Integration of Multi-omic Data in a Molecular Tumor Board Reveals EGFR-Associated ALK-Inhibitor Resistance in a Patient With Inflammatory Myofibroblastic Cancer

Abstract

Inflammatory myofibroblastic tumors (IMTs) are intermediate-grade mesenchymal neoplasms commonly characterized by chromosomal rearrangements causing constitutive activation of anaplastic lymphoma kinase (ALK) and/or ALK mutations causing reduced sensitivity to ALK tyrosine kinase inhibitors (TKI). We present a patient with an IMT who initially responded to first-line alectinib, but who later suffered disease relapse and presently survives with moderate residual disease after receiving second-line lorlatinib. Biopsy specimens were analyzed using next generation sequencing (DNA-seq and RNA-seq) and reverse phase protein microarray (RPPA) as part of an institutional Molecular Tumor Board (MTB) study. An EML4-ALK rearrangement and EGFR activation (pEGFRY1068) were present in both the primary and recurrent tumors, while a secondary ALK I1171N mutation was exclusive to the latter. EGFR signaling in the background of a secondary ALK mutation is correlated with reduced ALK TKI sensitivity in vitro, implicating an important mechanism of drug resistance development in this patient. The RPPA results also critically demonstrate that ALK signaling (ALKY1604) was not activated in the recurrent tumor, thereby indicating that standard-of-care use of third- or fourth-line ALK TKI would not likely be efficacious or durable. These results underscore the importance of real-time clinical integration of functional protein drug target activation data with NGS in the MTB setting for improving selection of patient-tailored therapy.

Quantitative proteomic analysis of HER2 protein expression in PDAC tumors

“…Tumor epithelium was harvested via LMD for RPPA quantification of total HER2 (HER2 Total ), phosphorylated (p)HER2 Y1248 , and pHER3 Y1289 as part of a 32-protein/phosphoprotein biomarker, research use only (RUO) panel in a commercial CLIA/CAP-accredited laboratory (Theralink Technologies, Inc.) examining the abundances of targets with known relevance in solid tumors, as previously described (Fig. 1 B, C) [ 7 , 16 – 18 , 28 , 29 , 32 , 33 ]. Protein and phosphoprotein-level expression was determined by comparing the RPPA-derived quantitative values from each patient LMD tumor sample in our cohort against an existing and extensively validated RPPA reference dataset derived from LMD enriched tumor epithelium from 400 breast cancer specimens with centrally-determined HER2-positive/amplified (IHC 3+ /2+ , FISH-positive) and HER2-negative/unamplified (IHC 0/1+ /2+ , FISH-negative) status.…”

“…RPPA analysis was performed as previously described [ 32 ] using a 32-marker, CLIA certified and CAP accredited commercial calibrated assay panel for examination of the total and phosphoprotein abundances of several targets with known relevance in solid tumors [ 29 , 33 ]. The CLIA and CAP accredited RPPA assay is a calibrated immunoassay format that has been previously described [ 34 – 36 ], and uses a series of calibrators and controls along with a reference population data set of levels of expression of each of the 32 analytes including total and phosphorylated HER2, phosphorylated HER3, etc.…”

“…Moreover, recent studies have shown that upfront cellular enrichment of tumor epithelium from tissue biopsies is required for accurate determination of total and phosphoprotein expression in tumor epithelium [ 27 , 28 ]. Selective harvest of discrete cellular subpopulations from the complex milieu of the tumor microenvironment (TME) via laser microdissection (LMD) prior to reverse phase protein array (RPPA) analysis allows for the quantitative measurement and functional assessment of the activation state of protein drug targets and other known cancer-related proteins directly from enriched populations of microdissected tumor cells [ 28 , 29 ]. IHC-based determinations of HER2 expression in the HER2-low (IHC 1+ /2+ , FISH-negative) or HER2-ultra low (IHC 0) were not analytically accurate when compared to a CLIA quantitative RPPA-based HER2 assay to determine protein expression [ 7 ].…”

Integration of proteomics in the molecular tumor board

“…Therefore, the implementation of proteomics for individual patients remains a challenge, which is addressed by only a few current studies [118,129]. The integration of proteomics into the MTB is an emerging and highly relevant topic, as illustrated by the More recently, the Reverse Phase Protein Array (RPPA) approach has been implemented for precision oncology and supported clinical decision-making in MTBs [119,129,135,136]. In RPPA, protein samples are arrayed as microspots on a solid phase and subsequently probed with a specific antibody to detect protein abundances and PTMs [137].…”

Unraveling the spectrum of inflammatory myofibroblastic tumors in the lung: A comprehensive case series highlighting endobronchial, pleural, and lung parenchymal tumors

“… 1 , 23 Crizotinib, a tyrosine kinase inhibitor targeting ALK, MET, ROS1, and RON, has only recently been approved by the Food and Drug Administration (July 2022) as monotherapy in adult and pediatric patients older than 1 year with unresectable, recurrent, or refractory ALK-positive IMTs. 24 Unfortunately, recurrent IMT with ALK gene rearrangements often develop resistance to crizotinib, which is why second- or even third-line ALK inhibitors are used, with different levels of response. Approximately one-quarter of surgically treated tumors may experience a recurrence.…”