Jürgen Wolf

Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell Lung Cancer

FGFR1 amplification provides a therapeutic target for squamous cell lung cancer, which is resistant to other targeted lung cancer drugs. A Smoking Gun for Lung Cancer Detectives and scientists alike need strong evidence to take their cases to the judge, who for scientists is often a patient with a deadly disease. Yet, new culprits are sometimes found that can break a case wide open. Lung cancer, which accounts for more than 10% of the global cancer burden, has a poor prognosis and inadequately responds to chemotherapy and radiotherapy. New targeted treatments for lung adenocarcinomas inhibit the oncogenic versions of signaling protein kinases that arise from mutations typically found in lung cancer patients who have never smoked. However, smokers frequently suffer from a different deviant, squamous cell lung cancers, for which there are no known molecular genetic targets for therapy. Now, Weiss et al. have fingered a new suspect in smoking-related lung cancer: amplification of the FGFR1 gene, which encodes the fibroblast growth factor receptor 1 tyrosine kinase (FGFR1). To identify therapeutically viable genetic alterations that may influence squamous cell lung cancer, Weiss et al. performed genomic profiles on a large set of lung cancer specimens. Squamous cell lung cancer samples showed FGFR1 amplification, which was not found in other lung cancer subtypes. The authors then determined that a molecule that broadly inhibits FGF receptor function could block tumor growth and cause cell death in the cancers that expressed high amounts of the FGFR1 gene product in a manner that was dependent on FGFR1 expression. Moreover, FGFR1 inhibition resulted in a considerable decrease in tumor size in a mouse model of FGFR1-amplified lung cancer. This culmination of evidence implies that inhibition of this receptor tyrosine kinase should be explored as a candidate therapy for corralling squamous cell lung cancer in smokers. Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Involved-Field Radiotherapy Is Equally Effective and Less Toxic Compared With Extended-Field Radiotherapy After Four Cycles of Chemotherapy in Patients With Early-Stage Unfavorable Hodgkin’s Lymphoma: Results of the HD8 Trial of the German Hodgkin’s Lymphoma Study Group

PURPOSE To investigate whether radiotherapy can be reduced without loss of efficacy from extended field (EF) to involved field (IF) after four cycles of chemotherapy. PATIENTS AND METHODS Between 1993 and 1998, patients with newly diagnosed early-stage unfavorable HD were enrolled onto this multicenter study. Patients were randomly assigned to receive cyclophosphamide, vincristine, procarbazine, and prednisone (COPP) + doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) for two cycles followed by radiotherapy of 30 Gy EF + 10 Gy to bulky disease (arm A) or 30 Gy IF + 10 Gy to bulky disease (arm B). RESULTS Of 1,204 patients randomly assigned to treatment, 1,064 patients were informative and eligible for the arm comparison (532 patients in arm A; 532 patients in arm B). The median observation time was 54 months. Five years after random assignment, the overall survival (OSran) for all eligible patients was 91% and freedom from treatment failure (FFTFran) was 83%. Survival rates at 5 years after start of radiotherapy revealed no differences for arms A and B, respectively, in terms of FFTF (85.8% and 84.2%) and OS at 5 years (90.8% and 92.4%). There also were no differences between arms A and B, respectively, in terms of complete remission (98.5% and 97.2%), progressive disease (0.8% and 1.9%), relapse (6.4% and 7.7%), death (8.1% and 6.4%), and secondary neoplasia (4.5% and 2.8%). In contrast, acute side effects including leukopenia, thrombocytopenia, nausea, gastrointestinal toxicity, and pharyngeal toxicity were more frequent in the EF arm. CONCLUSION Radiotherapy volume size reduction from EF to IF after COPP + ABVD chemotherapy for two cycles produces similar results and less toxicity in patients with early-stage unfavorable HD.

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Vandetanib Plus Pemetrexed for the Second-Line Treatment of Advanced Non–Small-Cell Lung Cancer: A Randomized, Double-Blind Phase III Trial

PURPOSE Vandetanib is a once-daily oral inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling. This randomized, placebo-controlled phase III study assessed the efficacy of vandetanib plus pemetrexed as second-line therapy in advanced non-small-cell lung cancer. PATIENTS AND METHODS Patients (N = 534) were randomly assigned to receive vandetanib 100 mg/d plus pemetrexed 500 mg/m(2) every 21 days (n = 256) or placebo plus pemetrexed (n = 278). Progression-free survival (PFS) was the primary end point; overall survival, objective response rate, disease control rate, time to deterioration of symptoms, and safety were secondary assessments. RESULTS There was no significant difference in PFS between treatment arms (hazard ratio [HR], 0.86; 97.58% CI, 0.69 to 1.06; P = .108). Overall survival was also not significantly different (HR, 0.86; 97.54% CI, 0.65 to 1.13; P = .219). Statistically significant improvements in objective response rate (19% v 8%; P < .001) and time to deterioration of symptoms (HR, 0.71; P = .0052; median, 18.1 weeks for vandetanib and 12.1 weeks for placebo) were observed in patients receiving vandetanib. Adding vandetanib to pemetrexed increased the incidence of some adverse events, including rash, diarrhea, and hypertension, while showing a reduced incidence of nausea, vomiting, anemia, fatigue, and asthenia with no reduction in the dose intensity of pemetrexed. CONCLUSION This study did not meet the primary end point of statistically significant PFS prolongation with vandetanib plus pemetrexed versus placebo plus pemetrexed. The vandetanib combination showed a significantly higher objective response rate and a significant delay in the time to worsening of lung cancer symptoms versus the placebo arm as well as an acceptable safety profile in this patient population.

Predicting drug susceptibility of non–small cell lung cancers based on genetic lesions

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non-small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.

Vaccination of multiple myeloma patients with idiotype-pulsed dendritic cells: immunological and clinical aspects

Multiple myeloma (MM) is characterized by a clonal proliferation of malignant plasma cells in the bone marrow secreting a monoclonal immunoglobulin (paraprotein) with specific antigenic determinants, the idiotype (Id), which can be regarded as a tumour‐associated antigen (TAA). In order to analyse the impact of a dendritic cell (DC)‐based vaccine, 11 patients with advanced MM were treated with CD34 stem cell‐derived dendritic cells that were pulsed with Id peptides. Subsequently, the patients received three boost immunizations every other week with a combination of Id and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (nine patients) or with Id peptide‐pulsed dendritic cells again (two patients). The treatment was well tolerated with no side‐effects. The present clinical study was a proof of concept analysis of dendritic cell‐based vaccines in MM. The capacity of the dendritic cells to activate idiotype‐specific T cells was verified by in vitro stimulation experiments before the vaccination therapy. Immunological effects of the Id vaccination were analysed by monitoring changes in anti‐idiotype antibody titres and idiotype‐specific T‐cell activity. After vaccination, three out of 10 analysed patients showed increased anti‐idiotype antibody serum titres, indicating the induction of an idiotype‐specific humoral immune response. The idiotype‐specific T‐cell response analysed by ELISpot was increased in four out of 10 analysed patients after vaccination, and one patient had a decreased plasma cell infiltration in the bone marrow. In conclusion, five out of 11 patients showed a biological response after vaccination. Thus, our data indicate that immunotherapy with Id‐pulsed DCs in MM patients is feasible and safe. DC generated from CD34+ progenitor cells can serve as a natural adjuvant for the induction of clinically relevant humoral and cellular idiotype‐specific immune responses in patients suffering from advanced MM.

Activity and safety of ceritinib in patients with ALK-rearranged non-small-cell lung cancer (ASCEND-1): updated results from the multicentre, open-label, phase 1 trial

SUMMARY Background ALK-rearranged non-small-cell lung cancer (NSCLC) is sensitive to ALK tyrosine kinase inhibitors (ALKi) such as crizotinib, but resistance invariably develops, often with progression in the brain. Ceritinib is a more potent ALKi than crizotinib in vitro, crosses the blood-brain barrier in vivo and shows clinical responses in crizotinib-resistant disease. Here, we assessed whole-body and intracranial activity of ceritinib in both ALK-pretreated and ALKi-naïve patients with ALK-rearranged NSCLC. Methods The primary objective (to determine the maximum tolerated dose of ceritinib) of this first-in-human, phase I, open-label ASCEND-1 trial has been reported previously. In the analysis reported here, antitumour efficacy of ceritinib was evaluated in all patients with ALK-rearranged NSCLC (n=246) treated with ceritinib at the recommended dose of 750 mg/day. Additionally, as patients with untreated or locally treated neurologically stable brain metastases at baseline were permitted in this study, intracranial efficacy was retrospectively confirmed by independent neuroradiologists for 94 patients with baseline brain metastases and at least one post-baseline MRI/CT tumour assessment. This study is no longer recruiting patients; however, treatment and follow-up are ongoing. This study is registered with ClinicalTrials.gov, number NCT01283516. Findings Median follow-up at the time of this report was 11 1 months (interquartile range 6·7–15·2). Patients were mainly heavily pretreated (105/246 [42·7%] at least three prior regimens). The overall response rate was 72·3% (60/83; 95% confidence interval [CI] 61·4–81·6) for ALKi-naïve (n=83) and 56·4% (92/163; 95% CI 48·5–64·2) for ALKi-pretreated (n=163) patients. Median progression-free survival in ALKi-naïve and ALKi-pretreated patients was 18·4 (95% CI 11·1-non-estimable) and 6·9 (95% CI 5·6–8·7) months, respectively. Brain metastases by investigator assessment were reported at study entry in 124 patients. Of these, 94 (n=19 ALKi-naïve and n=75 ALKi-pretreated) were included in the retrospective analysis; intracranial disease control rate was 78·9% (15/19; 95% CI 54·4– 93·9) in ALKi-naïve patients and 65·3% (49/75; 95% CI 53·5–76·0) in ALKi-pretreated patients. Of the 94 patients included in the retrospective analysis, 11 had measurable brain lesions and no prior radiotherapy to the brain: 6 of these achieved a partial intracranial response. Safety was evaluated for all 246 patients with ALK-rearranged NSCLC. Serious adverse events were recorded for 117 (47·6%) patients. The most common grade 3/4 laboratory abnormalities were increased alanine aminotransferase and increased aspartate aminotransferase, occurring in 73 (29·7%) and 25 (10·2%) patients, respectively. The most common grade 3/4 non-laboratory adverse events were diarrhoea and nausea, both of which occurred in 15 (6.1%) patients. Two on-treatment deaths in the study were considered to be related to study drug by the investigators, one due to interstitial lung disease and one as a result of multi-organ failure that occurred in the context of infection and ischaemic hepatitis. Interpretation This study demonstrated clinically meaningful and durable responses in mainly heavily pretreated patients with ALK-rearranged NSCLC (ALKi-naïve and ALKi-pretreated) receiving ceritinib 750 mg/day. Treatment with ceritinib also achieved both whole-body and intracranial efficacy in patients with brain metastases at baseline, a common site of disease progression in patients with NSCLC. The durable whole-body responses reported, together with the intracranial efficacy, support a clinical benefit for treatment with ceritinib in patients post-crizotinib, or as an alternative to crizotinib in patients with ALK-rearranged NSCLC. Funding Sponsored by Novartis Pharmaceuticals Corporation.BACKGROUND ALK-rearranged non-small-cell lung cancer (NSCLC) is sensitive to ALK tyrosine kinase inhibitors (ALK inhibitors) such as crizotinib, but resistance invariably develops, often with progression in the brain. Ceritinib is a more potent ALK inhibitor than crizotinib in vitro, crosses the blood-brain barrier in vivo, and shows clinical responses in patients with crizotinib-resistant disease. We aimed to assess whole-body activity of ceritinib in both ALK inhibitor-pretreated and ALK inhibitor-naive patients with ALK-rearranged NSCLC. METHODS ASCEND-1 was an open-label, phase 1 trial that recruited patients from 20 academic hospitals or cancer centres in 11 countries in Europe, North America, and Asia-Pacific. Eligible patients were aged 18 years or older with ALK-rearranged locally advanced or metastatic cancer that had progressed despite standard therapy (or for which no effective standard therapy existed), who had at least one measurable lesion at baseline. The primary objective (to determine the maximum tolerated dose) has been reported previously. This updated analysis includes all patients with ALK-rearranged NSCLC given oral ceritinib at the recommended dose of 750 mg/day in the dose-escalation and expansion phases. Here we report the secondary outcomes of overall response, duration of response, and progression-free survival, analysed in all patients who received at least one 750 mg dose of ceritinib. Exploratory analyses included retrospective analysis of intracranial activity by independent neuroradiologists, in patients with untreated or locally treated neurologically stable brain metastases at baseline. Safety was assessed in all patients who received at least one dose of ceritinib. This study is no longer recruiting patients; however, treatment and follow-up are ongoing. This study is registered with ClinicalTrials.gov, number NCT01283516. FINDINGS Between Jan 24, 2011, and July 31, 2013, 255 patients were enrolled and received at least one dose of ceritinib 750 mg/day, of whom 246 had ALK-rearranged NSCLC. At data cutoff (April 14, 2014), median follow-up was 11·1 months (IQR 6·7-15·2) and 147 (60%) patients had discontinued treatment, 98 (40%) as a result of disease progression. An overall response was reported in 60 (72% [95% CI 61-82]) of 83 ALK inhibitor-naive patients and 92 (56% [49-64]) of 163 ALK inhibitor-pretreated patients. Median duration of response was 17·0 months (95% CI 11·3-non-estimable [NE]) in ALK inhibitor-naive patients and 8·3 months (6·8-9·7) in ALK inhibitor-pretreated patients. Median progression-free survival was 18·4 months (95% CI 11·1-NE) in ALK inhibitor-naive patients and 6·9 months (5·6-8·7) in ALK inhibitor-pretreated patients. Of 94 patients with retrospectively confirmed brain metastases and at least one post-baseline MRI or CT tumour assessment, intracranial disease control was reported in 15 (79% [95% CI 54-94]) of 19 ALK inhibitor-naive patients and in 49 (65% [54-76]) of 75 ALK inhibitor-pretreated patients. Of these 94 patients, 11 had measurable brain lesions and no previous radiotherapy to the brain, six of whom achieved a partial intracranial response. Serious adverse events were recorded in 117 (48%) of 246 patients. The most common grade 3-4 laboratory abnormalities were increased alanine aminotransferase (73 [30%] patients) and increased aspartate aminotransferase (25 [10%]). The most common grade 3-4 non-laboratory adverse events were diarrhoea and nausea, both of which occurred in 15 (6%) patients. Two on-treatment deaths during the study were deemed to be related to study drug by the investigators, one due to interstitial lung disease and one as a result of multiorgan failure that occurred in the context of infection and ischaemic hepatitis. INTERPRETATION The durable whole-body responses reported, together with the intracranial activity, support a clinical benefit for treatment with ceritinib in patients with ALK-rearranged NSCLC who have received crizotinib, or as an alternative to crizotinib. A confirmatory phase 2 clinical trial is ongoing to assess ceritinib activity in patients with ALK-rearranged NSCLC and brain or leptomeningeal metastases. FUNDING Novartis Pharmaceuticals Corporation.

Crizotinib Therapy for Advanced Lung Adenocarcinoma and a ROS1 Rearrangement: Results From the EUROS1 Cohort

PURPOSE Approximately 1% of lung adenocarcinomas are driven by oncogenic ROS1 rearrangement. Crizotinib is a potent inhibitor of both ROS1 and ALK kinase domains. PATIENTS AND METHODS In the absence of a prospective clinical trial in Europe, we conducted a retrospective study in centers that tested for ROS1 rearrangement. Eligible patients had stage IV lung adenocarcinoma, had ROS1 rearrangement according to fluorescent in situ hybridization, and had received crizotinib therapy through an individual off-label use. Best response was assessed locally using RECIST (version 1.1). All other data were analyzed centrally. RESULTS We identified 32 eligible patients. One patient was excluded because next-generation sequencing was negative for ROS1 fusion. Median age was 50.5 years, 64.5% of patients were women, and 67.7% were never-smokers. Thirty patients were evaluable for progression-free survival (PFS), and 29 patients were evaluable for best response. We observed four patients with disease progression, two patients with stable disease, and objective response in 24 patients, including five complete responses (overall response rate, 80%; disease control rate, 86.7%). Median PFS was 9.1 months, and the PFS rate at 12 months was 44%. No unexpected adverse effects were observed. Twenty-six patients received pemetrexed (either alone or in combination with platinum and either before or after crizotinib) and had a response rate of 57.7% and a median PFS of 7.2 months. CONCLUSION Crizotinib was highly active at treating lung cancer in patients with a ROS1 rearrangement, suggesting that patients with lung adenocarcinomas should be tested for ROS1. Prospective clinical trials with crizotinib and other ROS1 inhibitors are ongoing or planned.