Article

Identification of Genetic Alterations by Circulating Tumor DNA in Leiomyosarcoma: A Molecular Analysis of 73 Patients

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Abstract

Background Leiomyosarcoma is a malignant mesenchymal tumor of cells of smooth muscle lineage arising commonly in retroperitoneum, uterus, large veins, and the limbs. The genetics of leiomyosarcomas are complex and there is very limited understanding of common driver mutations. Circulating tumor DNA (ctDNA) offers a rapid and noninvasive method of next-generation sequencing (NGS) that could be used for diagnosis, therapy, and detection of recurrence. Methods ctDNA testing was performed using Guardant360, which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in 73 genes using NGS. Results Of 73 patients, 59 were found to have one or more cancer-associated genomic alteration. Forty-five (76%) were female with a median age of 63 (range, 38–87) years. All samples were designated metastatic. The most common alterations were detected in Tp53 (65%), BRAF (13%), CCNE (13%), EGFR (12%), PIK3CA (12%), FGFR1 (10%), RB1(10%), KIT (8%), and PDGFRA (8%). Some of the other alterations included RAF1, ERBB2, MET, PTEN TERT, APC, and NOTCH1. Potentially targetable mutations, by Food and Drug Administration–approved or clinical trials, were found in 24 (40%) of the 73 patients. Four patients (5%) were found to have incidental germline TP53 mutations. Conclusion NGS of ctDNA allows identification of genomic alterations in plasma from patients with leiomyosarcoma. Unfortunately, there is limited activity of current targeted agents in leiomyosarcomas. These results suggest opportunities to develop therapy against TP53, cell cycle, and kinase signaling pathways. Further validation and prospective evaluation is warranted to investigate the clinical utility of ctDNA for patients with leiomyosarcoma.

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... The identification of molecular alterations can help develop therapy, targeting TP53, cell cycle, and kinase signaling pathways [120] ...
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Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.
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Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas.
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TP53 is the most commonly mutated gene in cancer, with over half of all human cancers harboring a mutation in the gene. The p53 protein is a transcription factor that functions as a tumor suppressor, and a subset of its numerous roles include the arrest of proliferation, promotion of DNA repair, and induction of apoptosis in cells with severe DNA damage or stress. The vast majority of p53 mutations are single amino acid substitutions within the DNA binding domain, which either directly impede the protein's ability to bind DNA or destabilize the structure, resulting in misfolding. These missense mutant proteins are found at high levels due to loss of the MDM2 mediated regulation, and consequently serve as potential drug targets. Numerous pharmacological approaches have been investigated to restore wild type p53 function to these mutants (so-called reactivating mutant p53) with some entering in clinical trials while most have failed in early development. Recently, the field of cancer drug development has produced a number of new compounds that continue to advance this field, each with a different mechanism of action. Here we sought to review these compounds and approaches to reactivating mutant p53. Given the large number of patients with missense mutant p53 mutations, reactivating mutant p53 remains a highly sought after goal in developmental therapeutics.
Article
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In a study of 65 primary cutaneous leiomyosarcomas and 15 primary superficial subcutaneous leiomyosarcomas, tumors occurred at any age but were more common in middle age and most common on the extremities. They developed as solitary painful or tender intracutaneous or subcutaneous nodules. Microscopically, the cutaneous leiomyosarcomas consist of a poorly delineated proliferation of spindle-shaped atypical myomatous cells arranged in interlacing fascicles which merge into collageous stroma. Subcutaneous leiomyosarcomas are more sharply circumscribed and typically include a vascular pattern. About 40% of the cutaneous leiomyosarcomas recurred, but none metastasized despite a high mitotic frequency and marked cytologic atypia. Among the 12 patients with subcutaneous leiomyosarcomas, one-half of the tumors recurred and one-third eventuated in metastasis or tumor-related death. Cutaneous leiomyosarcomas have a relatively benign biologic course and may be excised conservatively, but are less likely to recur if the local excision is wide enough to require a skin graft for closure of the surgical defect. For primary subcutaneous leiomyosarcoma, early wide local excision with adequate clear histologic borders constitutes rational treatment.
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Fibroid most commonly presents in the reproductive age group and presence of fibroid with postmenopausal bleeding is a rare entity and all investigations and measures should be done to rule out leiomyosarcoma. A 45-year-old female had attained menopause 3 year back and developed postmenopausal bleeding since 2 months, with palpable mass, of 24 weeks size. Ultrasonography showed multiple whorled mass lesions, endometrium and myometrium could not be seen separately. Total abdominal hysterectomy with bilateral salpingoophorectomy was performed. Intraoperative findings showed 24 weeks uterine mass with size 17.5×15.5×11.5 cm and weight 1.9 kg with multiple, intramural fibroids. Cut section of removed specimen showed black and yellow necrotic and haemorrhagic areas with degenerative changes suggestive of malignancy. Histopathology reported epithelioid leiomyosarcoma.
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Adult patients with advanced soft tissue sarcomas (STS) are generally treated similarly, regardless of great differences between STS subtypes, disease presentation and patients' characteristics. As ifosfamide is frequently applied in first line systemic therapy, we aimed to establish prognostic and predictive factors for outcome to ifosfamide-based therapy. A retrospective, exploratory analysis was performed on data from 1337 advanced STS patients who received first-line ifosfamide-containing chemotherapy. For predictive factor analysis, 660 patients treated with doxorubicin monotherapy served as comparators. Independent favourable prognostic factors for overall survival (OS) were good performance status, female gender, low histological grade, extremity primary tumour site and locally advanced disease; for progression-free survival (PFS), the combination of doxorubicin and ifosfamide, locally advanced disease, and tumour entity with a lower risk to progress for synovial sarcoma patients compared to leiomyosarcoma. For response, independent favourable prognostic factors were doxorubicin combined with ifosfamide, higher histological grade, and histology with synovial sarcoma patients having the highest chance to respond. Predictive factor analysis showed that compared to doxorubicin monotherapy, patients who benefited less from ifosfamide-based therapies were leiomyosarcoma patients in terms of OS, and patients with liposarcoma for response. No predictive factors were found for PFS. In this study, we established an independent set of prognostic and predictive factors for outcome to ifosfamide-based chemotherapy in advanced STS patients. This study provides important information for the interpretation and design of clinical trials for specific STS entities and may contribute to further treatment individualisation of advanced STS patients.
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Forty-four cases of leiomyosarcoma arising in the retroperitoneum (33 cases) and mesentery (11 cases) were analyzed clinicopathologically, and four were examined ultrastructurally. These tumors were more common in women (61%) than in men (39%), and the median age of the patients was 59 years. Except for three tumors, most were large and over 10 cm in the greatest diameter. Twenty-seven tumors were classical leiomyosarcomas, while seven were composed predominantly of epithelioid cells and classified as malignant leiomyoblastoma (epithelioid leiomyosarcoma). In addition to the classical microscopic features, dedifferentiated areas resembling malignant fibrous histiocytoma were noted in three cases. Ultrastructurally, the tumors were characterized by smooth muscle tumor cells showing a spectrum of differentiation of varying degrees. Of the 42 patients for whom follow-up information could be obtained, 33 (79%) died within a period of five years after the initial treatment. Mitotic activity showed no significant correlation with biological behavior; ten of the 15 patients in whom the mitotic counts were less than five per 10 high-power fields (HPF), expired of the tumor.
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DNA copy number changes were investigated in 29 leiomyosarcomas by comparative genomic hybridization. The most frequent losses were detected in 10q (20 cases, 69%) and 13q (17 cases, 59%). The most frequent gains were detected in 17p (16 cases, 55%). The most frequent high-level amplifications were detected in 17p (7 cases, 24%) and 8q (6 cases, 21%). A total of 137 losses and 204 gains were detected. Small tumors (less than 5 cm in diameter) displayed fewer changes per sample (3 to 11; mean, 7) than the other tumors (4 to 22; mean, 13). There was an increase in the number of gains from small tumors (mean, 4) to very large tumors (>20 cm; mean, 10). However, the number of losses was similar in small, large, and very large tumors (mean, 4.5). Tumor size-related aberrations were observed. Gains in 16p were detected in all small tumors but were infrequent in large and very large tumors (27% and 11%, respectively). Similarly, gains and high-level amplifications in 17p were more common in small (80%) than in very large tumors (33%). Gains in 1q, 5p, 6q, and 8q were not seen in any of the small tumors but were detected in large and very large tumors. Gains in 6q and 8q occurred in 8 of 9 cases (89%) of very large tumors, 5 of them with a high-level amplification in 8q.
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Few chemotherapy agents are active in leiomyosarcoma (LMS), particularly LMS that has progressed after doxorubicin treatment. We sought to determine the response to gemcitabine plus docetaxel among patients with LMS. Patients with unresectable LMS of uterine (n = 29) or other (n = 5) primary sites who did not respond to zero to two prior chemotherapy regimens were enrolled onto a phase II study of gemcitabine 900 mg/m(2) intravenously (i.v.) on days 1 and 8 plus docetaxel 100 mg/m(2) i.v. on day 8 with granulocyte colony-stimulating factor given subcutaneously on days 9 to 15, delivered every 21 days. Patients with prior pelvic radiation received 25% lower doses of both agents. Gemcitabine was delivered over 30 or 90 minutes in cycles 1 and 2 and by 90-minute infusion in all subsequent cycles. Pharmacokinetic studies assessed in vivo differences in gemcitabine concentrations with different rates of infusion. Thirty-four patients (median age, 55 years; range, 32 to 74 years) have enrolled. Fourteen had received prior pelvic radiation. Sixteen of 34 patients had progressed after doxorubicin-based therapy; 18 had no prior chemotherapy. Among 34 patients, complete response was observed in three patients and partial response in 15, for an overall response rate of 53% (95% confidence interval, 35% to 70%). Seven patients had stable disease. Fifty percent of patients previously treated with doxorubicin responded. Hematologic toxicity was common (neutropenia: grade 3, 15%; grade 4, 6%; thrombocytopenia: grade 3, 26%; grade 4, 3%), but neutropenic fever (6%) and bleeding events (0%) were rare. The median time to progression was 5.6 months (range, 4 to 10 months). Gemcitabine plus docetaxel is tolerable and highly active in treated and untreated patients with LMS.
Liquid biopsies come of age: towards implementation of circulating tumour DNA
  • Jcm Wan
  • C Massie
  • J Garcia-Corbacho
Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17:223-238.