Carol Beadling

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.

Imatinib for Melanomas Harboring Mutationally Activated or Amplified KIT Arising on Mucosal, Acral, and Chronically Sun-Damaged Skin

PURPOSE Amplifications and mutations in the KIT proto-oncogene in subsets of melanomas provide therapeutic opportunities. PATIENTS AND METHODS We conducted a multicenter phase II trial of imatinib in metastatic mucosal, acral, or chronically sun-damaged (CSD) melanoma with KIT amplifications and/or mutations. Patients received imatinib 400 mg once per day or 400 mg twice per day if there was no initial response. Dose reductions were permitted for treatment-related toxicities. Additional oncogene mutation screening was performed by mass spectroscopy. RESULTS Twenty-five patients were enrolled (24 evaluable). Eight patients (33%) had tumors with KIT mutations, 11 (46%) with KIT amplifications, and five (21%) with both. Median follow-up was 10.6 months (range, 3.7 to 27.1 months). Best overall response rate (BORR) was 29% (21% excluding nonconfirmed responses) with a two-stage 95% CI of 13% to 51%. BORR was significantly greater than the hypothesized null of 5% and statistically significantly different by mutation status (7 of 13 or 54% KIT mutated v 0% KIT amplified only). There were no statistical differences in rates of progression or survival by mutation status or by melanoma site. The overall disease control rate was 50% but varied significantly by KIT mutation status (77% mutated v 18% amplified). Four patients harbored pretreatment NRAS mutations, and one patient acquired increased KIT amplification after treatment. CONCLUSION Melanomas that arise on mucosal, acral, or CSD skin should be assessed for KIT mutations. Imatinib can be effective when tumors harbor KIT mutations, but not if KIT is amplified only. NRAS mutations and KIT copy number gain may be mechanisms of therapeutic resistance to imatinib.

How do viral infections predispose patients to bacterial infections

Purpose of review Bacterial sepsis is a leading cause of death in the United States, accounting for over 200 000 fatalities annually. Approximately half of bacterial sepsis cases occur following acute respiratory infections, and the lungs are the most common organs to fail. Notably, outbreaks of respiratory viral infections are associated with an increased incidence or severity of bacterial co-infections, with normally innocuous infections often becoming fatal. Understanding the ‘lethal synergism’ associated with concomitant infections may point the way toward improved anti-sepsis treatments. Recent findings Murine models of viral and bacterial co-infection mimic the lethal synergism observed in humans and reveal at least two mechanisms of interaction. First, bacterial infiltration is heightened during acute viral infection. Secondly, the nature of responding cell populations is dramatically altered during concomitant infections. Although natural killer cells and macrophages are predominant cell populations responding to bacterial infection in a naïve host, there is also a large T cell component that is activated upon viral infection. Inflammatory cytokines produced by these cells contribute to lethal immunopathology, and therapeutic strategies need to target the initial causative microbes as well as subsequent inflammatory responses. Current therapies directed only at the host immune response have not been overly successful, owing largely to difficulties in reversing the severe immunopathology associated with sepsis. Summary Respiratory viral infections may facilitate secondary bacterial infections and increase host immunopathology through the overproduction of inflammatory cytokines. Preventive measures, including vaccination and aggressive antimicrobial therapy early in the course of infection, may significantly reduce the morbidity and mortality of sepsis.

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.

Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping

There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000× can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels ≤12 bp long were detected by the variant caller software either exactly or as mismatched nucleotides within the indel region. The rapid turnaround time and low input DNA requirements make the multiplex PCR and semiconductor-based sequencing approach a viable option for mutation detection in a clinical laboratory.

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.

Regulation of innate and adaptive immune responses by the related cytokines IL-12, IL-23, and IL-27

Abstract.The functional characterization and subsequent purification of T cell growth factor/interleukin (IL)-2 in the early 1980s established this secreted protein as a key mediator of immune cell activation and provided the prototype that enabled the discovery of numerous cytokines over the ensuing two decades. While soluble immunoregulatory factors were initially identified functionally as biological activities present in the culture supernatants of activated lymphocytes/monocytes, this methodology shifted radically following the completion of the human genome sequence. Computer-generated structural modeling algorithms have replaced functional assays and biochemical purification as the initial means of discovering new cytokines. To date, a total of 31 interleukins, as well as over a dozen other related hematopoietic factors, have been identified. These cytokines and their receptors may be grouped on the basis of structural homologies as well as by shared ligand and receptor subunits. The challenge now at hand is to define the biological functions of the newly identified cytokines and to elucidate the common and divergent roles of related family members. This point is well illustrated by the IL-12/IL-23/IL-27 family, whose members share ligand and receptor subunits and play somewhat overlapping roles in innate and adaptive immune responses. These three cytokines are not entirely redundant, as they may preferentially activate naïve or memory T cells, induce discrete T cell cytokine profiles, contribute to distinct stages of host immune responses to infectious agents, and differentially promote autoimmunity. Further elucidation of the unique functions of the IL-12 family members may lead to improved immunodiagnostics and therapies.

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

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.