Samuel Singer

Terminal Differentiation of Human Breast Cancer through PPARγ

We have previously demonstrated that PPAR gamma stimulates the terminal differentiation of adipocyte precursors when activated by synthetic ligands, such as the antidiabetic thiazolidinedione (TZD) drugs. We show here that PPAR gamma is expressed at significant levels in human primary and metastatic breast adenocarcinomas. Ligand activation of this receptor in cultured breast cancer cells causes extensive lipid accumulation, changes in breast epithelial gene expression associated with a more differentiated, less malignant state, and a reduction in growth rate and clonogenic capacity of the cells. Inhibition of MAP kinase, shown previously to be a powerful negative regulator of PPAR gamma, improves the TZD ligand sensitivity of nonresponsive cells. These data suggest that the PPAR gamma transcriptional pathway can induce terminal differentiation of malignant breast epithelial cells and thus may provide a novel, nontoxic therapy for human breast cancer.

KIT Extracellular and Kinase Domain Mutations in Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms arising in the gastrointestinal tract. GISTs express the KIT receptor tyrosine kinase, and many cases have activating mutations in the KIT juxtamembrane region. We now report an analysis of KIT cDNA and genomic sequences in eight GISTs that lack juxtamembrane region mutations. Six cases contained heterozygous exon 9 mutations in which six nucleotides, encoding Ala-Tyr, were duplicated. The other two cases contained homozygous exon 13 missense mutations, resulting in substitution of Glu for Lys(642), that were associated with constitutive KIT tyrosine phosphorylation. Sequence analysis of DNAs from nonneoplastic companion tissues revealed that both the exon 9 and exon 13 mutations were somatic. These are the first descriptions, in any tumor, of mutations in KIT exons encoding the C-terminal end of the extracellular domain and the first part of the split kinase domain. These findings indicate that KIT may be activated by mutations in at least three domains-extracellular, juxtamembrane, and kinase-in GISTs.

Prognostic Value of KIT Mutation Type, Mitotic Activity, and Histologic Subtype in Gastrointestinal Stromal Tumors

PURPOSE Previous studies have reported clinical correlates for KIT mutations in GISTs, but in most of those studies the KIT mutations were found in less than 50% of the GISTs. The aim of this study was to evaluate the prognostic relevance for KIT mutations in a series of GISTs in which the mutations were evaluated intensively by genomic and cDNA sequencing. PATIENTS AND METHODS A comprehensive clinical and pathologic analysis of 48 patients with GISTs who had snap-frozen tissue was performed. The median tumor size was 10 cm (range, 2 to 30 cm). Median follow-up for disease-free patients was 48 months. KIT genomic and cDNA was sequenced by using nucleic acid templates isolated from frozen tumors. RESULTS The overall 5-year recurrence-free survival was 41% +/- 6%. Five-year recurrence-free survival for patients with tumors that had mitotic counts of three mitoses or fewer per 30 high-power fields (HPF), more than three to <or= 15 mitoses per 30 HPF, and more than 15 mitoses per 30 HPF were 89% +/- 7%, 49% +/- 12%, and 16% +/- 6%, respectively (P =.0001). The 32 patients with spindle-cell histology had a 49% +/- 7% 5-year recurrence-free survival; in contrast, the 16 patients with epithelioid or mixed histology had a 23% +/- 11% 5-year recurrence-free survival (P =.01). Five-year recurrence-free survival for patients with tumors less than 5 cm, 5 to 10 cm, and more than 10 cm were 82% +/- 12%, 45% +/- 9%, and 27% +/- 8%, respectively (P =.03). Prognostic associations were found with particular KIT mutation types, and patients with missense exon 11 mutations had a 5-year recurrence-free survival of 89% +/- 11% compared with 40% +/- 8% for GISTs with other mutation types (P =.03). The independent predictors for disease-free survival were the presence of deletion/insertion exon 11 mutations (hazard ratio [HR] = 4; P =.006), more than 15 mitoses per 30 HPF (HR = 18; P =.0001), mixed histology (HR = 21; P =.0001), and male sex (HR = 3; P =.05). CONCLUSION In this series of KIT-expressing GISTs, tumor mitotic activity and histologic subtype were the most important prognostic features. The majority of GISTs contain KIT-activating mutations with the type/location of mutation serving as an independent predictor for disease-free survival. These results suggest that KIT mutation and activation are important in GIST pathogenesis and also may provide important prognostic information.

PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft-tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1 associated) or after radiotherapy. Using comprehensive genomic approaches, we identified loss-of-function somatic alterations of the Polycomb repressive complex 2 (PRC2) components (EED or SUZ12) in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the lost PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 levels and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), both of which significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2 components, NF1 and CDKN2A highlights their critical and potentially cooperative roles in MPNST pathogenesis.

Management of soft-tissue sarcomas: an overview and update.

Soft-tissue sarcomas (STS) are relatively uncommon, especially when considered as individual histological subtypes (of which there are more than 50). Their incidence increases with age, although they are disproportionately common among children. When diagnosed and managed in a non-specialist environment, outcome is generally significantly poorer than if patients are managed by a multidisciplinary team in a tertiary centre of excellence. Prompt referral of patients with clinically suspicious masses is strongly advocated, before any type of intervention is attempted. This brief, opinion-based overview emphasises the team approach and provides a synopsis of the strategies used at our institution for pre-operative assessment and biopsy, surgical management, and the delivery of radiation therapy when appropriate (focusing on limb preservation and optimisation of function). Predictable variations in the natural history of these tumours, based on accurate histological subclassification, merit wider recognition. The role of systemic chemotherapy for soft-tissue sarcoma is still evolving, but at present the main aims are improved local control, disease-free survival, and quality of life. There are overall survival benefits for specific histological types, but this is a relatively small subgroup. Novel therapies, based on disease mechanisms at the molecular level, show promise for future advances.

Response to chemotherapy and predictors of survival in adult rhabdomyosarcoma.

ObjectiveTo assess outcome and identify predictors of survival of adults with rhabdomyosarcoma. Summary Background DataThe literature on adult rhabdomyosarcoma is limited. Few studies have identified predictors of long-term survival in this patient population. MethodsThirty-nine adults with rhabdomyosarcoma were treated between 1973 and 1996 and prospectively followed. Outcomes were assessed with respect to patient and tumor characteristics, local treatment, and response to chemotherapy. ResultsTwenty-six patients had localized/locoregional disease and 13 patients had metastatic disease at presentation. Twenty-one patients underwent attempted curative resection, 27 received radiotherapy, and 37 received chemotherapy. Median follow-up for surviving patients was 152 months. The overall 5- and 10-year survival rates were 31% and 27%, respectively. Five-year survival rates for patients with tumors less than 5 cm, 5 to 10 cm, and more than 10 cm were 60%, 14%, and 0%, respectively. Patients with localized/locoregional disease at presentation had a 44% 5-year survival rate; there were no 5-year survivors among patients with metastatic disease. Patients who had a complete response to chemotherapy had a 5-year survival rate of 57%, compared with a rate of only 7% for poor responders. Metastatic disease at presentation and poor response to chemotherapy were independent predictors of death on multivariate analysis. ConclusionsAge, location, nodal status, and histologic subtype do not appear be associated with survival in adults with rhabdomyosarcoma treated with multimodal therapy. Metastatic disease at presentation and poor response to chemotherapy are strongly associated with poor prognosis. Future systemic therapies should be targeted to patients with localized/locoregional disease and partial responders to conventional chemotherapy.

Adults with Ewing's sarcoma/primitive neuroectodermal tumor: adverse effect of older age and primary extraosseous disease on outcome.

OBJECTIVE To assess outcome and prognostic factors for survival of adults with Ewings sarcoma/primitive neuroectodermal tumor (PNET). BACKGROUND Ewings sarcoma/PNET is a disease of childhood rarely seen in adults. Accordingly, there is a relative paucity of published literature pertaining to outcome for adults with this disease. METHODS Between 1979 and 1996, 37 patients with newly diagnosed Ewings sarcoma/PNET were evaluated and treated at the Adult Sarcoma Program at Dana-Farber Cancer Institute and Brigham & Womens Hospital. Twenty-six patients had localized disease at presentation and 11 had metastatic disease. All but two patients received multiagent chemotherapy. Local treatment consisted of surgery (7 patients), surgery and radiation therapy (19), radiation therapy (6), or no local treatment (5). Median follow-up for living patients was 100 months (range 8 to 199). RESULTS The 5-year survival rate for the group overall was 37%+/-9%. The 5-year local control rate was 85%+/-7%. Significant favorable predictors for survival on univariate analysis included localized disease at presentation, primary origin in bone, primary size <8 cm, and a favorable objective response to chemotherapy. Patients with localized disease had a 5-year survival rate of 49%+/-11% compared with 0% for those with metastatic disease at presentation. Multivariate analysis showed three significant independent predictors for death: metastatic disease at presentation, primary origin in extraosseous tissue versus bone, and age 26 years or older. CONCLUSION Adult patients with Ewings sarcoma/PNET at highest risk for death are those who are older than 26 years and have metastatic disease or an extraosseous primary tumor. The development of novel therapies should target these high-risk groups.

Classification of human liposarcoma and lipoma using ex vivo proton NMR spectroscopy

Prognostication in patients with liposarcoma is a complex and controversial subject based on recognition of lipoblasts, adipocyte nuclear atypia, and qualitative estimations of cellularity and cell size. We show here that for 30 patients with liposarcoma and 5 patients with lipoma, spectral differences on high‐resolution, magic angle spinning proton nuclear magnetic resonance (hr‐MAS 1H‐NMR) spectroscopy relate to known biochemical changes and correlate with adipocyte tissue differentiation, histologic cell type, and cellularity. The NMR‐visible level of triglyceride is shown to correlate with liposarcoma differentiation, since the triglyceride level in well‐differentiated liposarcoma is 33‐fold higher on average than for myxoid/round cell liposarcoma, which in turn is 6‐fold higher than the dedifferentiated and/or pleomorphic subtypes. The NMR‐visible phosphatidylcholine level serves as an estimate of total tissue cell membrane phospholipid mass and was found to correlate with liposarcoma subtype. Pleomorphic liposarcoma, the most aggressive and metastatic subtype, was found to have a threefold increase in NMR‐visible phosphatidylcholine level compared with dedifferentiated liposarcoma. The level of NMR‐visible phosphatidylcholine was twofold greater in well‐differentiated liposarcoma compared with lipoma and was threefold larger for the hypercellular myxoid/round cell subtype compared with the pure myxoid histology. Thus, NMR‐derived parameters of tissue lipid may be used for objective distinction of liposarcoma histologic subtype/grade and lipoma from liposarcoma. These biochemical parameters may ultimately improve prognostication in patients with liposarcoma. Magn Reson Med 41:257–267, 1999.

Spindle cell rhabdomyosarcoma (so-called) in adults: Report of two cases with emphasis on differential diagnosis

Spindle cell rhabdomyosarcoma (RMS) is a recently described variant of embryonal RMS that carries a relatively favorable prognosis when compared with other forms of RMS. To date, spindle cell RMS has been described only in children. The authors have identified two unusual cases occurring in adults using the following criteria: tumors composed mainly of fascicular, relatively monomorphic spindle-shaped cells that show unequivocal immunohistochemical and ultrastructural evidence of myogenic differentiation. The tumors were identified in a 38-year-old woman and a 56-year-old man, arising in the cheek and left hemidiaphragm, respectively. Both were treated with surgical resection and chemotherapy. The first patient died of uncontrolled local recurrence of her tumor at 27 months after diagnosis, and the second died of metastatic disease at 13 months follow-up. The tumors were composed mainly of fascicles of spindle cells with palely eosinophilic cytoplasm admixed diffusely with sparse polygonal, rounded, or strap-shaped rhabdomyoblasts with brightly eosinophilic cytoplasm and with cross-striations in the first case only. Immunostaining for muscle-related antigens showed staining for smooth-muscle actin (focal), pan-actin HHF-35, desmin, fast myosin, myoglobin, and MyoD1. Both cases were negative for S-100 protein. On electron microscopy, both cases showed neoplastic rhabdomyoblasts with clear-cut sarcomeric differentiation in many of the tumor cells. Spindle cell RMS poses special problems in differential diagnosis when arising in adults and should be distinguished from leiomyosarcoma, malignant peripheral nerve sheath tumor with heterologous rhabdomyoblastic differentiation (malignant Triton tumor), and fibrosarcoma. In view of the good prognosis afforded children with spindle cell RMS and in light of the chemoresponsive behavior of RMS in general, we feel that it is important to identify tumors that meet the criteria for spindle cell RMS occurring in the adult population. However, based on these two cases, it is possible that spindle cell RMS occurring in adults may not be associated with such a favorable outcome.

The cyclin-dependent kinase inhibitor flavopiridol potentiates doxorubicin efficacy in advanced sarcomas: preclinical investigations and results of a phase I dose-escalation clinical trial

Purpose: Dysregulated cyclin-dependent kinases are important to the growth of some sarcomas. Flavopiridol is a pan-CDK inhibitor that has been shown to potentiate chemotherapy. As such, we explored the potentiation of doxorubicin by flavopiridol in sarcoma, in vitro and in vivo, and conducted a phase I trial of flavopiridol with doxorubicin in patients with advanced sarcomas. Experimental Design: Sarcoma cell lines and xenografts were treated with flavopiridol alone and in combination with doxorubicin. In the phase I study, doxorubicin and flavopiridol were administered on two flavopiridol schedules; a 1-hour bolus and split dosing as a 30-minute bolus followed by a 4-hour infusion. Results: Preclinically, flavopiridol potentiated doxorubicin. In vivo, doxorubicin administered 1 hour before flavopiridol was more active than doxorubicin alone. Clinically, 31 patients were enrolled on protocol and flavopiridol was escalated to target dose in two schedules (90 mg/m2 bolus; 50 mg/m2 bolus + 40 mg/m2 infusion) both in combination with doxorubicin (60 mg/m2). Dose-limiting toxicities were neutropenia, leukopenia, and febrile neutropenia but no maximum tolerated dose was defined. Flavopiridol pharmacokinetics showed increasing Cmax with increasing dose. Response Evaluation Criteria in Solid Tumors (RECIST) responses included two partial responses, however, stable disease was seen in 16 patients. Of 12 evaluable patients with progressive well- and dedifferentiated liposarcoma, eight had stable disease greater than 12 weeks. Conclusions: The sequential combination of doxorubicin followed by flavopiridol is well tolerated on both schedules. Disease control was observed in well- and dedifferentiated liposarcoma specifically, a disease in which CDK4 is known to be amplified. Clin Cancer Res; 18(9); 2638–47. ©2012 AACR.