Additional file 1: of MET alterations detected in blood-derived circulating tumor DNA correlate with bone metastases and poor prognosis

Table S1. 54-gene panel (N = 122 patients)— identifies potential tumor-related genomic alterations within 54 cancer-related genes including amplifications in ERBB2, EGFR, and MET. Only non-synonymous alterations were analyzed. Table S2. 68-gene panel (N = 272 patients), comprising amplifications in 16 genes as well as some fusions and indels. Only non-synonymous alterations were analyzed. Table S3. 70-gene panel (N = 22 patients). Only non-synonymous alterations were analyzed. Table S4. Comparison of clinical characteristics in 438 patients with or without MET alterations (univariate analysis). (DOCX 20 kb

Squamousness: Next-generation sequencing reveals shared molecular features across squamous tumor types

<p>In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were <i>TP53</i> (64.5% of patients), <i>PIK3CA</i> (28.5%), <i>CDKN2A</i> (24.4%), <i>SOX2</i> (17.7%), and <i>CCND1</i> (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that <i>CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1</i>, and <i>FBXW7</i> were significantly more frequently altered, unlike <i>KRAS</i>, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified “squamousness” gene signatures (<i>TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1</i>, and <i>FBXW7</i> aberrations, and absence of <i>KRAS</i> alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom <i>TP53</i> and cyclin pathway (<i>CDKN2A</i> and <i>CCND1)</i> alterations strongly correlated but in whom <i>PIK3CA</i> aberrations were less frequent; and (ii) patients with <i>PIK3CA</i> alterations in whom <i>TP53</i> mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for “squamousness.” Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.</p

Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

<p>Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (<i>P</i> < 0.0001), <i>PTEN</i> (<i>P</i> < 0.0001), and <i>CDKN2A</i> alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), <i>APC</i> (P = 0.008), <i>PTEN</i> (P = 0.026) and <i>TP53</i> (P = 0.044) alterations. <i>TP53</i> (P = 0.003) and <i>PTEN</i> (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (<i>PTEN, CDKN2A, APC</i>, and/or <i>TP53</i>) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.</p

GDC0941 and AZD6244 <i>in vivo</i> treatment inhibits pAKT and pERK respectively.

<p>Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o., or AZD6244 50 mg/kg p.o., or with vehicle control. Animals were sacrificed 1 h after treatment, plasma samples were collected, analyzed and quantified by mass spectrometry for GDC0941 (A) or AZD6244 (B). Tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) for the model Rat1-myr-p110α (C) or the model Panc 10.05 (D).</p

K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition <em>In Vivo</em>

<div><p>Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.</p> </div

K-RAS mutant pancreatic models show stronger response to MEK than to PI3K inhibition <i>in vivo</i>.

<p>(A/B). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 50 mg/kg p.o. twice a day, or with vehicle control, with at least 5 mice per group. Tumor volumes were measured twice a week for the indicated period of time, and antitumor activity was plotted and quantified.</p

Combining MEK and PI3K inhibition <i>in vivo</i> is superior to single agent treatment.

<p>(A). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 5 mg/kg p.o. once a day, or with the combination of both, or with vehicle control, with 8 mice per group. Tumor volumes were measured twice a week, for the indicated period of time, and antitumor activity was plotted and quantified. (B). Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o. or of AZD6244 5 mg/kg p.o., with the combination of both or with vehicle control. Animals were sacrificed 3 h after treatment, tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204).</p

K-RAS knock down impairs tumor growth of pancreatic models <i>in vivo</i>.

<p>(A) For each xenograft model indicated, tumors were grown subcutaneously in female nude mice and groups of at least 4 mice each were formed once tumors had reached a size of 200–300 mm³. The first group was given normal drinking water, whereas the second was given drinking water containing 2 mg/ml doxycycline and 10% sucrose. Mice were sacrificed after one week of treatment (after 18 days in case of the K-RAS wild type model), and tumors were analyzed by qPCR for K-RAS. K-RAS levels were normalized to ribosomal protein s18. Obtained p-values were as follows: Capan-1 shNT: p = 0.35, Capan-1 sh236: p = 0.011, Panc 10.05 sh236: p = 0.009, AsPC-1 sh236: p = 0.002, L3.3 sh236: p = 0.004, NCI-H1437 sh236: p = 0.007. (B/C) As in (A), except that mice were randomized to groups of at least 6 mice each, with the exception of the Panc 10.05 model, where the group size was n = 4. Treatment was started once tumors had reached a size of 100 mm³, tumor size was followed over time, and mice were sacrificed once tumors of the control group reached a size of 1000 mm³ at most. Statistically significant differences of tumor volumes between groups (*) as well as the area under the curve (AUC/mm³ x treatment days) are indicated. Obtained p-values for AUC at the end of the study were as follows: Capan-1 shNT: p = 0.57, Capan-1 sh236: p = 0.04, Panc 10.05 sh236: p = 0.01, AsPC-1 sh236: p = 0.01, L3.3 sh236: p = 0.0003, NCI-H1437: p = 0.22. The PANC-1 cells could not be grown <i>in vivo</i>, and for this reason this model was only examined <i>in vitro</i>.</p

K-RAS knock down impairs proliferation in pancreatic lines <i>in vitro</i>.

<p>(A) Indicated cell lines (NT: non-targeting shRNA; 236 and 562: shRNAs targeting K-RAS) were either treated for 7 days with 200 ng/ml doxycycline (dox) or left untreated (no dox), followed by preparation of cell lysates. Corresponding cell extracts were then analyzed for K-RAS, total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) levels by Western Blot. (B) As in (A), except that cells were fixed on day 1 and day 7, followed by determination of relative cell number. Each cell line was tested in at least two independent experiments, and untreated samples were set to 100% of growth. Statistically significant differences (p<0.05) are indicated (*). Obtained p-values were as follows: Capan-1 shNT: p = 1, Capan-1 sh236: p = 0.002, Capan-1 sh562: p = 0.029; Panc 10.05 shNT: p = 0.33, Panc 10.05 sh236: p<0.001, Panc 10.05 sh562: p = 0.029; AsPc1 shNT: p = 0.33, AsPc1 sh236: p = 0.002, AsPc1 sh562: p = 0.029; L3.3 shNT: p = 0.187, L3.3 sh236: p<0.001, L3.3 sh562: p = 0.333; PANC-1 shNT: p = 1, PANC-1 sh236: p<0.001, PANC-1 sh562: p = 0.002.</p

K-RAS mutant pancreatic lines are independent of AKT <i>in vitro</i>.

<p>(A). Indicated cell lines were treated for 72 h with the AKT inhibitor MK2206, and effects on proliferation were determined by calculation of respective GI₅₀ values. (B/C). As in (A), except that indicated cell lines were treated for 62 h with either the PI3K inhibitor GDC0941 (B) or with the MEK inhibitor AZD6244 (C), and effects on proliferation were determined by calculation of respective GI₅₀ values. MCF7 cells were used as control for cells sensitive to GDC0941 and insensitive to AZD6244 and A375 cells were used as control for cells sensitive to AZD6244 and insensitive to GDC0941.</p