Identification of usual interstitial pneumonia pattern using RNA-Seq and machine learning: challenges and solutions.

BACKGROUND: We developed a classifier using RNA sequencing data that identifies the usual interstitial pneumonia (UIP) pattern for the diagnosis of idiopathic pulmonary fibrosis. We addressed significant challenges, including limited sample size, biological and technical sample heterogeneity, and reagent and assay batch effects. RESULTS: We identified inter- and intra-patient heterogeneity, particularly within the non-UIP group. The models classified UIP on transbronchial biopsy samples with a receiver-operating characteristic area under the curve of ~ 0.9 in cross-validation. Using in silico mixed samples in training, we prospectively defined a decision boundary to optimize specificity at ≥85%. The penalized logistic regression model showed greater reproducibility across technical replicates and was chosen as the final model. The final model showed sensitivity of 70% and specificity of 88% in the test set. CONCLUSIONS: We demonstrated that the suggested methodologies appropriately addressed challenges of the sample size, disease heterogeneity and technical batch effects and developed a highly accurate and robust classifier leveraging RNA sequencing for the classification of UIP.

Analytical performance of Envisia: a genomic classifier for usual interstitial pneumonia.

BACKGROUND: Clinical guidelines specify that diagnosis of interstitial pulmonary fibrosis (IPF) requires identification of usual interstitial pneumonia (UIP) pattern. While UIP can be identified by high resolution CT of the chest, the results are often inconclusive, making surgical lung biopsy necessary to reach a definitive diagnosis (Raghu et al., Am J Respir Crit Care Med 183(6):788-824, 2011). The Envisia genomic classifier differentiates UIP from non-UIP pathology in transbronchial biopsies (TBB), potentially allowing patients to avoid an invasive procedure (Brown et al., Am J Respir Crit Care Med 195:A6792, 2017). To ensure patient safety and efficacy, a laboratory developed test (LDT) must meet strict regulatory requirements for accuracy, reproducibility and robustness. The analytical characteristics of the Envisia test are assessed and reported here. METHODS: The Envisia test utilizes total RNA extracted from TBB samples to perform Next Generation RNA Sequencing. The gene count data from 190 genes are then input to the Envisia genomic classifier, a machine learning algorithm, to output either a UIP or non-UIP classification result. We characterized the stability of RNA in TBBs during collection and shipment, and evaluated input RNA mass and proportions on the limit of detection of UIP. We evaluated potentially interfering substances such as blood and genomic DNA. Intra-run, inter-run, and inter-laboratory reproducibility of test results were also characterized. RESULTS: RNA content within TBBs preserved in RNAprotect is stable for up to 14 days with no detectable change in RNA quality. The Envisia test is tolerant to variation in RNA input (5 to 30 ng), with no impact on classifier results. The Envisia test can tolerate dilution of non-UIP and UIP classification signals at the RNA level by up to 60% and 20%, respectively. Analytical specificity studies utilizing UIP and non-UIP samples mixed with genomic DNA (up to 30% relative input) demonstrated no impact to classifier results. The Envisia test tolerates up to 22% of blood contamination, well beyond the level observed in TBBs. The test is reproducible from RNA extraction through to Envisia test result (standard deviation of 0.20 for Envisia classification scores on > 7-unit scale). CONCLUSIONS: The Envisia test demonstrates the robust analytical performance required of an LDT. Envisia can be used to inform the diagnoses of patients with suspected IPF.

A retrospective study of induction chemotherapy with docetaxel, cisplatinum, and 5-fluorouracil followed by concurrent radiotherapy with cetuximab in locally advanced head and neck cancer.

BACKGROUND: The objective was to study the results of induction chemotherapy followed by external beam radiation therapy with concurrent cetuximab in the treatment of locally advanced head and neck cancer. METHODS: Seventeen patients with stage III or IV squamous cell carcinomas of the head and neck who received docetaxel, cisplatinum, and 5-fluorouracil followed by radiation therapy with concurrent cetuximab were retrospectively analyzed. All radiation was delivered with image-guided intensity-modulated radiation treatments. Primary end points analyzed were local control and overall survival. RESULTS: With a median follow-up of 17 months, the approximate 2-year local control was 85%, with overall survival being 91%. At time of last follow-up, only 1 death was observed, with the cause of death unknown. Two local failures were observed, and the patients were under active management for their recurrences at time of last follow-up. No distant metastatic failures were noted among the patients. CONCLUSIONS: Induction chemotherapy with docetaxel, cisplatinum, and 5-fluorouracil followed by concurrent radiation with cetuximab provides for excellent locoregional control of disease. Future prospective studies can better establish the efficacy of this treatment regimen to current favored protocols.

Use of a molecular classifier to identify usual interstitial pneumonia in conventional transbronchial lung biopsy samples: a prospective validation study.

BACKGROUND: In the appropriate clinical setting, the diagnosis of idiopathic pulmonary fibrosis (IPF) requires a pattern of usual interstitial pneumonia to be present on high-resolution chest CT (HRCT) or surgical lung biopsy. A molecular usual interstitial pneumonia signature can be identified by a machine learning algorithm in less-invasive transbronchial lung biopsy samples. We report prospective findings for the clinical validity and utility of this molecular test. METHODS: We prospectively recruited 237 patients for this study from those enrolled in the Bronchial Sample Collection for a Novel Genomic Test (BRAVE) study in 29 US and European sites. Patients were undergoing evaluation for interstitial lung disease and had had samples obtained by clinically indicated surgical or transbronchial biopsy or cryobiopsy for pathology. Histopathological diagnoses were made by experienced pathologists. Available HRCT scans were reviewed centrally. Three to five transbronchial lung biopsy samples were collected from all patients specifically for this study, pooled by patient, and extracted for transcriptomic sequencing. After exclusions, diagnostic histopathology and RNA sequence data from 90 patients were used to train a machine learning algorithm (Envisia Genomic Classifier, Veracyte, San Francisco, CA, USA) to identify a usual interstitial pneumonia pattern. The primary study endpoint was validation of the classifier in 49 patients by comparison with diagnostic histopathology. To assess clinical utility, we compared the agreement and confidence level of diagnosis made by central multidisciplinary teams based on anonymised clinical information and radiology results plus either molecular classifier or histopathology results. FINDINGS: The classifier identified usual interstitial pneumonia in transbronchial lung biopsy samples from 49 patients with 88% specificity (95% CI 70-98) and 70% sensitivity (47-87). Among 42 of these patients who had possible or inconsistent usual interstitial pneumonia on HRCT, the classifier showed 81% positive predictive value (95% CI 54-96) for underlying biopsy-proven usual interstitial pneumonia. In the clinical utility analysis, we found 86% agreement (95% CI 78-92) between clinical diagnoses using classifier results and those using histopathology data. Diagnostic confidence was improved by the molecular classifier results compared with histopathology results in 18 with IPF diagnoses (proportion of diagnoses that were confident or provisional with high confidence 89% vs 56%, p=0·0339) and in all 48 patients with non-diagnostic pathology or non-classifiable fibrosis histopathology (63% vs 42%, p=0·0412). INTERPRETATION: The molecular test provided an objective method to aid clinicians and multidisciplinary teams in ascertaining a diagnosis of IPF, particularly for patients without a clear radiological diagnosis, in samples that can be obtained by a less invasive method. Further prospective clinical validation and utility studies are planned. FUNDING: Veracyte.

Performance of a Genomic Sequencing Classifier for the Preoperative Diagnosis of Cytologically Indeterminate Thyroid Nodules.

Importance: Use of next-generation sequencing of RNA and machine learning algorithms can classify the risk of malignancy in cytologically indeterminate thyroid nodules to limit unnecessary diagnostic surgery. Objective: To measure the performance of a genomic sequencing classifier for cytologically indeterminate thyroid nodules. Design, Setting, and Participants: A blinded validation study was conducted on a set of cytologically indeterminate thyroid nodules collected by fine-needle aspiration biopsy between June 2009 and December 2010 from 49 academic and community centers in the United States. All patients underwent surgery without genomic information and were assigned a histopathology diagnosis by an expert panel blinded to all genomic information. There were 210 potentially eligible thyroid biopsy samples with Bethesda III or IV indeterminate cytopathology that constituted a cohort previously used to validate the gene expression classifier. Of these, 191 samples (91.0%) had adequate residual RNA for validation of the genomic sequencing classifier. Algorithm development and independent validation occurred between August 2016 and May 2017. Exposures: Thyroid nodule surgical histopathology diagnosis by an expert panel blinded to all genomic data. Main Outcomes and Measures: The primary end point was measurement of genomic sequencing classifier sensitivity, specificity, and negative and positive predictive values in biopsies from Bethesda III and IV nodules. The secondary end point was measurement of classifier performance in biopsies from Bethesda II, V, and VI nodules. Results: Of the 183 included patients, 142 (77.6%) were women, and the mean (range) age was 51.7 (22.0-85.0) years. The genomic sequencing classifier had a sensitivity of 91% (95% CI, 79-98) and a specificity of 68% (95% CI, 60-76). At 24% cancer prevalence, the negative predictive value was 96% (95% CI, 90-99) and the positive predictive value was 47% (95% CI, 36-58). Conclusions and Relevance: The genomic sequencing classifier demonstrates high sensitivity and accuracy for identifying benign nodules. Its 36% increase in specificity compared with the gene expression classifier potentially increases the number of patients with benign nodules who can safely avoid unnecessary diagnostic surgery.

Patient-specific vaccines derived from autologous tumor cell lines as active specific immunotherapy: results of exploratory phase I/II trials in patients with metastatic melanoma.

Seventy-four (74) patients with metastatic melanoma were treated with patient-specific vaccines derived from autologous tumor cell lines. Cryopreserved irradiated tumor cells were injected weekly for 3 weeks, then monthly for 5 months. At a median follow up >6 years, the median event-free survival (EFS) was 4.5 months, with 13 patients alive and progression free 6-12 years later. Median overall survival (OS) was 20.5 months, with 29% 5-year OS. Tumor response rate was 9% among the 35 patients with evaluable disease who received at least 3 injections. Better survival was observed for patients who had minimal rather than clinically evident metastatic disease at the time vaccine therapy was initiated (5-yr OS 47% vs. 13%; p < 0.0001), received granulocyte-macrophage colony-stimulating factor and/or interferon gamma as an adjuvant (5-yr EFS 26% vs. 0%; p < 0.0001) or received an average of <7 million cells for each of the first 3 injections, compared to those who received 7-11.9 million or >12 million cells per injection (5-yr EFS OS 35% vs. 24%; p = 0.041 and p = 0.034). There was a trend toward better EFS for those who had a positive delayed type hypersensitivity (DTH) reaction to an intradermal injection of 1 million irradiated tumor cells at baseline, or converted to positive after 3 injections, compared to those whose DTH remained negative (5-yr EFS 39% vs. 18%; p = 0.159). This treatment approach is feasible, produces minimal toxicity, and is associated with longterm survival in a significant proportion of patients.

Usual Interstitial Pneumonia Can Be Detected in Transbronchial Biopsies Using Machine Learning.

RATIONALE: Usual interstitial pneumonia (UIP) is the histopathologic hallmark of idiopathic pulmonary fibrosis. Although UIP can be detected by high-resolution computed tomography of the chest, the results are frequently inconclusive, and pathology from transbronchial biopsy (TBB) has poor sensitivity. Surgical lung biopsy may be necessary for a definitive diagnosis. OBJECTIVES: To develop a genomic classifier in tissue obtained by TBB that distinguishes UIP from non-UIP, trained against central pathology as the reference standard. METHODS: Exome enriched RNA sequencing was performed on 283 TBBs from 84 subjects. Machine learning was used to train an algorithm with high rule-in (specificity) performance using specimens from 53 subjects. Performance was evaluated by cross-validation and on an independent test set of specimens from 31 subjects. We explored the feasibility of a single molecular test per subject by combining multiple TBBs from upper and lower lobes. To address whether classifier accuracy depends upon adequate alveolar sampling, we tested for correlation between classifier accuracy and expression of alveolar-specific genes. RESULTS: The top-performing algorithm distinguishes UIP from non-UIP conditions in single TBB samples with an area under the receiver operator characteristic curve (AUC) of 0.86, with specificity of 86% (confidence interval = 71-95%) and sensitivity of 63% (confidence interval = 51-74%) (31 test subjects). Performance improves to an AUC of 0.92 when three to five TBB samples per subject are combined at the RNA level for testing. Although we observed a wide range of type I and II alveolar-specific gene expression in TBBs, expression of these transcripts did not correlate with classifier accuracy. CONCLUSIONS: We demonstrate proof of principle that genomic analysis and machine learning improves the utility of TBB for the diagnosis of UIP, with greater sensitivity and specificity than pathology in TBB alone. Combining multiple individual subject samples results in increased test accuracy over single sample testing. This approach requires validation in an independent cohort of subjects before application in the clinic.

Dendritic Versus Tumor Cell Presentation of Autologous Tumor Antigens for Active Specific Immunotherapy in Metastatic Melanoma: Impact on Long-Term Survival by Extent of Disease at the Time of Treatment.

In patients with metastatic melanoma, sequential single-arm and randomized phase II trials with a therapeutic vaccine consisting of autologous dendritic cells (DCs) loaded with antigens from self-renewing, proliferating, irradiated autologous tumor cells (DC-TC) showed superior survival compared with similar patients immunized with irradiated tumor cells (TC). We wished to determine whether this difference was evident in cohorts who at the time of treatment had (1) no evidence of disease (NED) or (2) had detectable disease. Eligibility criteria and treatment schedules were the same for all three trials. Pooled data confirmed that overall survival (OS) was longer in 72 patients treated with DC-TC compared with 71 patients treated with TC (median OS 60 versus 22 months; 5-year OS 51% versus 32%, p=0.004). Treatment with DC-TC was associated with longer OS in both cohorts. Among 70 patients who were NED at the time that treatment was started, OS was better for DC-TC: 5-year OS 73% versus 43% (p=0.015). Among 73 patients who had detectable metastases, OS was better for DC-TC: median 38.8 months versus 14.7 months, 5-year OS 33% versus 20% (p=0.025). This approach is promising as an adjunct to other therapies in patients who have had metastatic melanoma.

Does continuous-infusion interleukin-2 increase survival in metastatic melanoma?

Pharmacy logbooks and clinical trial records were used to identify all 60 patients with metastatic melanoma who were treated as inpatients with intermediate-dose, continuous-infusion interleukin-2 (IL-2) in Hoag Hospital during 1987 to 1998. The hospital tumor registry was used to identify contemporary controls who had not received inpatient IL-2, matched for having distant metastatic melanoma, and by year and stage at original diagnosis, gender, and age. The mean time from original diagnosis to the documentation of distant metastatic disease was similar in both groups, 24 to 26 months. From the date of starting IL-2 therapy, patients had a median survival of 8.8 months, 38% 1-year survival, and 20% 5-year survival, with 8 patients alive beyond 5 years. However, there was no difference in survival from the first date of distant metastatic disease (median 25.8 months for IL-2 versus 31.5 months for controls, with survival rates 5 years after metastatic disease of 26% versus 31%). There was also no difference in overall survival from the date of original diagnosis (60.1 months for the IL-2 group versus 86.3 months for controls, with 5-year survival rates of 51% versus 64%, and 10-year survival rates of 29% versus 33%). This single-institution study failed to establish a survival advantage for patients with metastatic melanoma who received intermediate-dose, continuous-infusion IL-2 administered in the inpatient setting, compared to contemporary, matched-control patients who never received inpatient IL-2 therapy. However, the 5-year survival rates after a diagnosis of distant metastatic disease were a surprisingly high 26% to 31% in both groups. In the absence of a control group, the survival impact of IL-2 has probably been overestimated from single-arm phase II and III trials.

Tumor-infiltrating lymphocytes and interleukin-2: dose and schedules of administration in the treatment of metastatic cancer.

PURPOSE: The potential for therapeutic use of tumor-infiltrating lymphocytes (TIL), as adoptive cellular therapy has been touted for many years with some encouraging reports in patients with metastatic melanoma. MATERIALS AND METHODS: We previously described methodologies for TIL production and phenotypic characterization of TIL generated in our laboratory between 1991 and 1995 in semipermeable bags and between 1996 and 2000 in bioreactors. Patients treated in the earlier era were to have received a hybrid bolus and a 12-hour continuous infusion of interleukin (IL)-2 (total, 48 MIU), while in the latter era 4 days of interferon- alpha preceded the TIL and IL-2; which was given by a hybrid schedule that included bolus and 72- hour continuous IL-2 (total, 96 MIU). There were 55 patients, including 23 patients with melanoma, 9 patients with renal cell carcinoma, and 8 patients with colorectal cancer. There was only 1 objective tumor response, which was noted in a patient with renal cell carcinoma. The 55 patients who received these products were grouped in cohorts by treatment era, quantity of TIL received, amount of IL-2 intended, and different combinations of TIL and IL-2. RESULTS: There was no difference in survival by production method (treatment era), or amount of IL-2 given with TIL, but 33 patients who received an intermediate or higher dose of TIL (mean = 54.4 x 10(9)) had a median survival of 11.8 months, compared to 6.4 months for 22 patients who received 1 low-dose TIL (mean = 6.48 x 10(9)) (p = 0.059, log rank test). The objective response rate in this heterogeneous group of patients was not encouraging. The data suggest there may be a dose/benefit relationship between the total number of TIL infused and survival.

Autologous tumor cell line-derived vaccine for patient-specific treatment of advanced renal cell carcinoma.

AIM: We previously reported the laboratory methodology for producing patient-specific irradiated autologous tumor-cell products derived from short-term cultured tumor cells from resected renal cell carcinoma, and described preliminary clinical results. In this study, we report the final clinical results and efforts to define vaccine potency on the basis of clinical outcome for these 25 patients with advanced renal cell carcinoma. MATERIALS AND METHODS: Approximately 10(8) cells from successful short-term cell lines were irradiated, frozen in aliquots of 10(7) cells, then thawed and administered subcutaneously (s.c.) once a week for 3 weeks, then once a month for 5 months. Patients included 19 men and 6 women, who were 43-82 years of age. Six (6) patients had a large primary lesion, 2 patients had regionally advanced disease, 3 patients had been rendered disease-free by surgical resection of distant metastases, and 14 patients had measurable distant metastatic disease. RESULTS: The vaccines were well tolerated, and no delayed autoimmune effects were documented. Delayed-type hypersensitivity (DTH) tests of irradiated tumor cells were positive in only 1 of 25 patients at week 0, but converted to positive in 6 of 18 patients of DTH-negative patients who were retested at week 4. Objective response rate in patients who had measurable metastatic disease was 0 of 14 patients. With a median follow-up of greater than 7 years from the date of the first DTH test, median survival is 33.4 months, 5-year survival is 43%, and 10 patients are alive 3-12 years later. The 7 DTH+ patients survived a median of 2.5 years, and 3 patients are alive after 3, 4, and 7 years. There was no correlation between the number of irradiated cells or viable irradiated cells injected and tumor DTH reactivity or survival. CONCLUSION: This approach is feasible and the therapy is well tolerated, but clinical benefit was not established in this trial. Any further exploration of this product should be limited to the adjuvant setting in a randomized trial.

Phase I/II trial of autologous tumor cell line-derived vaccines for recurrent or metastatic sarcomas.

AIM: We previously reported the laboratory methodology for producing patient-specific irradiated autologous tumor-cell products derived from short-term cultured tumor cells. We attempted to determine the feasibility, safety, and clinical effects of autologous tumor vaccine-derived sarcomas. PATIENTS AND METHODS: Efforts were made to establish tumor cell lines in tissue culture with expansion to 100 million cells for patients who were candidates for therapy. Cells were irradiated and cryopreserved in aliquots of 10 million cells for subcutaneous (s.c.) injections, once a week for 3 weeks, then once a month for 5 months. RESULTS: Efforts were made to establish short-term tumor cell lines from 86 fresh sarcoma specimens (10 primary, 14 recurrent, and 62 metastatic). Initial growth was successful for 48 patients (56%), and cultures were expanded for 36 patients, with 25 patients treated. There were 23 evaluable patients, including 12 women and 11 men, with a median age of 52 years and a range from 16-79 years. Vaccine therapy was well tolerated. Delayed-type hypersensitivity (DTH) tests to irradiated tumor cells were positive in 0 of 20 patients tested at baseline, but converted to positive after 3 weekly vaccinations in 8 of 16 patients who were retested. Median survival for the 8 DTH converters was 16.6 months versus 8.2 months for the 8 responders whose tumor DTH test remained negative, and 6.0 months for the 7 patients who were not tested. No objective responses were recorded among 12 evaluable patients with measurable disease; 10 patients have survived more than 1 year. CONCLUSIONS: This approach is feasible, well tolerated, and the resulting DTH conversion rate is of interest. Patients with minimal tumor burden would be preferred for further future testing.

Phase I/II trial of melanoma patient-specific vaccine of proliferating autologous tumor cells, dendritic cells, and GM-CSF: planned interim analysis.

AIM: The aim of this study was to investigate the feasibility, safety, and clinical efficacy of patient-specific dendritic cell vaccines in patients with metastatic melanoma. A planned interim analysis was conducted on the first 20 patients. METHODS: Tumor cell lines were established from metastatic tumor, expanded to 200 million cells, and then incubated with interferon-gamma for patients who were candidates for therapy. Cells were irradiated and cryopreserved. Patients underwent leukapheresis to obtain mononuclear cells that were cultured in the presence of IL-4 and GM-CSF to produce dendritic cells, which were incubated with cryopreserved, irradiated tumor cells, and then stored in aliquots of about 20 million cells for subcutaneous (s.c.) injections with GM-CSF once a week for 3 weeks, then once a month for 5 months. RESULTS: The first 20 eligible patients included 10 men and 10 women, with a median age of 48 years (range, 16-79 years). Three (3) patients had brain metastases, and 13 patients had experienced disease progression after biochemotherapy. At the time of vaccine treatment, 6 patients had evaluable metastatic disease, while 14 patients lacked measurable disease. Vaccine therapy was well tolerated, except for what appeared to be GM-CSF-related allergic reactions in 2 patients. Delayed-type hypersensitivity (DTH) tests to irradiated tumor cells were positive in 0 of 20 patients tested at baseline, but converted to positive in 8 patients (40%). At a median follow-up of 13.8 months, there is a 95% overall survival and a 48% progression-free survival. Four (4) patients have already survived more than 3.0 years since starting the vaccine. CONCLUSION: Based on tolerability, rate of tumor DTH conversion, and encouraging survival, the trial will continue accrual to at least 19 patients with measurable disease and 40 patients who lack measurable disease at the time of treatment.

Irradiated cells from autologous tumor cell lines as patient-specific vaccine therapy in 125 patients with metastatic cancer: induction of delayed-type hypersensitivity to autologous tumor is associated with improved survival.

OBJECTIVE: We established short-term cultures of pure tumor cells for use as autologous tumor cell vaccines in an effort to study the effects of patients-specific immunotherapy. PATIENTS AND METHODS: Surgically resected fresh tumor was obtained from patients with metastatic cancer. Successful tumor cell lines (5 x 10(7)) were expanded to 10(8) cells, irradiated, and cryopreserved for clinical use. Following a baseline test of delayed-type hypersensitivity (DTH) to an i.d. injection of 10(6) irradiated autologous tumor cells, patients received 3 weekly s.c. injections of 10(7) cells, had a repeat DTH test at week-4, then received monthly vaccinations for 5 months. A positive DTH test was defined as > or = 10 mm induration; survival was determined from the first DTH test. RESULTS: Short-term cell lines were successfully established for 299/695 patients (43%). Vaccines were prepared for 231 patients, 142 of whom were treated, and 125 had a baseline DTH test recorded. Median follow up at the time of analysis was greater than 5 years. There was no difference in survival for any of the following: gender, age > 50 years, melanoma histology, anergy to common recall antigens or baseline DTH test result. Only 17 patients had a positive DTH at baseline (14%), but DTH converted from negative to positive in 31/80 (39%) of those who were tested, and in 31/108 (29%) of all patients (intent-to-convert analysis). For the 48 patients who were DTH-positive at entry, or converted to DTH-positive, the median survival was 30.5 months and 5-year survival 41% compared to 11.4 months and 9% 5-year survival for 77 patients whose DTH was never positive (P2 = 0.003). However, survival was even better for patients whose DTH test converted to positive compared to patients who were DTH-positive at baseline (median 37.5 vs 11.9 mos, P2 = 0.066). CONCLUSION: This patient-specific, cell culture-derived, autologous tumor cell vaccine induced anti-tumor immune reactivity that was associated with improved survival in patients with advanced cancer.

MammaPrint molecular diagnostics on formalin-fixed, paraffin-embedded tissue.

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Drug-Addicted Oncology Practice: The Drug Test.

PURPOSE: The buy-and-bill model of pharmaceuticals has been an economic reality for medical oncology practices for more than 30 years. Infusion services, once just a supportive service, grew exponentially in most practices between 1991 and 2000, to the point at which revenue from these services created a high, which made all things possible, including care of underinsured or uninsured patients. This altered state was further enhanced in the early part of this past decade when the introduction of high-cost chemotherapeutics and biologic agents created the euphoria of big business and a true dollar distortion for even the smallest oncology practice. RESULTS: In 2005, government and payer intervention forced average wholesale price to average sales price conversion to reduce the drug effect. Concurrently, payer scrutiny of utilization and medical necessity with further dose reductions, equating to margins, led many practices to experience painful drug withdrawal symptoms. Despite the enormous financial risk for unreimbursed expense and acknowledged spiraling decline of drug margins of greater than 30% since 2002, the seductive possibility of a financial fix keeps the practices hooked. CONCLUSION: Many practices will make poor financial choices and will gamble with their financial health by consuming precious resources to support the habit. We propose the drug test: Run your practice financials without drugs. Can you cover the total practice expense without drugs? If not, determine your level of dependency on the drug margin, and adjust your service lines and salaries accordingly. We issue the following warning: Drug dependencies may be hazardous to the financial health of your organization.

Should high-dose interleukin-2 still be the preferred treatment for patients with metastatic melanoma?

For more than 20 years interleukin-2 (IL2) was the preferred treatment for medically fit metastatic melanoma patients, but recently two new agents, ipilimumab and vemurafenib, were approved for stage IV disease. Single-institution data were used to determine the long-term survival rate for IL2-treated melanoma patients, and whether use of inpatient IL2 had declined recently. Between May 1987 and April 2010, 150 patients were hospitalized for high-dose, intravenous (i.v.) IL2. The average number of IL2 patients increased from 5.4 per year during 1987-1991 to 5.8 during 1992-1997 after regulatory approval of IL2, to 8.3 during 1998-2006 after a marketing indication in metastatic melanoma was granted, but dropped to 3.0 during 2007-2010. At the time of treatment, median age was 52 years; 27% were 60 years of age or older. At the time of analysis 122 patients were deceased. Median survival from the start date of IL2 treatment was 15.6 months, with a 20% 5-year survival. Among patients enrolled in clinical trials, there were as many nonresponders who survived 5 years as responders, which is consistent with a delayed immunotherapy benefit. In the absence of long-term survival data for these newer agents, IL2 probably should still be the preferred initial treatment for most patients with metastatic melanoma who are medically fit.

Should high-dose interleukin-2 still be the preferred treatment for patients with metastatic renal cell cancer?

Interleukin-2 (IL-2) was the preferred treatment for medically fit patients with advanced kidney cancer, but recently, several targeted therapies have been approved for metastatic renal cell carcinoma. We wished to determine the long-term survival rate for patients with kidney cancer treated with IL-2 and whether the use of intense inpatient IL-2 has declined since the introduction of targeted therapies. Patients who received IL-2 were identified from clinical trial enrollment, pharmacy logs, and financial billing records. Survival was determined from the earliest date of IL-2 therapy. There were 79 patients hospitalized for high-dose infusional IL-2 between March 1989 and March 2009. Median age was 58 years, and 27% were older than 65 years at the time of treatment. At the time of this analysis, 72 patients had deceased. Median survival was 9.9 months, but 5-year survival was 19.4%. The average number of patients with IL-2 increased from 2.2 per year during 1989-1992 to 5.6 during 1993-2001 after FDA approval, but dropped to 2.0 during 2002-2009. High-dose IL-2 is associated with a 5-year survival rate that is higher than objective response rates, suggesting a delayed immunotherapy benefit for some patients. The use of intensive IL-2 has declined dramatically in recent years, but unless a long-term survival benefit can be shown for these new targeted products, we feel that inpatient IL-2 remains the preferred initial treatment.