Andrea Warrick

KIT Gene Mutations and Copy Number in Melanoma Subtypes

Purpose: We recently identified a KIT exon 11 mutation in an anorectal melanoma of a patient who had an excellent response to treatment with imatinib. To determine the frequency of KIT mutations across melanoma subtypes, we surveyed a large series of tumors. Experimental Design: One hundred eighty-nine melanomas were screened for mutations in KIT exons 11, 13, and 17. KIT copy number was assessed by quantitative PCR. A subset of cases was evaluated for BRAF and NRAS mutations. Immunohistochemistry was done to assess KIT (CD117) expression. Results:KIT mutations were detected in 23% (3 of 13) of acral melanomas, 15.6% (7 of 45) of mucosal melanomas, 7.7% (1 of 13) of conjunctival melanomas, 1.7% (1 of 58) of cutaneous melanomas, and 0% (0 of 60) of choroidal melanomas. Almost all the KIT mutations were of the type predicted to be imatinib sensitive. There was no overlap with NRAS mutations (11.1% of acral and 24.3% of mucosal tumors) or with BRAF mutations (absent in mucosal tumors). Increased KIT copy number was detected in 27.3% (3 of 11) of acral and 26.3% (10 of 38) of mucosal melanomas, but was less common among cutaneous (6.7%; 3 of 45), conjunctival (7.1%; 1 of 14), and choroidal melanomas (0 of 28). CD117 expression, present in 39% of 105 tumors representing all melanoma types, did not correlate with either KIT mutation status or KIT copy number. Conclusions: Our findings confirm that KIT mutations are most common in acral and mucosal melanomas but do not necessarily correlate with KIT copy number or CD117 expression. Screening for KIT mutations may open up new treatment options for melanoma patients.

Molecular Profiling and Targeted Therapy for Advanced Thoracic Malignancies: A Biomarker-Derived, Multiarm, Multihistology Phase II Basket Trial

PURPOSE We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. PATIENTS AND METHODS We enrolled patients with advanced non-small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. RESULTS Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). CONCLUSION This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies.

High prevalence of PIK3CA/AKT pathway mutations in papillary neoplasms of the breast.

Papillary lesions of the breast have an uncertain relationship to the histogenesis of breast carcinoma, and are thus diagnostically and managerially challenging. Molecular genetic studies have provided evidence that ductal carcinoma in situ and even atypical ductal hyperplasia are precursors of invasive carcinoma. However, papillary lesions have been seldom studied. We screened papillary breast neoplasms for activating point mutations in PIK3CA, AKT1, and RAS protein-family members, which are common in invasive ductal carcinomas. DNA extracts were prepared from sections of 89 papillary lesions, including 61 benign papillomas (28 without significant hyperplasia; 33 with moderate to florid hyperplasia), 11 papillomas with atypical ductal hyperplasia, 7 papillomas with carcinoma in situ, and 10 papillary carcinomas. Extracts were screened for PIK3CA and AKT1 mutations using mass spectrometry; cases that were negative were further screened for mutations in AKT2, BRAF, CDK, EGFR, ERBB2, KRAS, NRAS, and HRAS. Mutations were confirmed by sequencing or HPLC assay. A total of 55 of 89 papillary neoplasms harbored mutations (62%), predominantly in AKT1 (E17K, 27 cases) and PIK3CA (exon 20 >exon 9, 27 cases). Papillomas had more mutations in AKT1 (54%) than in PIK3CA (21%), whereas papillomas with hyperplasia had more PIK3CA (42%) than AKT1 (15%) mutations, as did papillomas with atypical ductal hyperplasia (PIK3CA 45%, AKT1 27%, and NRAS 9%). Among seven papillomas with carcinoma in situ, three had AKT1 mutations. The 10 papillary carcinomas showed an overall lower frequency of mutations, including 1 with an AKT1 mutation (in a tumor arising from a papilloma), 1 with an NRAS gene mutation (Q61H), and 2 with PIK3CA mutations (1 overlapping with the NRAS Q61H). These findings indicate that approximately two-thirds of papillomas are driven by mutations in the PI3CA/AKT pathway. Some papillary carcinomas may arise from these lesions, but others may have different molecular origins.

Multiplex Mutation Screening by Mass Spectrometry: Evaluation of 820 Cases from a Personalized Cancer Medicine Registry

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.

Adoption of a clinical pharmacogenomics implementation program during outpatient care–initial results of the University of Chicago “1,200 Patients Project”

Pharmacogenomic testing is viewed as an integral part of precision medicine. To achieve this, we originated The 1,200 Patients Project which offers broad, preemptive pharmacogenomic testing to patients at our institution. We analyzed enrollment, genotype, and encounter‐level data from the first year of implementation to assess utility of providing pharmacogenomic results. Results were delivered via a genomic prescribing system (GPS) in the form of traffic lights: green (favorable), yellow (caution), and red (high risk). Additional supporting information was provided as a virtual pharmacogenomic consult, including citation to relevant publications. Currently, 812 patients have participated, representing 90% of those approached; 608 have been successfully genotyped across a custom array. A total of 268 clinic encounters have occurred at which results were accessible via the GPS. At 86% of visits, physicians accessed the GPS, receiving 367 result signals for medications patients were taking: 57% green lights, 41% yellow lights, and 1.4% red lights. Physician click frequencies to obtain clinical details about alerts varied according to color severity (100% of red were clicked, 72% yellow, 20% green). For 85% of visits, clinical pharmacogenomic information was available for at least one drug the patient was taking, suggesting relevance of the delivered information. We successfully implemented an individualized health care model of preemptive pharmacogenomic testing, delivering results along with pharmacogenomic decision support. Patient interest was robust, physician adoption of information was high, and results were routinely utilized. Ongoing examination of a larger number of clinic encounters and inclusion of more physicians and patients is warranted.

Newly described activating JAK3 mutations in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease that accounts for 10–15% of adult and 25% of childhood ALL cases. Despite advances in therapy of T-ALL, relapsed disease remains a leading cause of death. The genetic and biological determinants of treatment failure and clinical outcomes remain incompletely understood.

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ∼25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.

Frequent phosphatidylinositol-3-kinase mutations in proliferative breast lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly altered molecular pathways in invasive breast carcinoma, with phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) mutations in 25% of invasive carcinomas. Ductal carcinoma in situ (DCIS), benign papillomas, and small numbers of columnar cell lesions harbor an analogous spectrum of PIK3CA and AKT1 mutations, yet there is little data on usual ductal hyperplasia and atypical ductal and lobular neoplasias. We screened 192 formalin-fixed paraffin-embedded breast lesions from 75 patients for point mutations using a multiplexed panel encompassing 643 point mutations across 53 genes, including 58 PIK3CA substitutions. PIK3CA point mutations were identified in 31/62 (50%) proliferative lesions (usual ductal hyperplasia and columnar cell change), 10/14 (71%) atypical hyperplasias (atypical ductal hyperplasia and flat epithelial atypia), 7/16 (44%) lobular neoplasias (atypical lobular hyperplasia and lobular carcinoma in situ), 10/21 (48%) DCIS, and 13/37 (35%) invasive carcinomas. In genotyping multiple lesions of different stage from the same patient/specimen, we found considerable heterogeneity; most notably, in 12 specimens the proliferative lesion was PIK3CA mutant but the concurrent carcinoma was wild type. In 11 additional specimens, proliferative epithelium and cancer contained different point mutations. The frequently discordant genotypes of usual ductal hyperplasia/columnar cell change and concurrent carcinoma support a role for PIK3CA-activating point mutations in breast epithelial proliferation, perhaps more so than transformation. Further, these data suggest that proliferative breast lesions are heterogeneous and may represent non-obligate precursors of invasive carcinoma.

Mucinous breast carcinomas lack PIK3CA and AKT1 mutations

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma.

Immunohistochemical and molecular markers in breast phyllodes tumors.

Phyllodes tumors of the breast are diagnostically and managerially enigmatic, as their malignant potential is difficult to predict based on the standard morphologic criteria. Thus, there is a need for additional markers of biologic potential. Although a number of ancillary tests have been reported, consensus in the literature is lacking. We studied 38 cellular fibroadenomas and phyllodes tumors of various grade (World Health Organization benign, borderline, and malignant) with a panel of immunohistochemical stains (p53, CD117, phospho-Histone3, mdm2, cdk4) and screened 26 of the tumors for mutations across 30 cancer-related genes using PCR and mass-spectrometry based methods. p53 and phospho-Histone3 (mitotic marker) showed increased staining in higher grade phyllodes tumors. CD117, mdm2, and cdk4 showed no difference in expression across different grades of phyllodes tumors. Mutational analysis revealed an S8R substitution in FBX4 (an E3 ubiquitin ligase) in 3 cases: 1 benign and 2 borderline. The S8R substitution seems to be more common in phyllodes tumors (11.5%) as compared with other cancers. FBX4 S8R cases had high cyclin D1 expression, but this finding was not specific. Our data support earlier studies showing that p53 has potential use in pathologic assessment of phyllodes tumors, and we newly characterized phospho-Histone3 for this application. Further studies are needed to characterize the molecular pathogenesis of the phyllodes tumors, as we were unable to identify activating mutations despite screening for a large panel of activating hotspot mutations. The significance of the FBX4 substitution deserves further investigation.