Evaluation of Barriers to Referral for Cancer Predisposition Syndromes in Pediatric Oncology Patients

Genomic medicine and the use of genetic information in pediatric oncology has provided insight on the molecular underpinnings of childhood cancers and has demonstrated that cancer predisposition syndromes (CPS) are underdiagnosed. Diagnosis of a CPS has important implications for the patient and their family. In childhood, CPS are often diagnosed by geneticists or oncologists with expertise in CPS following a malignancy, however, this requires a member of the care team, most commonly, the treating oncologist to suspect a CPS and refer them for assessment. We sought to understand current referral practices of pediatric oncology healthcare providers and barriers to referral for evaluation of a CPS. An online survey was sent to members of the Children’s Oncology Group. Of the 189 respondents, 80.4% were pediatric hematologists/oncologists and most (69%) used formal guidelines to aid in referral assessment. Guideline use was associated with a higher proportion (>5%) of patients with a CPS in the respondent’s practice. Participants were more likely to refer patients with malignancy and additional features of a CPS than for a specific type of cancer, despite the use of guidelines. Most respondents indicated they would rarely refer patients with tumors highly associated with CPS. Patient/parent knowledge of family history was considered the most challenging barrier to obtaining a family history, though a thorough three-generation pedigree was not consistently attempted. Overall, participants indicated the most significant barrier to referral was priority given the patient’s more immediate care needs. Other barriers to genetics referral identified elsewhere by primary care physicians were unlikely to be considered barriers in this population. These data suggest that pediatric hematologists/oncologists experience different barriers than other specialties and may benefit from further education about CPS, and clearer referral guidelines. Furthermore, utilization of a genetic counselor within the pediatric oncology clinic may encourage CPS assessment but allow oncologists to focus on the patient’s immediate care needs. These findings may facilitate increased referrals for CPS evaluation which improve public health by identifying patients and families who may benefit from preventative care measures

PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma.

BackgroundLeiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth.MethodsLMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts.ResultsCompounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values <1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone.ConclusionsIn summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients

PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma

Abstract Background Leiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth. Methods LMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts. Results Compounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values <1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone. Conclusions In summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients

PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma.

BackgroundLeiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth.MethodsLMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts.ResultsCompounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values &lt;1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone.ConclusionsIn summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients

MOESM1 of PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma

Additional file 1: Figure S1. Characterisation of LMS cell lines: A. Heat map of copy number variations between STS39 tumor, STS39 cell lines passages 4, 9, 14, 24, 34, SKLMS1 and HUVEC (Human umbilical vein endothelial cells) as a control, showing genomic stability of cell lines over time. The scale represents the percentage of genetic differences, where white represents minimal to no genetic change and dark purple represents maximum genetic change. For example, the genetic difference between HUVEC and STS39 is 23%. B. Immunocytochemistry of both cell lines using DAPI, Desmin, Smooth Muscle Actin (SMA) and mouse IgG (msIgG) as an isotype control. SKLMS1 demonstrates focally positive SMA staining, while STS39 shows focal positivity for Desmin and SMA (n=3). C. Immunoblot analysis showing protein stability of PI3K pathway proteins with serial passaging of STS39 and SKLMS1 cells. Increased phosphorylation of p85, a subunit of the PI3K receptor, was seen in SKLMS1 cells. In addition, elevated levels of RICTOR, a binding partner of mTOR, were observed. Both of these modifications can potentially lead to increased pathway activation. HeLa and Jurkat cell lines were used as controls for protein expression. siRNA against 4EBP1 was used to create a negative control for 4EBP1 and p-4EBP1 antibodies. Figure S2. Treatment with BEZ235 and/or Dox induces cell death via apoptosis. A. Cells were treated with BEZ235 (500nM), Dox (500nM) and BEZ/Dox for 72h and then analysed for apoptosis by flow cytometry for Annexin V and 7-ADD staining. Combination of BEZ235 and Dox significantly induced apoptosis in SKLMS1 and STS-39 cells (data not shown). B. Quantification of apoptotic cells at 72h post-treatment of SKLMS1 cells (n=3) and STS39 cells (n=3). Table S1. Sequencing primers (5′-3′) used to determine the presence of mutations in mTOR and in exon 9 and 20 of the kinase domain of PI3K. Table S2. Combination Index (CI) tables with BEZ235, BKM120 and/or Dox at 3 dosing schedules. Viability was determined using an ATPlite assay and analysed using CalcuSyn software. Treatment with BEZ235 (15-240nM) and Dox (125-2000nM) showed synergy in all 3 schedules (CI<0.9), while the combination of BKM120 and Dox was not synergistic (n=3)