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Comparative Study
. 2016 Oct;19(4):1066-1079.
doi: 10.1007/s10120-015-0566-0. Epub 2015 Nov 18.

Mass-spectrometry-based Quantitation of Her2 in Gastroesophageal Tumor Tissue: Comparison to IHC and FISH

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Free PMC article
Comparative Study

Mass-spectrometry-based Quantitation of Her2 in Gastroesophageal Tumor Tissue: Comparison to IHC and FISH

Daniel V T Catenacci et al. Gastric Cancer. .
Free PMC article

Abstract

Background: Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results.

Methods: We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/μg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification.

Results: After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r (2) = 0.9842; FFPE r (2) = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of <450 amol/μg for identifying HER2 FISH-amplified tumors. An "equivocal zone" of 450-750 amol/µg of Her2-SRM protein was analogous to IHC2+ but represented fewer cases (9-16 % of cases versus 36-41 %).

Conclusions: Compared to IHC, targeted SRM-Her2 proteomics provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with its multiplex capability for other relevant oncoproteins, these results demonstrate a refined HER2 protein expression assay for clinical application.

Keywords: Clinical biomarker assay; Companion diagnostic; Esophageal; Gastric; Gastroesophageal adenocarcinoma; HER2 (ERBB2) amplification; Her2 expression; Multiplex protein expression analysis in FFPE tissue; SRM-MS; Selected reaction monitoring mass spectrometry; Stomach cancer.

Figures

Figure 1
Figure 1. Development of HER2 SRM assay
(A) The fragmentation spectrum for heavy ELVSEFSR peptide and (B) the standard curve generated in human PC3 cell lysate; inset: the standard curve generated without the highest two spiking points (5000 and 25000 amol). Each point injected contained 5000 amol heavy (ELVSEFSR [13C6,15N4]) on column. (C) The total ion chromatograms for the light and heavy isotopically labeled peptides, with (D) the transition ions used to identify and quantitate each peptide. (E) Precision assessment for measuring Her2 level in 8 breast cancer (red) and 11 GEC (blue) FFPE tissues. (F) Temporal reproducibility of FFPE sections processed and analyzed using LT-SRM at two time points over one year apart (blue, GEC (n=18); red, NSCLC (n=9)).
Figure 2
Figure 2. Her2 expression levels using LT-SRM and correlation with HER2 gene amplification (by FISH) in 27 cell lines
(A) Quantification of Her2-SRM (amol/ug) for 27 cell lines including GM15677 lymphoblast control. HER2 amplified cell lines (HER2/CEP7 ratio ≥2) indicated in dark red, and heterogenous HER2 amplification in pink (see methods for hetero+ scoring). (B) Her2-SRM and FISH gene copy number (GCN) univariate correlations: The left y-axis (blue plus sign) represents the mean HER2 GCN per nucleus and the right y-axis (red triangle) indicates HER2:CEP17 ratio (see text for multivariate analysis including Egfr-, Her3-, and Met-SRM coexpression, GCN R2=0.8829 and Ratio R2= 0.9824) and (C) scatter plot of HER2 GCN/ratio (by FISH) (blue diamond, red square, respectively) and Her2-SRM expression in samples where Her2 expressions are < 1500 amol/ug represented by the black box in (3B). A preliminary Her2-SRM cut-off, from these cell line data, correlating with HER2 amplification was determined to be ≥1150 amol/ug. (D) Her2-SRM (red) and Her3-SRM (blue) levels in a cell line mixing study (OE-19: HER2 amplified / MKN-1: HER2 non-amplified), modeling intra-tumor clonal heterogeneity.
Figure 3
Figure 3. Absolute levels of Her2 in GEC tissues and correlation of Her2-SRM levels with HER2 gene amplification
(A) Her2-SRM analysis of clinical FFPE GEC tissues (n=139) ranging <150-24617 amol/ug. Red highlighted samples are verified by FISH to be HER2 amplified (HER2/CEP17 ratio≥2); non-amplified samples are labeled as green, and samples in black were not FISH tested. (B) Univariate correlation of Her2-SRM and HER2 FISH GCN (n=42, blue, r2 = 0.3615) and ratio (n=54, red, r2=0.5354). Multivariate analysis revealed stronger correlations when incorporating Met-SRM, Egfr-SRM, Her3-SRM and HER2 FISH heterogeneity in the model: Her2-SRM:HER2 GCN r2 = 0.7345 and Her2-SRM:HER2/CEP17 ratio r2=0.7643 (54 Her2-SRM cases had absolute FISH ratio available). (C) Optimal Her2-SRM cutoff values determined by receiver operating characteristics (ROC) curve with respect to HER2/CEP17 ratio ≥2 . Using one cut-off level, a value of 450 amol/ug was 75% sensitive and 93% specific to identify ‘amplification’; alternatively, a cut-off level of 750 amol/ug was 55% sensitive and 100% specific. (D) Using two cut-points (analogous to IHC 0/1+ = Her2 negative, and IHC 3+ = Her2 positive), with values in between (analogous to IHC 2+) = ‘equivocal’, an upper SRM level bound of 750 amol/ug and lower bound of 450 amol/ug created an equivocal range 450-750 amol/ug. The two red lines (1 and 2) represent the Her2-SRM expression falling into this equivocal range (n=9/54, 16%). (54 cases were available with binary FISH ratio data (≥2 or <2). Currently, it is recommended that these SRM-equivocal cases undergo confirmatory FISH testing. (E) Among tumors exhibiting Her2 IHC 2+ with FISH results (n=20), 15 tumors (75%) were FISH- and 5 (25%) were FISH+. Her2-SRM expression levels are superimposed, demonstrating that the majority (18, 90%) of these IHC2+ samples were below the 450 amol/ug SRM cut-off.
Figure 4
Figure 4. Her2 IHC correlations with FISH assay or SRM
Correlation of Her2 IHC to (A) FISH GCN in 42 GEC tumors (r2=0.1959), (B) HER2/CEP17 ratio in n=52 GEC tumors (r2=0.1242) (52 cases with IHC results had absolute FISH ratio results); and (C) Her2-SRM (amol/μg) in 122 GEC tumors (r2=0.0355). (D) IHC compared to Her2-SRM with inset 2X2 table; and (E) three-way comparison of Her2-IHC, Her2-SRM and HER2/CEP17 ratio by FISH in 51 GEC tumors where all three values were available. Inset tables demonstrate the comparison of the lower boundary for each assay (left, IHC2+ versus SRM 450amol/ug) and IHC2+ versus FISH (right). Red bar-SRM, green bar-IHC, and black dot-HER2/CEP17 ratio by FISH. Inset tables assess sensitivity/specificity of IHC assuming SRM (D,E) and FISH (B,E) as the comparative standards.
Figure 5
Figure 5. Her2 status assessment of GEC cases by IHC (A) and Her2-SRM (B) to identify HER2 ‘positive’ and ‘negative’ cases, as determined by underlying FISH HER2/CEP17 ratio
Both assays resulted in an equivocal zone in identifying underlying HER2 amplification (by ratio>2). However, the incidence of the Her2-SRM equivocal zone (450-750amol/ug) was much lower (9.4%) compared to IHC2+ (36%). Within these Her2-SRM equivocal cases, the PPV was relatively high (57%) compared to IHC2+ (PPV 37.5%). Her2-SRM cases >750amol/ug demonstrated a high mean HER2/CEP17 ratio (9.28) and all were FISH amplified, compared to IHC3+ (4.16); mean ratio for Her2-SRM equivocal cases that were FISH positive was 3.04, compared to 5.15 in IHC2+/FISH+. Fewer equivocal cases requiring less reflex FISH testing along with better stratification of HER2/CEP17 ratio demonstrated superiority of Her2-SRM over IHC. (C) Depicting these same IHC, SRM and FISH results now sorted by IHC category demonstrated the wide range of SRM expression within each IHC category, particularly the large proportion of samples with very low SRM expression within IHC2+ and IHC3+ groups, potentially leading to better predictive capacity of Her2-SRM with respect to benefit from anti-HER2 therapy. Four cases were scored clinically as IHC2-3+ cases (represented with ‘*) (Supplementary Table 4), and all four had FISH ratio >2, with three cases having Her2-SRM >750 amol/ug and one case between 450-750 amol/ug; these cases were included in the IHC 2+ category given the pathologist’s uncertainty of scoring (ie equivocal) and requirement for reflex FISH.
Figure 6
Figure 6. (A). Molecular profiling and a call for treatment prioritization based on degree of genomic/proteomic aberration
A GEC patient diagnosed clinically as ‘HER2+#x2019; based on IHC (2+) and FISH, yet a more appropriate drug pairing was suggested by SRM multiplex testing (Fgfr2). Her2-SRM was observed to be <150 amol/ug (the range observed in all preclinical/clinical samples to date is <150-26170 amol/ug) while Fgfr2-SRM was observed to be 384 amol/ug (above the 95 percentile of documented Fgfr2 expression). NGS, next generation sequencing. (B) Serial Her2-SRM, Her3-SRM, and Egfr-SRM levels over time/treatment of a Her2 overexpressing and HER2 gene amplified esophageal tumor of a 39 year old man. Segment 1 to 2: First line cisplatin/irinotecan-trastuzumab 6 cycles (D1,D8 q 21 days) not well tolerated; RT to lumbar metastases; Second line FOLFIRI – trastuzumab 12 doses, SD but increasing tumor markers/bleeding; Segment 2 to 3: Embolization to bleeding primary tumor; Third Line –anti-PD1 antibody (PDL1+ IHC), 3 biweekly doses then PD (new multiple liver metastases); Segment 3 to 4: Fourth Line docetaxel-trastuzumab 10 cycles, then recurrent primary tumor bleeding but SD; Segment 4 to 5: RT 20Gy 1/2014-2/2014 to bleeding primary tumor. Fifth Line lapatinib/paclitaxel plus trastuzumab (15 biweekly cycles then PD on CT as well as PD clinically and by tumor markers); Segment 5 to 6: Sixth line FOLFOX-trastuzumab/pertuzumab – stable disease on CT with slight progression of tumor markers. Segment 6 to present: FOLFIRINOX-trastuzumab/pertuzumab for three cycles, response in tumors markers and PR on CT. Biopsy time points (all via EGD of primary tumor, except #6 – liver core biopsy): 1) 5/22/2012 2) 6/10/2013 3) 10/30/2013 4) 1/14/2014 5) 10/7/2014 6) 12/17/15. Diagnosed with symptoms 1/2012, ultimate tissue diagnosis 5/2012 with stage IV disease (bone, M1 lymph nodes) - now almost 36 months from onset of symptoms, 33 months from biopsy (as of 2/2015).

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