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Dermatofibrosarcoma protuberans (DFSP) is a translocation-associated, low-grade sarcoma with fibroblastic differentiation. It is the most common superficial sarcoma, almost exclusively arising within the dermis. In a minority of cases, there is a transition from the conventional morphology to a fibrosarcomatous pattern, known as a fibrosarcomatous DFSP (FS-DFSP). Although a number of different molecular alterations have been described to account for this transformation, it remains poorly understood. Herein we report the first case of a FS-DFSP with a fusion between ERG, an ETS family transcription factor, and MAP3K7CL, a kinase gene rarely observed in fusion gene events.

Next generation sequencing (NGS) has introduced new types of data, such as variant allele frequencies (VAFs), into the workup of acute myeloid leukemias (AML). There is interest in using NGS to prognosticate disease behavior and monitor residual disease, but the attribution of sequencing data entirely to the leukemic clone may be confounded by VAF contribution from background non-leukemic populations and undetected copy number aberrations. Sixty-eight patients with AML were evaluated by a clinically validated gene sequencing panel at our institution from 2015 to 2018. No correlation was found with a direct comparison of blast counts and VAFs in both primary- and secondary-AML (

Malignant melanoma (MM) may display highly variable phenotypic diversity, sometimes associated with loss of immunohistochemical melanocytic markers and acquisition of nonmelanocytic lineage of differentiation. Primary cutaneous MM with rhabdomyosarcomatous differentiation is extremely rare with only 5 reported cases in the literature. To date, a chronological progression of a MM to rhabdomyosarcoma has not been conclusively documented. A 96-year-old man underwent a re-excision of an "atypical fibroxanthoma" of the forearm, which revealed a small lentigo maligna melanoma associated with a dominant dermal high-grade spindle cell nodule admixed with a population of malignant polygonal epithelioid cells. On immunohistochemical studies, the spindle cells were completely negative for all melanocytic markers, whereas a small population of polygonal neoplastic cells at the periphery was positive for Desmin and Myo-D1, supporting early rhabdomyosarcomatous transformation. Several subsequent re-excisions demonstrated merely nodules of malignant pleomorphic epithelioid cells with rhabdomyosarcomatous differentiation and devoid of melanocytic markers. In addition, both rhabdomyosarcomatous component and original MM displayed identical mutations. Therefore, the histologic, immunohistochemical, and molecular findings documented for the first time a chronological progression from an invasive MM to a pleomorphic rhabdomyosarcoma through an intermediate stage of undifferentiated sarcoma/atypical fibroxanthoma. Interestingly, subsequent recurrences of pure rhabdomyosarcomatous component displayed skip lesions/microsatellitosis, marked tumor-infiltrative lymphocytes, and rare junctional nests of rhabdomyosarcomatous cells in the epidermis, histologic features that were not described in primary cutaneous rhabdomyosarcoma and therefore could serve as morphologic clues to the diagnosis of rhabdomyosarcomatous transformation in an MM.

Xanthomas are histiocytic lesions of the skin, soft tissue, and bone and are generally considered to be reactive in nature. When they arise in the bones of the jaw, they are referred to as central xanthomas. New evidence supports the hypothesis that central xanthomas are a separate and distinct entity from their extragnathic counterparts. Noonan syndrome (NS) is an autosomal dominant disorder that has been associated with giant cell lesions, which also commonly occur in the jaw. We present a case of a 15-year-old boy with NS who presented with a radiolucent lesion of the mandible that on excision was found to be a central xanthoma. Although giant cell lesions have been well described in NS, xanthomas of the jaw have not been reported. We will also discuss the entities that must be excluded before making a diagnosis of central xanthoma, as this can affect both treatment and follow-up.

Pulmonary adenofibroma (PAF) is a rare neoplasm that may be related to solitary fibrous tumor (SFT). A subset of PAFs harbor the NAB2-STAT6 fusion that is typical of SFT, but a significant proportion do not. Their distinction is clinically important as SFTs can potentially have an aggressive clinical course, while there has been no report of a PAF behaving in a malignant fashion. We report a case of a 60-year-old male who developed a SFT and PAF in the same lung. The SFT harbored a NAB2-STAT6 fusion, while the PAF did not have any identifiable fusion. This case represents the first instance of a single patient with both of these tumors occurring simultaneously in the same lung.

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are important metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations are associated with the development of multiple malignancies. In this study, we examine the prevalence and features of non-small cell lung cancers (NSCLC) with IDH1/2 mutations. From May 2013 to March 2017, 800 lung cancer samples were successfully sequenced for somatic mutations on the Ion Torrent PGM with the 50-gene AmpliSeq Cancer Hotspot Panel v2 on the Ion Torrent PGM (318 chip). Variants were identified using the Ion Torrent Variant Caller Plugin and reference genome hg19. Golden Helix's SVS software was used for variant annotation and prediction of significance. Nine samples (1.1%) from 8 patients harbored an IDH1 (3 p.R132C and 2 p.R132L) or IDH2 mutation (p.R140W, p.R172S, and p.R172T). All patients' tumors had adenocarcinoma histology, and all patients had a smoking history. Eighty-eight percent of patients' tumors had a coexisting KRAS mutation and 6 of 8 were diagnosed at greater than 70 years of age. Five patients presented with stage IV disease and 3 with stage I. After comparison to a cohort of KRAS-mutated NSCLC with a history of smoking, IDH-mutated cases were significantly older but presented with similar rates of advanced-stage disease and sex distribution. Additional studies are needed to understand the role of IDH1/2 mutations in the development of NSCLC, but such patients who have poor prognostic indicators (KRAS mutation and advanced stage at presentation) may benefit from IDH1/2-directed therapies.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%), non–small cell lung carcinoma (NSCLC) (36%), and glioma/glioblastoma (28%) at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA.

Presently, the ability to study disease at the most fundamental molecular level has led to a reclassification of human cancers into numerous subtypes that vary in disease progression and response to therapy. Similar to most solid tumors, breast cancer is a heterogeneous disease with considerable variation in histologic and biological features. Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the estrogen receptor and progesterone receptor are not expressed, and human epidermal growth factor-receptor 2 is not amplified or overexpressed. Data derived from highly complex molecular technologies, such as microarrays and next-generation sequencing, have identified gene expression and somatic mutation profile subsets of TNBC that reflect biological behavior more accurately and may lead to further effective therapeutic targets, better prognosis, and improved outcomes. Herein, we review the genomic findings of TNBC and discuss current efforts in precision medicine as they relate to TNBC.

Matching of actionable tumor mutations with targeted therapy increases response rates and prolongs survival in lung cancer patients. Drug development and trials targeting genetic alterations are expanding rapidly. We describe the role of a Molecular Tumor Board (MTB) in the design of molecularly informed treatment strategies in our lung cancer patient population. Tumor DNA was sequenced using a 50-gene targeted next-generation sequencing panel. Cases were evaluated by a multidisciplinary MTB who suggested a course of treatment based on each patient’s molecular findings. During a three-year period, 21 lung cancer patients were presented at the MTB. All patients lacked common activating EGFR mutations and ALK rearrangements. One patient had Stage IIIb disease; all others were Stage IV; 18 patients had received ≥1 prior line of therapy (range 0–5). Suggestions for treatment with a targeted therapy were made for 19/21 (90.5%) patients, and four patients (21%) underwent treatment with a targeted agent, two as part of a clinical trial. Identified barriers to treatment with targeted therapy included: ineligibility for clinical trials (n ​= ​2), lack of interest in study/distance to travel (n ​= ​2), lack of disease progression (n ​= ​2), poor performance status (n ​= ​5), decision to treat next with immunotherapy (n ​= ​3), and unknown (n ​= ​1). For the majority of lung cancer patients, the MTB provided recommendations based on tumor genetic profiles. Identified barriers to treatment suggest that presentation to the MTB at earlier stages of disease may increase the number of patients eligible for treatment with a genetically informed targeted agent.

Objectives: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. Methods: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing. Results: Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2 , BCR-ABL , and CALR mutations. Conclusions: Screening for CALR mutations is essential in BCR-ABL –negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.

Detection of somatic mutations in non–small cell lung cancers (NSCLCs), especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM) and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips). We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate). In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7) variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%), EGFR (11.1%), BRAF (4.8%), and PIK3CA (4.3%). In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

Accurate identification of somatic mutations in formalin-fixed, paraffin-embedded tumor tissue is required for enrollment into clinical trials for many novel targeted therapeutics, including trials requiring EGFR mutation status in non–small-cell lung carcinomas. Central clinical trial laboratories contracted to perform this analysis typically rely on US Food and Drug Administration–approved targeted assays to identify these mutations. We present two cases in which central laboratories inaccurately reported EGFR mutation status because of improper identification and isolation of tumor material and failure to accurately report assay limitations, resulting in enrollment denial. Such cases highlight the need for increased awareness by clinical trials of the limitation of these US Food and Drug Administration–approved assays and the necessity for a mechanism to reevaluate discordant results by alternative laboratory-developed procedures, including clinical next-generation sequencing.

Advanced stage malignant melanoma often responds poorly to therapy with low survival rates. New therapeutic approaches are based upon a growing understanding of the underlying molecular abnormalities. We demonstrate the feasibility of a next generation sequencing (NGS) assay, which targets hotspots in 50 cancer genes, to assess genotypes that may influence therapeutic selection and response. DNA was extracted from formalin fixed paraffin embedded (FFPE) melanoma specimens to create multiplexed libraries which were sequenced. Of the 121 cases, BRAF mutations were present in 48 cases (40%) and NRAS mutations in 24 cases (20%). We identified other gene variants in 20 BRAF-mutated cases. Additional gene variants were also identified in the 57 BRAF wild-type cases. Four patients harbored different gene mutations at metastatic sites as compared to their primary lesions or metastasis from different sites. Concurrent gene variants may provide additional targets for future therapies and may suggest alternative mechanisms of secondary resistance.

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.

Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non–muscle-invasive urothelial carcinoma of the bladder (NMIUC) population. Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut−, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3–4 event. Six-month CR rate was 8% (0% in IHC+ Mut−; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94–5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment. Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003–11. ©2016 AACR.

Background Metastatic breast cancer is a genetically heterogeneous disease and effective therapies for advanced stage disease are limited. Methods In this study, distant metastases of 22 formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were sequenced using the Ion Torrent PGM and the 50 gene AmpliSeq Cancer Hotspot Panel v2 from 10 ng of extracted DNA using 318 chips. Data analysis was performed with the Ion Torrent Variant Caller Plugin (hg19) and Golden Helix's SVS software for annotation and prediction of the significance of the variants. Results All patients were female with a median age of 61 years (range 37–85 years). Metastatic sites included liver (n = 6, 27%), skin (n = 5, 23%), brain (n = 4, 18%), lymph node (n = 3, 14%), lung (n = 2, 9%), retroperitoneum (n = 1, 4.5%), and colon (n = 1, 4.5%). Overall, 28 variants in 11 genes were observed. Five (23%) samples showed no alterations and 17 (77%) showed at least one potentially biologically significant variant (BSV) defined as having FDA-approved drugs or clinical trials evaluating their significance. BSVs included mutations in the following genes: TP53 (n = 8), APC (n = 4), PIK3CA (n = 5), MET (n = 2), ERBB2 (n = 2), AKT1 (n = 1), CDKN2A (n = 1), KRAS (n = 1), and FGFR3 (n = 1). Conclusions Potentially actionable mutations were identified in the majority of breast cancer metastases. Evaluating metastatic breast tumors using a NGS approach provides a better understanding of the mechanisms behind tumor progression and evolution and also identifies additional potentially beneficial therapeutic targets for patient management or eligibility for clinical trials.

Chlamydia causes enteritis in young pigs and calves, and granulomas within intestinal lymphatics. Recently we called attention to the lymphangitis and lymphatic obstruction that occurs in Crohn’s disease. We searched resected tissues from patients for evidence of chlamydia and sera for evidence of previous exposure to chlamydia. Immunohistochemistry and real-time PCR were employed to seek chlamydia in preserved tissues. In the IHC, antibody to C. trachomatis served as primary antibody; in the PCR, primers specific for Chlamydiaceae were employed. Commercial ELISA kits measured anti-chlamydia IgG and IgA against C. trachomatis antigen in sera derived from a population of patients different from that which yielded the tissue specimens. IHC revealed focal positive staining for chlamydia in tissues of 5 of 19 patients. Positive reacting cells occurred within dense inflammation, in sparsely scattered macrophages in the submucosa and subserosa. Tissues from 3 of 22 control subjects were positive. Real-time PCR done on ileal, colonic, and regional lymph node tissues revealed evidence of chlamydia in 3 of 33 patients. Serology for anti-chlamydia IgG revealed 2 positive values in 24 patients, while serology for anti-chlamydia IgA revealed 4 positives among the 24 patients, and 1 positive in the 15 controls. One patient and one control had both elevated IgG and IgA titers. The 4 patients with elevated IgA titers were from a single family of 6 with Crohn’s disease, which had been previously described. Additional consideration needs to be given to the chlamydia species, including those of animal origin, which leave behind little evidence of their previous involvement.

KRAS mutant non-small cell lung cancers (NSCLCs) vary in clinical outcome depending on which specific KRAS mutation is present. Shorter progression free survival has been associated with KRAS variants G12C and G12V. Cell lines with these variants depend to a greater extent on the RAS/RAF/MEK/ERK signaling pathway and become more susceptible to MEK inhibition. Because different KRAS mutations may lead to altered drug sensitivity, we aimed to determine specific KRAS mutation status in a NSCLC patient cohort at our institution. A total of 502 NSCLC samples were screened for somatic mutations using the 50 gene AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2). However only samples positive for variants in the KRAS gene were included in this study. Variants identified in the KRAS genes were curated using publicly available databases. The overall mutation rate in the KRAS gene was 32.7% (164/502). The most common KRAS mutations were G12C (41%), G12V (19%), and G12D (14%) along with less frequent variants. After re-mining our sequencing data, we found that more than a half of our KRAS mutant NSCLC patients could potentially benefit from the addition of a MEK inhibitor such as selumetinib to standard chemotherapeutic agents. Due to mutated KRAS, these patients will likely fail traditional anti-EGFR therapies but be eligible for newer combination therapies.

N-of-1 trials target actionable mutations, yet such approaches do not test genomically-informed therapies in patient tumor models prior to patient treatment. To address this, we developed patient-derived xenograft (PDX) models from fine needle aspiration (FNA) biopsies (FNA-PDX) obtained from primary pancreatic ductal adenocarcinoma (PDAC) at the time of diagnosis. Here, we characterize PDX models established from one primary and two metastatic sites of one patient. We identified an activating KRAS G12R mutation among other mutations in these models. In explant cells derived from these PDX tumor models with a KRAS G12R mutation, treatment with inhibitors of CDKs (including CDK9) reduced phosphorylation of a marker of CDK9 activity (phospho-RNAPII CTD Ser2/5) and reduced viability/growth of explant cells derived from PDAC PDX models. Similarly, a CDK inhibitor reduced phospho-RNAPII CTD Ser2/5, increased apoptosis, and inhibited tumor growth in FNA-PDX and patient-matched metastatic-PDX models. In summary, PDX models can be constructed from FNA biopsies of PDAC which in turn can enable genomic characterization and identification of potential therapies.

e23168Background: Genetic profiling of tumors is quickly increasing as are trials targeting genetic alterations. Furthermore, genetic alterations are a relatively permanent part of a tumor profile,...