Abstract B21: Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue

Pathway-specific therapy is the future of cancer management. The oncogenic phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in solid tumors; however, currently, no reliable test for PI3K pathway activation exists for human tumors. Taking advantage of the observation that loss of PTEN, the negative regulator of PI3K, results in robust activation of this pathway, we developed and validated a microarray gene expression signature for immunohistochemistry (IHC)-detectable PTEN loss in breast cancer (BC). The most significant signature gene was PTEN itself, indicating that PTEN mRNA levels are the primary determinant of PTEN protein levels in BC. Some PTEN IHC-positive BCs exhibited the signature of PTEN loss, which was associated to moderately reduced PTEN mRNA levels cooperating with specific types of PIK3CA mutations and/or amplification of HER2. This demonstrates that the signature is more sensitive than PTEN IHC for identifying tumors with pathway activation. In independent data sets of breast, prostate, and bladder carcinoma, prediction of pathway activity by the signature correlated significantly to poor patient outcome. Stathmin, encoded by the signature gene STMN1, was an accurate IHC marker of the signature and had prognostic significance in BC. Stathmin was also pathway-pharmacodynamic in vitro and in vivo. Thus, the signature or its components such as stathmin may be clinically useful tests for stratification of patients for anti-PI3K pathway therapy and monitoring therapeutic efficacy. This study indicates that aberrant PI3K pathway signaling is strongly associated with metastasis and poor survival across carcinoma types, highlighting the enormous potential impact on patient survival that pathway inhibition could achieve.

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EIT PACMAN Study preliminary results: OncoSignal pathway analysis identifies actionable cancer targets.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15606 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15606-e15606

Author(s):

Patricia Martin ◽

Sigi Neerken ◽

Anja Van De Stolpe ◽

Eveline den Biezen-Timmermans ◽

Martijn Akse ◽

...

Keyword(s):

Precision Medicine ◽

Pathway Analysis ◽

Drug Response ◽

Tumor Tissue ◽

Ovarian Tissue ◽

Metastatic Tumor ◽

Pi3k Pathway ◽

Expression Array ◽

Tissue Samples ◽

Targeted Drug

e15606 Background: Precision medicine refers to tailoring of treatment to each individual patient, although identifying tumor driving signaling pathways (SP) that are functionally active is still a challenge. OncoSignal pathway tests quantitatively measure activity of SP such as estrogen receptor (ER), androgen receptor (AR), PI3K, MAPK, Hedgehog (HH), TGF-β, Notch on fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples. OncoSignal pathway analysis aimed at assessing clinically actionable SP and retrospectively predicting targeted drug response on a series of patients’ (pts) samples from the MOSCATO trial run at Gustave Roussy. Methods: OncoSignal pathway analysis (ER, AR, PI3K, MAPK, HH, Notch, TGF-β) was performed blinded by Molecular Pathway Dx (Philips, Eindhoven) on metastatic tumor tissue samples from breast cancer (BC), prostate (PC), and high grade serous ovarian cancers (OC). Using Affymetrix expression array data from public GEO datasets, SP activity was analyzed in healthy prostate, breast, and ovarian tissue to define abnormal SP activity thresholds for tumor tissue pathway analysis. For each individual sample, SP alterations were considered tumor driving SP if sample SP activity exceeded the 95th percentile of SP activity within healthy tissue. Results by OncoSignal were also combined with clinical characteristics and molecular alterations identified in the MOSCATO trial. Results: Identified tumor driving SP were ER, AR, MAPK-AP1, HH, PI3K pathway in BC (n = 5), AR in PC (n = 30); AP1, Notch, TGFβ in OC (n = 17). OncoSignal identified clinically actionable tumor driving pathways in all BC samples (median tumor cellularity [MTC]: 40%, range 15-80%); 30/31 PC samples (MTC: 62%, range 25-90%), 16/17 OC samples (MTC 62%, range 15-80%). Actionable mutations were previously identified in 4/5 BC; 13/31 PC; 6/17 OC. Seven pts with BC and PC were treated with targeted therapy. OncoSignal pathway analysis correctly predicted response/resistance in 4 of these pts (57%). Conclusions: OncoSignal pathway analysis correctly identified SP activity alterations and predicted targeted drug response in this series of patients. OncoSignal will be further validated prospectively in precision medicine studies at Gustave Roussy in which patients are stratified for targeted treatment by mutation analysis.

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Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1

Sarcoma ◽

10.1155/2015/839182 ◽

2015 ◽

Vol 2015 ◽

pp. 1-20 ◽

Cited By ~ 2

Author(s):

Georg F. Weber

Keyword(s):

Transcription Factor ◽

Drug Transport ◽

Wnt Pathway ◽

Tumor Tissue ◽

Molecular Therapy ◽

Specific Gene ◽

Gene Products ◽

Pathway Activity ◽

Suitable Target ◽

Exome Sequence

A patient presented with a recurrent sarcoma (diagnosed as leiomyosarcoma) 12 years after the removal of an initial cancer (diagnosed as extracompartmental osteosarcoma) distally on the same limb. Following surgery, the sarcoma and unaffected muscle and bone were subjected to measurements of DNA exome sequence, RNA and protein expression, and transcription factor binding. The investigation provided corroboration of the diagnosis leiomyosarcoma, as the major upregulations in this tumor comprise muscle-specific gene products and calcium-regulating molecules (calcium is an important second messenger in smooth muscle cells). A likely culprit for the disease is the point mutation S181G in FAF1, which may cause a loss of apoptotic function consecutive to transforming DNA damage. The RNA levels of genes for drug transport and metabolism were extensively skewed in the tumor tissue as compared to muscle and bone. The results suggest that the tumor represents a recurrence of a dormant metastasis from an originally misdiagnosed neoplasm. A loss of FAF1 function could cause constitutive WNT pathway activity (consistent with the downstream inductions of IGF2BP1 and E2F1 in this cancer). While the study has informed on drug transport and drug metabolism pharmacogenetics, it has fallen short of identifying a suitable target for molecular therapy.

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OS5.3 Quantitative signaling pathway analysis of Diffuse Intrinsic Pontine Glioma identifies two subtypes, respectively high TGFβ/MAPK-AP1 and high PI3K/HH pathway activity, which are potentially clinically actionable

Neuro-Oncology ◽

10.1093/neuonc/noz126.036 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii11-iii11

Author(s):

A van de Stolpe ◽

W Verhaegh ◽

L Holtzer

Keyword(s):

Signal Transduction ◽

Signaling Pathways ◽

Signaling Pathway ◽

Pi3k Pathway ◽

Diffuse Intrinsic Pontine Glioma ◽

Low Grade Glioma ◽

Signal Transduction Pathways ◽

Low Grade ◽

Pontine Glioma ◽

Pathway Activity

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a pediatric brain tumor (glioma), resistant to chemotherapy, with only a temporary response to radiotherapy and an extremely bad prognosis. Genomic abnormalities have been found, indicating abnormal activation of certain growth factor signaling pathways, while expression analysis suggests involvement of developmental signaling pathways.10–15 signal transduction pathways can drive cancer growth and metastasis. We have developed, and biologically validated, a method which enables quantitative measurements of functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. A major envisioned clinical utility is prediction of therapy response. MATERIAL AND METHODS For signaling pathway analysis, Affymetrix expression microarray data were available (GEO dataset GSE26576) from 2 normal brain stem samples and from 6 low grade glioma and 26 DIPG samples (post-mortem after therapy). Of one DIPG patient samples were available before and after therapy. Signaling pathway activity scores were calculated for estrogen and androgen receptor, PI3K-FOXO, MAPK-AP1, JAK-STAT, NFκB, Hedgehog (HH), TGFβ, NOTCH and Wnt pathways. PI3K pathway activity is the reverse of FOXO activity, in the absence of oxidative stress (measured by SOD2 expression). Pathway activity scores were compared between normal tissue and low grade glioma samples and DIPG, and k-means cluster analysis was performed on the DIPG pathway activity scores. RESULTS After treatment, HH pathway activity was increased in DIPG compared to low grade glioma (p=0.0003), PI3K pathway activity scores showed large variations in activity in the DIPG group. Tumors with cell cycle (CDK4/6, CCND1-3) or Receptor Tyrosine Kinase-related gene amplifications had higher PI3K and HH pathway activity compared to tumors without identified amplifications (p<0.05) which, in contrast, had higher MAPK-AP1 pathway activity (p<0.002). Pathway-based clustering analysis revealed two DIPG clusters, C1: high TGFβ/MAPK-AP1 and low PI3K/HH pathway activity; C2: low TGFβ/MAPK-AP1, high PI3K/HH pathway activity. C1 best resembled low grade glioma. In the patient with pre/post treatment samples, a C1 pathway profile switched to a C2 profile after treatment. CONCLUSION Using our quantitative analysis of signaling pathway activity in post-treatment DIPG, two pathway activity subtypes were identified, of which the HH/PI3K high, TGFβ low activity subtype was associated with defined gene amplifications, and may have been induced by chemoradiation therapy. Clusters are supported by a clear biological rationale. Identified signaling pathways are potentially drug targetable.

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Buparlisib in Patients With Recurrent Glioblastoma Harboring Phosphatidylinositol 3-Kinase Pathway Activation: An Open-Label, Multicenter, Multi-Arm, Phase II Trial

Journal of Clinical Oncology ◽

10.1200/jco.18.01207 ◽

2019 ◽

Vol 37 (9) ◽

pp. 741-750 ◽

Cited By ~ 30

Author(s):

Patrick Y. Wen ◽

Mehdi Touat ◽

Brian M. Alexander ◽

Ingo K. Mellinghoff ◽

Shakti Ramkissoon ◽

...

Keyword(s):

Phase Ii ◽

Tumor Tissue ◽

Phase Ii Trial ◽

Pi3k Pathway ◽

Recurrent Glioblastoma ◽

Phosphatidylinositol 3 Kinase ◽

Open Label ◽

Brain Penetration ◽

Pathway Activation ◽

Pathway Inhibition

PURPOSE Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation. METHODS This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway–activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2. RESULTS Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKTS473 immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6S235/236 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]). CONCLUSION Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.

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Heterogeneity in signaling pathway activity within primary and between primary and metastatic breast cancer

10.1101/2020.07.27.223834 ◽

2020 ◽

Author(s):

Márcia A. Inda ◽

Paul van Swinderen ◽

Anne van Brussel ◽

Cathy B. Moelans ◽

Wim Verhaegh ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Signaling Pathway ◽

Metastatic Breast ◽

Pi3k Pathway ◽

Pathway Activity ◽

Micro Scale ◽

Scale Heterogeneity ◽

Macro Scale ◽

Heterogeneity Analysis

AbstractBackgroundTargeted drug treatment aims to block tumor driving signaling pathways, and is generally based on analysis of one primary tumor (PT) biopsy. Phenotypic heterogeneity within primary and between primary and metastatic lesions was investigated.MethodsActivity of androgen and estrogen receptor, PI3K-FOXO, Hedgehog, TGFβ, and Wnt signaling pathways was measured in breast cancer samples using a novel mRNA-based assay platform. Macro-scale heterogeneity analysis was performed on multiple spatially distributed PT tissue blocks from 17 luminal A-like, 9 luminal B-like, and 9 ER-negative primary breast cancers; micro-scale heterogeneity analysis was performed on four “quadrant” samples of a single tissue block of respectively 9, 4, and 4 matched PT. Samples from 6 PT with matched lymph node (LN, n=23) and 9 PT with distant metastatic sites (DS, n=12) were analyzed. Statistical variance analysis was performed with linear mixed models. A “checkerboard” model was introduced to explain the observed heterogeneity in PT.ResultsWithin PT, macro-scale heterogeneity in signaling pathway activity was similar to micro-scale heterogeneity, with a possible exception of the PI3K pathway. Variation was significantly higher on microscale for Hedgehog and TGFβ pathways. While pathway activity scores correlated significantly between different locations in the PT, positive correlations decreased between PT and LN, and even more between PT and DS metastases, including the emergence of a negative correlation for the ER pathway.ConclusionWith a possible exception of the PI3K pathway, variation in signaling pathway activity within a single PT tissue block was generally representative for the whole PT, but not for DS or LN metastases. The higher variation in TGFβ and HH pathway activity on microscale suggested the presence of multiple small cancer cell clones. While analysis of multiple sub-samples of a single biopsy block may be sufficient to predict PT response to some targeted therapies, such as hormonal therapy, metastatic breast cancer treatment requires analysis of metastatic biopsies. The findings on phenotypic intra-tumor heterogeneity are compatible with currently emerging ideas on a Big Bang type of cancer evolution.

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Relationship of PIK3CA Mutation and PI3K Pathway Activity with Antiproliferative Response to Anastrozole in Er+ Breast Cancer

Annals of Oncology ◽

10.1093/annonc/mdt084.5 ◽

2013 ◽

Vol 24 ◽

pp. iii29 ◽

Cited By ~ 1

Author(s):

C. Segal ◽

E. Lopez-Knowles ◽

V. Patel ◽

I. Garcia-Murillas ◽

N. Turner ◽

...

Keyword(s):

Breast Cancer ◽

Pik3ca Mutation ◽

Pi3k Pathway ◽

Pathway Activity ◽

Relationship Of

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Heterogeneity in Signaling Pathway Activity within Primary and between Primary and Metastatic Breast Cancer

Cancers ◽

10.3390/cancers13061345 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1345

Author(s):

Márcia A. Inda ◽

Paul van Swinderen ◽

Anne van Brussel ◽

Cathy B. Moelans ◽

Wim Verhaegh ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Tumor Heterogeneity ◽

Metastatic Breast ◽

Big Bang ◽

Pi3k Pathway ◽

Pathway Activity ◽

Micro Scale ◽

Macro Scale ◽

Scale Variation

Targeted therapy aims to block tumor-driving signaling pathways and is generally based on analysis of one primary tumor (PT) biopsy. Tumor heterogeneity within PT and between PT and metastatic breast lesions may, however, impact the effect of a chosen therapy. Whereas studies are available that investigate genetic heterogeneity, we present results on phenotypic heterogeneity by analyzing the variation in the functional activity of signal transduction pathways, using an earlier developed platform to measure such activity from mRNA measurements of pathways’ direct target genes. Statistical analysis comparing macro-scale variation in pathway activity on up to five spatially distributed PT tissue blocks (n = 35), to micro-scale variation in activity on four adjacent samples of a single PT tissue block (n = 17), showed that macro-scale variation was not larger than micro-scale variation, except possibly for the PI3K pathway. Simulations using a “checkerboard clone-size” model showed that multiple small clones could explain the higher micro-scale variation in activity found for the TGFβ and Hedgehog pathways, and that intermediate/large clones could explain the possibly higher macro-scale variation of the PI3K pathway. While within PT, pathway activities presented a highly positive correlation, correlations weakened between PT and lymph node metastases (n = 9), becoming even worse for PT and distant metastases (n = 9), including a negative correlation for the ER pathway. While analysis of multiple sub-samples of a single biopsy may be sufficient to predict PT response to targeted therapies, metastatic breast cancer treatment prediction requires analysis of metastatic biopsies. Our findings on phenotypic intra-tumor heterogeneity are compatible with emerging ideas on a Big Bang type of cancer evolution in which macro-scale heterogeneity appears not dominant.

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