and 69% at 24 months for responding patients, while it was nil at 12 months for progressing ones. Disease-specific survival at 12 months was 100% for responding patients and 60% for progressing ones. In responding cases, secondary progression was mainly related to postsurgical imatinib discontinuation, irrespective of pathologic or molecular variables. In progressing patients, secondary resistance was mainly related to acquired mutations. Conclusion:In advanced GIST patients who are responding to imatinib mesylate, the role of surgery is not formally demonstrated at the moment, but this option may well be considered investigational, or suitable for an individualized decision-making in the lack of evidence. In our series, patients progressing on imatinib mesylate did not seem to have any major benefit from surgery, although their number is low.

Molecular and Biochemical Analyses of Platelet-Derived Growth Factor Receptor (PDGFR) B, PDGFRA, and KIT Receptors in Chordomas

Purpose: We have previously shown the presence of an activated platelet-derived growth factor (PDGF) receptor (PDGFR) B and its ligand PDGFB in a limited number of patients with clinical and radiological responses to imatinib mesylate treatment. This article describes the results of comprehensive molecular/biochemical analyses of the three receptors targeted by the drug (PDGFRB, PDGFRA, and KIT) in a series of 31 chordoma patients. Experimental Design: The presence and activation status of PDGFRB, PDGFRA, and KIT receptors were investigated by means of immunoprecipitation and Western blot analyses complemented by immunohistochemistry, their expression level was analyzed by means of real-time PCR, and the occurrence of activating point mutations was investigated by means of cDNA sequencing. The PDGFB, PDGFA, and stem cell factor cognate ligands were investigated by reverse transcription-PCR, and gene status was assessed by fluorescence in situ hybridization. Results: The results show that PDGFRB was highly expressed and phosphorylated, whereas PDGFRA and KIT were less expressed but phosphorylated and thus activated. These findings, together with the absence of gain-of-function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism is the autocrine/paracrine loop. No role seems to be played by gene amplification. Conclusions: In the light of our findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be attributable to the switching off of all three receptors.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 μM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

c-Kit/PDGFRA gene status alterations possibly related to primary imatinib resistance in gastrointestinal stromal tumors.

Purpose: To correlate morphologic changes with molecular, biochemical, and cytogenetic profiles in gastrointestinal stromal tumor (GIST) patients before and after imatinib treatment. Experimental Design: We investigated 132 tumor samples obtained from 35 patients with advanced disease who underwent resective surgery after imatinib treatment according to the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group protocol. On the basis of imaging findings, 27 patients were responders and 8 progressors, and retaining this radiological subdivision, we analyzed posttreatment morphologic changes correlating them with molecular, biochemical, and cytogenetic analyses. Results: On the basis of morphology (residual viable cellularity/proliferation markers), three subgroups were identified showing high, moderate, or low response. All of the progressing cases clustered in the low-response subgroup, whereas the responding cases were distributed in all three subgroups. The correlation between morphology and the molecular findings showed that secondary mutations segregated with the low-response subgroup, whereas c-Kit primary resistance mutations were randomly distributed in the three subgroups. Fluorescence in situ hybridization analysis of c-Kit/PDGFRA genes showed that all of the progressing cases were disomic. Referring to morphology, among the responding cases, a disomic pattern was mainly restricted to the high responders, whereas the moderate and low responders were aneusomic. Comparison of post-imatinib genomic profiles with the 23 available primary tumors showed that 17 cases carried the same cytogenetic pattern. Overall, 12 of the 27 primary tumors presented a gain/loss of c-Kit/PDGFRA gene copy number. Conclusions: Our findings show that c-Kit/PDGFRA genomic alterations were present at disease onset in 1/3 of the examined cases. They therefore represent an early event possibly related to primary imatinib resistance in GISTs.

Cutaneous Melanoma in Childhood and Adolescence Shows Frequent Loss of INK4A and Gain of KIT

Childhood cutaneous melanoma is a rare disease with increasing incidence. It is not clear whether it differs from adult melanoma in etiology and clinical evolution. To genetically characterize childhood melanoma, 21 pediatric patients were studied by germ-line analysis of CDKN2A, CDK4, and MC1R genes. In addition, alterations in CDKN2A, c-Kit, BRAF, and NRAS genes were evaluated at the somatic level by direct gene sequencing, fluorescence in situ hybridization analysis, and immunohistochemistry. As a control group of susceptible patients, we studied patients from 23 melanoma-prone families. At the germ-line level, CDKN2A and MC1R gene variants were detected in 2/21 and 12/21 pediatric patients and in 9/23 and 19/22 in familial patients. At the somatic level, most lesions (9/14) from pediatric patients showed CDKN2A locus homozygous deletions and a null p16 immunophenotype, whereas most lesions (5/8) from familial patients were disomic and immunoreactive. A c-Kit low-polysomy profile seems to parallel CDKN2A homozygous deletions in pediatric melanoma whereas the single activating mutation observed segregates with familial patients. Loss of KIT protein expression was frequent (7/14) in pediatric melanomas, where metastatic cases were prevalent. BRAF(V600E) mutation occurred at a similar rate (approximately 50%) in lesions from pediatric and familial patients, whereas no NRAS mutations were detected.

PDGFRA immunostaining can help in the diagnosis of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are characterized by the presence of activating mutations affecting the c-Kit or the PDGFRA gene. Although these mutations are mutually exclusive, their proteins are coexpressed in many GISTs with various modulations of immunostaining depending on which gene is mutated. CD117 expression is currently considered a sensitive (although not entirely specific) marker of KIT activation, but there is no consensus concerning the reliability of PDGFRA antibody. Our database contains 236 molecularly analyzed GISTs, and we here describe the 180 cases that underwent KIT/PDGFRA immunophenotyping. By correlating the immunophenotype with the molecular status of the genes expected to be involved, we observed the coexpression of KIT and PDGFRA in the majority of the mutated c-Kit and wild-type c-Kit/PDGFRA GISTs, whereas the −/+ immunophenotype (0% vs. 48.6%) and PDGFRA dotlike immunostaining (P<0.005) segregated with the PDGFRA-mutated GISTs. Taking either the dotlike decoration (26 cases) or −/+ immunophenotype (5 cases) as hallmarks of PDGFRA mutation, the presence of a PDGFRA mutation was predicted in 31 (83.8%) of the 37 PDGFRA mutated GISTs. Our findings suggest that, when critically applied, the routine use of CD117/PDGFRA immunophenotyping is a useful diagnostic tool (especially in CD117-negative cases) as it correctly predicts the presence of PDGFRA mutations in most cases.

Analysis of potential receptor tyrosine kinase targets in intimal and mural sarcomas.

Primary sarcomas of the great vessels are very rare neoplasms and only a few cases have been reported. They are divided into the two broad categories of intimal or luminal and mural sarcomas. We analysed eight advanced high‐grade sarcomas originating from major vessels (seven intimal and one mural sarcoma) by means of immunohistochemistry and FISH analysis for PDGFRA, PDGFRB, EGFR and KIT receptor tyrosine kinases (RTKs), together with immunoprecipitation/western blotting, sequencing of the corresponding genes, and the search for cognate ligands. The intimal sarcomas showed a wide spectrum of morphologies and immunophenotypes, whereas the mural sarcoma had common leiomyosarcomatous features. Regardless of their category, all of the cases had a PDGFRA‐deregulated cytogenetic profile mainly consisting of an amplification cluster; five were also polysomic for PDGFRB, whereas three showed disomy. Six cases had a deregulated EGFR gene, and c‐Kit gene status was similar to that of PDGFRA. In one case, biochemical analysis revealed the presence of activated and highly expressed PDGFRA, PDGFRB and EGFR, whereas KIT was expressed at reference level. Sequencing of the corresponding genes revealed no activating mutations in any of the analysed receptors. The cognate ligands were detected in all cases. In predictive terms, the evidence of gene amplification/high polysomy of several RTKs, together with PDGFRA, PDGFRB and EGFR expression and phosphorylation, suggests that these tumours may be sensitive to RTK‐inhibiting treatments. Copyright

A sporadic multiple GIST with unusual pathologic, molecular, and genetic features.

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