Technical development and validation of a clinically applicable microenvironment classifier as a biomarker of tumour hypoxia for soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS) are rare, heterogeneous tumours and biomarkers are needed to inform management. We previously derived a prognostic tumour microenvironment classifier (24-gene hypoxia signature). Here, we developed/validated an assay for clinical application. METHODS: Technical performance of targeted assays (Taqman low-density array, nanoString) was compared in 28 prospectively collected formalin-fixed, paraffin-embedded (FFPE) biopsies. The nanoString assay was biologically validated by comparing to HIF-1α/CAIX immunohistochemistry (IHC) in clinical samples. The Manchester (n = 165) and VORTEX Phase III trial (n = 203) cohorts were used for clinical validation. The primary outcome was overall survival (OS). RESULTS: Both assays demonstrated excellent reproducibility. The nanoString assay detected upregulation of the 24-gene signature under hypoxia in vitro, and 16/24 hypoxia genes were upregulated in tumours with high CAIX expression in vivo. Patients with hypoxia-high tumours had worse OS in the Manchester (HR 3.05, 95% CI 1.54-5.19, P = 0.0005) and VORTEX (HR 2.13, 95% CI 1.19-3.77, P = 0.009) cohorts. In the combined cohort, it was independently prognostic for OS (HR 2.24, 95% CI 1.42-3.53, P = 0.00096) and associated with worse local recurrence-free survival (HR 2.17, 95% CI 1.01-4.68, P = 0.04). CONCLUSIONS: This study comprehensively validates a microenvironment classifier befitting FFPE STS biopsies. Future uses include: (1) selecting high-risk patients for perioperative chemotherapy; and (2) biomarker-driven trials of hypoxia-targeted therapies.

The hypoxia marker CAIX is prognostic in the UK phase III VorteX-Biobank cohort: an important resource for translational research in soft tissue sarcoma.

BACKGROUND: Despite high metastasis rates, adjuvant/neoadjuvant systemic therapy for localised soft tissue sarcoma (STS) is not used routinely. Progress requires tailoring therapy to features of tumour biology, which need exploration in well-documented cohorts. Hypoxia has been linked to metastasis in STS and is targetable. This study evaluated hypoxia prognostic markers in the phase III adjuvant radiotherapy VorteX trial. METHODS: Formalin-fixed paraffin-embedded tumour biopsies, fresh tumour/normal tissue and blood were collected before radiotherapy. Immunohistochemistry for HIF-1α, CAIX and GLUT1 was performed on tissue microarrays and assessed by two scorers (one pathologist). Prognostic analysis of disease-free survival (DFS) used Kaplan-Meier and Cox regression. RESULTS: Biobank and outcome data were available for 203 out of 216 randomised patients. High CAIX expression was associated with worse DFS (hazard ratio 2.28, 95% confidence interval: 1.44-3.59, P<0.001). Hypoxia-inducible factor-1α and GLUT1 were not prognostic. Carbonic anhydrase IX remained prognostic in multivariable analysis. CONCLUSIONS: The VorteX-Biobank contains tissue with linked outcome data and is an important resource for research. This study confirms hypoxia is linked to poor prognosis in STS and suggests that CAIX may be the best known marker. However, overlap between single marker positivity was poor and future work will develop an STS hypoxia gene signature to account for tumour heterogeneity.

Translation and preliminary validation of the English version of the DUX questionnaire for lower extremity bone tumor patients (Bt-DUX): a disease-specific measure for quality of life.

PURPOSE: To translate the Dutch DUX questionnaire for lower extremity bone tumor patients (Bt-DUX), a disease-specific quality of life (QoL) instrument, into the English (UK) language and preliminary validate the English version in patients who were treated for lower-extremity bone tumors. METHODS: Adaptation and translation process included forward translation, back-translation, and a review of the back-translation by an expert committee. Internal consistency and validity of the translated questionnaire were examined in a sample of adolescents treated for lower extremity osteosarcoma in the United Kingdom. Assessments included the Bt-DUX, the Toronto Extremity Salvage Score (TESS), the Short Form (SF)-36, and the TNO-AZL Questionnaire for Adult's Quality of Life (TAAQOL). RESULTS: Seventeen patients (7 ♂ and 10 ♀), median age 19.9 (range: 16-25) years completed the questionnaires. Mean Bt-DUX score was 38.8 (range: 23-78), with Cronbach's α being 0.95 domain-total correlations ranged between 0.84 and 0.93 (P < 0.01). Spearman's correlation coefficients between the Bt-DUX total and domain scores and corresponding TAAQOL and SF-36 scores were overall moderate to good and reaching statistical significance in a most cases. CONCLUSION: Preliminary evidence suggests that the English Bt-DUX translation is a valid disease-specific instrument for evaluating QoL of adolescents with lower extremity bone cancer.

Patient experience after lower extremity amputation for sarcoma in England: a national survey.

PURPOSE: After amputation, rehabilitation and limb fitting services are critically important to optimise outcomes. We investigated the reported patient experience and variation in limb fitting services after amputation for musculoskeletal tumours in England. METHODS: A postal survey instrument was developed following literature review, patient and clinician consultation and piloting. The survey was sent from each of the five bone tumour surgical centres in England. RESULTS: One hundred and five responses were received from 250 patients (42%). The number of limb fitting centres accessed by each surgical centre varied from 2 to 28. Many patients reported care falling short of national standards in areas including pre-amputation counselling, information provision, meeting someone with a similar amputation before surgery, psychological support and falls management. Patients were seen sooner where limb fitting services were on site. Many patients rely on being driven, ambulance and public transport to access services. CONCLUSION: This study demonstrates variation in the reported experience of limb fitting services by sarcoma patients. Areas for improvement include information provision, pre-amputation counselling, psychological support and falls management. Clinicians should be aware services are highly variable, and this may impact on outcomes. Patients treated in sarcoma centres with limb fitting services on site may experience better care. Implications for Rehabilitation Rehabilitation services should strive to meet agreed national standards consistently. Where preamputation counselling involving meeting someone with a similar amputation is not possible, good information including video could be helpful. Services should support rehabilitation in the form of early walking aids and efficient prosthetic repair and maintenance. Psychological support, occupational therapy and physiotherapy support must be provided in the acute and chronic phases, including access to long-term rehabilitation care. Rehabilitation programmes must include training to patients and families on reporting, prevention and management of falls. On site services may support better care. Mechanisms for delivering expert specialist care close to home are needed.

Detection of ASPL/TFE3 fusion transcripts and the TFE3 antigen in formalin-fixed, paraffin-embedded tissue in a series of 18 cases of alveolar soft part sarcoma: useful diagnostic tools in cases with unusual histological features.

Alveolar soft part sarcoma (ASPS) is a rare malignancy; diagnostic problems may occur when cases present as a metastasis or with unusual morphologic features. In this study, a series of 18 cases with follow-up information were analysed with regard to the ASPL/TFE3 fusion transcripts and immuno-detection of TFE3 using archival formalin-fixed, paraffin-embedded tissues. Novel primers to detect ASPL/TFE3 fusion transcripts, type 1 and 2, were designed. The patients, ten female and eight male, ranged in age from 3 to 46 years; 16 involved soft tissues of the extremities (nine, lower; seven, upper), one involved the uterine cervix and one was a primary bone tumour of the foot. Seven ASPS had unusual morphologic features lacking the typical alveolar pattern. Seven had lung metastases at the time of diagnosis, and three developed lung and brain metastases later. Four patients died of disease (after 1-5 years); four are alive with metastases (after 2-15 years), and ten are alive and well (after 1-10 years). Vascular invasion correlated with metastatic disease. All 18 ASPS, four granular cell tumours (one of which was malignant) and one adrenal cortical carcinoma showed TFE3 immuno-positivity. The 18/18 ASPS showed ASPL/TFE3 fusion transcripts (nine, type 1; nine, type 2), four of which had a balanced translocation. ASPL/TFE3 fusion transcripts were not detected in 25 controls. We conclude that immuno-detection of TFE3 and RT-PCR-based identification of ASPL/TFE3 fusion transcripts in formalin-fixed, paraffin-embedded tissues are powerful tools in the diagnosis of ASPS, particularly in cases with unusual morphologic features.

Extensible endoprostheses for bone tumors of the proximal femur in children.

Between 1983 and 1996, nine children were treated with extensible endoprosthetic replacements of the proximal femur after resection of primary bone tumors. Four patients died of their disease. The remaining five patients were observed for an average follow-up period of 7.6 years. In these five patients, the authors performed an average of 10.2 operations per patient, including five lengthenings and a mean total extension of 69.7 mm per patient. Acetabular loosening and hip dislocations were the most frequent complications. Only two patients have not had a revision or a major complication. Despite this, four children are alive with a functioning lower limb and a mean Musculoskeletal Tumor Society functional score of 77.6%. Extensible endoprosthesis of the proximal femur is a viable alternative to hip disarticulation and in selected children offers an opportunity for near-normal development of the lower limb by allowing equalization of limb length and the ability to walk without the use of mobility aids.