Patterns of HER2 expression and genomic correlates in lung cancer, with a focus on preanalytic variables impacting immunohistochemical staining results

Abstract

Aims:

Fam-trastuzumab deruxtecan-nxki (trastuzumab deruxtecan; T-DXd) was recently approved for advanced stage or metastatic solid tumors with HER2 immunohistochemical (IHC) 3+ staining. Data on standardized definitions of HER2 IHC testing and knowledge of genomic correlates in lung cancer are scarce. This study analyzes genomic characteristics of HER2 expressing tumors, and addresses issues with pre-analytic variables for lung cancer specimens.

Methods:

HER2 IHC staining was performed on selected archival cytology and surgical pathology lung cancer specimens for patients eligible for T-DXd therapy. Patient and tumor characteristics and next generation sequencing (NGS) data were correlated with HER2 IHC results.

Results:

166 patients with thoracic tumor samples had HER2 expression assessed: 46% were IHC 0, 28% were IHC 1+, 13% were IHC 2+, and 13% were IHC 3+. In both the entire tested cohort and for paired specimens, HER2 IHC scores were overall lower for cytology cell blocks as compared to surgical pathology specimens. Of specimens with HER2 IHC 3+ and NGS available, only 14% (3/21) had concomitant ERBB2 alterations. Among all specimens, ERBB2 point mutations were noted in 4% (4/110), and ERBB2 amplification in 3% (3/110). The majority of HER2 3+ cases with paired NGS (17/21, 81%) had non-ERBB2 genomic alterations, including: KRAS, TP53, and STK11 mutations.

Conclusions:

HER2 IHC 3+ is seen in a small but clinically significant proportion of samples, and is associated with a variety of co-occurring non-ERBB2 genomic alterations. Preanalytic variables including specimen fixation techniques can significantly impact assessment of HER2 expression via immunohistochemistry.

INTRODUCTION

Despite lung cancer remaining the leading cause of cancer-related deaths in the United States, significant progress has been made in recent years, with continued trends in declining mortality rates.¹ This progress is driven by rapid advancements in our understanding of the disease, including the identification of numerous biomarkers that provide critical information for selecting targeted therapies and improving patient outcomes. These biomarkers include gene mutations (such as EGFR, BRAF, KRAS, MET exon 14, and ERBB2/HER2 mutations) and gene rearrangements (ALK, ROS1, RET, and NTRK fusions), which are generally assessed using molecular techniques such as next generation sequencing (NGS).² Additionally, PD-L1 protein expression on tumor cells can be assessed using immunohistochemistry (IHC), the results of which direct the selection of immune checkpoint inhibitor therapy. The increasing complexity of these biomarkers underscores the crucial role of pathologists in accurately and efficiently analyzing tumor specimens to guide optimal treatment decisions.

On April 5, 2024, based on the results of the DESTINY trials,^3–6 the United States Food and Drug Administration (FDA) granted accelerated approval to the HER2 antibody-drug conjugate fam-trastuzumab deruxtecan-nxki (trastuzumab deruxtecan; TDX-d) for adult patients with unresectable or metastatic previously treated HER2-positive solid tumors without satisfactory alternative treatment options.⁷ In the DESTINY trials, HER2-positivity was defined as 3+ on immunohistochemical (IHC) staining of tumor cells, according to the 2016 HER2 Testing and Clinical Decision Making Guideline in Gastroesophageal Adenocarcinoma.⁸

Because HER2 IHC biomarker testing of lung cancer specimens has not routinely been performed prior to this approval by the FDA, our thoracic group sought to assess real-world patterns of HER2 IHC expression for archival patient samples based on eligibility for TDX-d therapy. These were then correlated with clinical, pathologic, and genomic alterations in this cohort of patients.

MATERIALS AND METHODS

Study cohort and specimen selection

Promptly following expanded approval for use of T-DXd in April 2024, patients with lung cancer being treated at the Beth Israel Deaconess Medical Center (BIMDC, Boston, Massachusetts, USA) were selected for HER2 IHC testing based on eligibility for T-DXd as determined by the treating thoracic oncologists (HLB, DR, HV, PCW). This patient list was evaluated by a thoracic pathologist (PVL), who reviewed the online medical records to select the most clinically appropriate specimen for HER2 IHC testing. The newest or most current specimen was chosen, provided there was sufficient tumor cellularity in the formalin-fixed, paraffin-embedded (FFPE) block. Surgical pathology resection or biopsy specimens were selected over a FFPE cytology cell block if there were concurrent specimens. When multiple tissue blocks were available, the one with the highest tumor cellularity was chosen. If insufficient tumor was present in the initially chosen block, then testing was performed on either a concurrent surgical pathology or cytology cell block or alternatively an older specimen as available. Acid decalcified specimens were avoided. For the cohort of tested specimens, patient demographics, tumor data, and comprehensive tumor molecular profiling results were also collected via retrospective chart review.

Specimen processing

Surgical pathology resection specimens (such as lung lobectomy or wedge resections, or excisions of metastases) and small biopsy specimens (including transthoracic core biopsies, transbronchial forceps or cryobiopsies, or pleural biopsy), were processed using standard histopathologic techniques: 10% neutral buffered formalin fixation and paraffin-embedding. Any surgical pathology specimen requiring decalcification was processed using ethylenediaminetetraacetic acid (EDTA) as the chelating agent. Cytology specimens including ultrasound-guided transbronchial needle aspirates (TBNA) and fine needle aspirates (FNA) were collected directly into a methanol-water fixative (CytoLyt, Hologic Corp., Marlborough, MA); upon receiving the specimen in the cytology division, this material was allocated for both a single ThinPrep slide as well as a cell block using either a plasma-thrombin or modified Histogel method prior to formalin-fixation and paraffin embedding.⁹ In contrast, cytology effusion specimens (pleural or pericardial) were received in the cytology laboratory fresh, with sequential 50mL aliquots centrifuged until a visible pellet was seen, at which time a ThinPrep slide was prepared along with a formalin-fixed, paraffin-embedded (FFPE) cell block.

HER2 IHC evaluation

HER2 IHC (Dako, HercepTest, Dako autostainer) was performed on freshly cut 5 μm unstained sections from archival formalin-fixed, paraffin-embedded (FFPE) tissue blocks and scored by a single thoracic pathologist (PVL) according to the 2016 HER2 testing guideline used in the DESTINY studies.⁸ Briefly, HER2 3+ was defined as strong, complete, basolateral or lateral membranous reactivity in ≥10% of tumor cells on a resection specimen, or in a tumor cell cluster (≥5 tumor cells) on a biopsy/cytology specimen. HER2 2+ defined as weak to moderate membranous reactivity, HER2 1+ as faint/barely perceptible membranous reactivity, and HER2 0 as no membranous reactivity; with similar surgical resection versus biopsy/cytology cutoffs as described for 3+ (Figure 1A). Only an IHC score of 3+ was considered positive; no reflex FISH testing was performed.

Figure 1.

Case breakdown by HER2 expression, tumor classification, and specimen type. A.) Representative examples of HER2 IHC expression in lung adenocarcinoma samples. B.) Distribution of HER2 expression status across all cases (n=166 total). C.) Distribution of thoracic tumor type stratified by HER2 IHC expression scores. D.) Distribution of HER2 IHC expression by tumor site (primary vs. metastasis).

Next generation sequencing and PD-L1 analysis

Comprehensive tumor molecular profiling consisted of next generation sequencing (NGS) using the FoundationOne CDx test and programmed cell death ligand (PD-L1) IHC (clone 22C3 pharmDx kit, Agilent Technologies, Santa Clara, CA), both performed by Foundation Medicine Inc. (Cambridge, MA). The database OncoKB (https://www.oncokb.org/) available through Memorial Sloan Kettering Cancer Center was utilized to interpret the effect of ERBB2 point mutations on protein function.

Statistical analysis

Statistical analysis was performed using Fisher’s exact test for all parameters examined with the exception of age (ANOVA) and tumor mutational burden (Kruskal-Wallis). When appropriate, the Benjamini-Hochberg method was used to obtain p-values adjusted for multiple comparisons. Significance level was set at p<0.05. This study was conducted under approval of the BIDMC Institutional Review Board.

RESULTS

HER2 IHC expression distribution across cohort

166 thoracic tumor samples had HER2 expression assessed via IHC, which was performed on a mixture of FFPE surgical pathology specimens (biopsy or resection) and FFPE cytology cell blocks (effusion, bronchoalveolar lavage, or fine needle aspiration specimens). Overall HER2 IHC scoring was as follows: 46% HER2 0, 28% HER2 1+, 13% HER2 2+, and 13% HER2 3+ (Table 1, Figure 1A–1B). No appreciable differences among HER2 IHC scoring groups were seen with respect to age, sex, smoking status, or race (Table 1). The most common tumor type tested was lung adenocarcinoma (118/166, 71%), and notably the HER2 3+ group was almost exclusively comprised of lung adenocarcinomas (19/21, 90%) (Figure 1C). There were no significant differences observed in tumor distribution site (primary lung vs. metastatic site) when stratifying by HER2 IHC score (Figure 1D, Supplemental Table 1).

Table 1.

Clinical and pathologic cohort characteristics by HER2 expression status. There were no statistically significant differences observed across any of the clinicopathologic variables when grouping by HER2 score.

Characteristics	HER2 0 (n=77)	HER2 1+ (n=47)	HER2 2+ (n=21)	HER2 3+ (n=21)
Age, years
Average (range)	66.8 (33–91)	68.4 (40–86)	63.6 (32–82)	67.2 (52–82)
Sex
Male	30 (39.0%)	23 (48.9%)	9 (42.9%)	8 (38.1%)
Female	47 (61.0%)	24 (51.1%)	12 (57.1%)	13 (61.9%)
Smoker	59 (76.6%)	35 (74.5%)	17 (81.0%)	18 (85.7%)
Race (self-reported)
White	43 (55.8%)	31 (66.0%)	14 (66.7%)	14 (66.7%)
African-American	6 (7.8%)	6 (12.8%)	2 (9.5%)	5 (23.8%)
Asian	20 (26.0%)	6 (12.8%)	4 (19.0%)	1 (4.8%)
Hispanic	1 (1.3%)	1 (2.1%)	0	0
NA/Other	7 (9.1%)	3 (6.4%)	1 (4.8%)	1 (4.8%)
Mean TMB (muts/Mb)	10.6 (n=58)	7.7 (n=37)	6.7 (n=18)	11.3 (n=20)
TMB low (<10 muts/Mb)	38 (65.5%)	26 (70.3%)	12 (66.7%)	12 (60.0%)
TMB high (≥10 muts/Mb)	20 (34.5%)	11 (29.7%)	6 (33.3%)	8 (40.0%)

Impact of specimen type on HER2 IHC expression

Previous studies have demonstrated that pre-analytic specimen processing variables can impact select IHC results, thus we next analyzed HER2 IHC expression stratified by cytologic and surgical specimen type. We observed that the cytologic specimens had a significantly larger overall proportion of HER2 0 expression (74%, n=35/47) compared to the surgical pathology specimen cohort (35%, n=42/119) (p<0.0001, Figure 2A). Next, we further broke down cytology specimens by type (TBNA/FNA, fluid, and BAL) and surgical specimens by type (resection and biopsy) and observed a higher proportion of biopsy specimens in the HER2 positive specimens (Figure 2B). When grouping by testing substrate for HER2 IHC expression (cytology vs. surgical specimen), we observed that overall, the cytology specimens had a higher proportion of metastases tested compared to primary tumors when compared to surgical specimens (70% vs. 45% respectively, p<0.0001, Supplemental Figure 1A, Supplemental Table 1). We next compared HER2 IHC expression based on testing substrate (cytology or surgical specimen) and sampling site (primary lung vs. metastatic site). We observed no differences in HER2 score breakdown when comparing the cytology primary vs. cytology metastasis group or the surgical primary vs. surgical metastasis group (p=0.67 and p=0.74, respectively). However, when comparing the cytology primary vs. surgical primary groups and the cytology metastasis vs. surgical metastasis groups, we noted a significantly higher frequency of HER2 0 cases in both cytology groups (p=0.01 and p<0.01, respectively; Supplemental Figure 1B).

Figure 2.

Comparison of HER2 IHC expression between cytology and surgical specimens. A.) Distribution of cytology vs. surgical specimens by HER2 IHC score. B.) Breakdown of specimen type by HER2 score. C.) Plot demonstrating the change in cytology specimen HER2 score (y-axis) vs. original surgical specimen score (x-axis). D.) Representative images of paired surgical and cytology specimen from the same patient and same anatomic location demonstrating a decrease in HER2 score from 2+ to 0 between the surgical and cytology specimen, respectively.

Given our observation that the cytology specimens tended to have lower HER2 IHC scores compared to surgical specimens, we sought to investigate this further. Therefore, we next identified patients within our study that had paired surgical pathology and cytology specimens available for testing (n=24). The criteria for paired specimens were those taken from the same anatomic site during either the exact same procedure (n=20) or separate procedures less than 2 months from each other with no intervening treatment (n=4). Of the 24 paired specimen cases, ten were HER2 0, five were HER2 1+, four were HER2 2+, and five were HER2 3+. All surgical specimens that were scored as HER2 0, remained unchanged at 0 in their paired cytology counterpart. Of the remaining 14 cases that scored 1+, 2+ or 3+, 11 cases (79%) had a decrease in their HER2 IHC score from their surgical specimen to their paired cytology specimen (Figure 2C and 2D). These striking findings raise concern about the impact of pre-analytic variables (namely, differences in specimen fixation solutions) and their consequences on assessing HER2 IHC expression.

ERBB2 alterations by HER2 expression

We next analyzed the cohort of HER2 IHC lung adenocarcinoma cases with available NGS results (110/118, 93%). Overall, the frequency of ERBB2 alterations (amplifications and point mutations) in this cohort was low at 6% (7/110, Figure 3A). In the HER2 0 group, the frequency of ERBB2 alterations was 9% (4/43), 0% in the HER2 1+ and 2+ groups, and 16% (3/19) in the HER2 3+ group. In the HER2 0 group, ERBB2 amplification was present in 1 tumor and ERBB2 point mutations were found in 3 others. In the HER2 3+ group, there were 2 tumors with ERBB2 amplification and 1 with an ERBB2 point mutation (Figure 3B–3D). Interestingly, of the three tumors amplified by NGS, the HER2 0 was a cytology specimen. The other two NGS-amplified tumors, which both scored HER2 3+, were surgical specimens. All point mutations identified had been previously classified as known oncogenic, gain of function mutations through OncoKB.

Figure 3.

ERBB2 alteration analysis. A.) Flowchart detailing workflow. B.) Frequency of ERBB2 alteration within the cohort stratified by HER2 expression demonstrates 9% of HER2 0 cases (n=4/43) and 16% of HER2 3+ cases (n=3/19) had ERBB2 alterations. No ERBB2 alterations were observed in the HER2 1+ or HER2 2+ groups. C.) ERBB2 alterations organized by type, surgical vs cytologic, and primary tumor vs. metastasis. D.) ERBB2 alteration details to include protein domain location and predicted effect on protein function.

Next generation sequencing of lung adenocarcinomas by HER2 expression

Finally, we compared the frequency of non-ERBB2 alterations by HER2 IHC expression among 10 genes commonly associated with lung cancer pathogenesis: TP53, KRAS, STK11, EGFR, CDKN2A/B, ALK, BRAF, MET, RET, and ROS1. Of these, alterations in KRAS, STK11, and TP53 mutations were most common, with a higher frequency of STK11 alterations in the HER2 3+ group (p=0.01, adjusted p=0.08 after Benjamini-Hochberg correction) (Figure 4A and 4B). PD-L1 TPS, tumor mutational burden (TMB), and smoking history across all available lung adenocarcinoma cases were also analyzed and showed no demonstrable differences across the HER2 IHC cohorts (Table 1, Figure 4B).

Figure 4.

Molecular analysis of lung adenocarcinoma cases stratified by HER2 expression status. A.) Mutation frequency by HER2 expression status for KRAS, EGFR, TP53, STK11, CDKN2A/B, and ALK demonstrates an increased frequency of STK11 alterations in the HER2 3+ group (p=0.01, adjusted p=0.08 after Benjamini-Hochberg correction). B.) Co-mutational profile stratified by HER2 IHC score across 11 different genes, PD-L1 TPS (<1, 1–49, or ≥50%), and smoking status (non-smoker or <100 cigarettes in lifetime vs. smoker). ERBB2 amplification denoted with red bar coloration. Boxes with an “X” denote results are unavailable.

DISCUSSION

To our knowledge, our study is one of the first to describe real-world applications of HER2 IHC testing in lung cancer specimens in the wake of the disease-agnostic approval of T-DXd for advanced and refractory solid tumors with HER2 3+ IHC.⁷ Rates of HER2 overexpression (IHC 2+ and 3+) were similar in our dataset (26%) compared to other prior studies (18.5–23%), though generally higher IHC 3+ was seen in our cohort (12.9%) as compared to others (1.7–4%).^8,9 Our real-world cohort also demonstrated a similar rate of ERBB2 genomic alterations (6%) as has been previously reported by others.¹⁰

Previously published data exploring the concordance between HER2 protein expression status and ERBB2 mutation status in lung cancers is low.¹¹ In our cohort, most ERBB2 alterations (4/7) were seen in the HER2 IHC 0 cohort (Figure 3). High-level ERBB2 amplification and HER2 overexpression by IHC appear more congruent, and our results are compatible with those reported by others.¹² Thus, HER2 overexpression as measured by IHC should not be conflated with ERBB2 amplification or mutation status. Our findings suggest that while there is a subset of tumors where HER2 protein overexpression is concordant with altered ERBB2 biology at the genomic level, there is an equally, perhaps more significant cohort where the mechanism and implications of HER2 overexpression are less clear. Perhaps even more strikingly, concomitant alterations in driver oncogenes other than ERBB2 were the most frequent genetic events in HER2 3+ tumors (Figure 3). The clinical and biological relevance of actionable driver oncogene events (KRAS, EGFR, ROS1, BRAF) in HER2 3+ expressing tumors and their impact on efficacy of HER2-directed therapies is as yet unknown, and will be important to understand given the high prevalence of these alterations in our HER2 3+ cohort and the often multiple lines of oncogene-directed targeted therapies available to some of these cohorts (e.g., EGFR-mutated NSCLC).

Rigorous standards and definitions for assessing HER2 overexpression (IHC and FISH) and its clinical implications have been best characterized in breast cancer. For these specimens, HER2 FISH has been routinely used to assess HER2-equivocal (2+ by IHC) cases.¹³ Such standards have not yet been established in most other solid tumors. Since the expanded, disease-agnostic FDA approval for T-DXd relies solely on HER2 overexpression as defined by IHC, particular care must be paid to pre-analytical specimen variables such as cold ischemic and formalin fixation times, which could impact this quantitative biomarker test. In contrast to the standard formalin fixation that surgical pathology specimens undergo, cytology cell blocks may have an alcohol pre-fixation step (as is the case with cell blocks in our laboratory), which has been shown to impact HER2 protein detection via IHC.^14,15

Our study highlights the potential influence of preanalytical variables on assessing HER2 IHC expression. In general, an actionable HER2 IHC score of 3+ was most frequently obtained from surgical pathology small biopsy specimens (Figure 2). This may relate to the scoring methodology mandated by this drug approval; small biopsy specimens only need strong, complete, basolateral or lateral membranous reactivity in a single tumor cell cluster (≥5 tumor cells), as compared to a potentially higher threshold of ≥10% of all tumor cells on a resection specimen.⁸ It remains to be seen in other cohorts if small biopsies are more frequently scored as a HER2 3+ compared to larger resection specimens, which would have implications for downstream patient eligibility for T-DXd therapy.

Beyond the size or amount of tissue obtained, the type of specimen tested may also impact HER2 IHC scoring. In our initial cohort of 47 cytology cell block specimens and 119 surgical pathology specimens, HER2 IHC scores were overall lower for cytology compared to surgical pathology specimens (Figure 2). Although the cytology cell block group had a higher proportion of metastases sampled compared to the surgical pathology group (70.2% vs. 45.4% respectively, p<0.0001, Supplemental Figure 1A), it does not appear that HER2 IHC expression of metastasis vs. primary site alone can explain the observed difference of cytology cell block vs surgical pathology specimen testing (Supplemental Figure 1B). Although HER2 expression by tumor cells may differ between the primary site and a metastasis, a previous study of metastatic endometrial cancer specimens demonstrated both higher or lower expression of HER2 as compared to the primary tumor without a clearly delineated pattern in one direction.¹⁶

Perhaps the strongest explanation for the lower observed HER2 IHC scores for cytology cell block specimens as compared to surgical pathology specimens in this cohort is secondary to the slightly divergent tissue processing itself. As gleaned from subsequent analysis of paired surgical pathology and cytology cell block specimens (same patient, same anatomic site), 79% of surgical pathology specimens that scored HER2 positive (1+, 2+, or 3+) had a decrease in their paired cytology specimen HER2 IHC score. These results raise concern that the differences in processing between surgical pathology and cytology cellblock specimens can result in discordant HER2 IHC expression, even though both are FFPE specimens. For patients undergoing consideration for T-DXd treatment, which currently requires a HER2 IHC score of 3+, falsely low or negative results can have a significant clinical impact through rendering them ineligible for T-DXd therapy.

The methanol-based buffered preservative solution Cytolyt¹⁷ used in our laboratory as a pre-fixative prior to FFPE cell block processing has previously been shown to negatively impact the staining characteristics of different antibodies, such as for c-KIT in gastrointestinal stromal tumors,¹⁸ the MIB-1 clone of the proliferation marker Ki-67,¹⁹ the neuroendocrine marker insulinoma-associated protein 1 (INSM-1),²⁰ and 8G7G3/1 clone of TTF-1 and the MOC-31 clone of EPCAM.²¹ Previous studies have compared the differences in HER2 expression assessed via IHC in unpaired specimens following alcohol-based fixation compared to direct formalin fixation. Two studies found that cytology cell block breast cancer samples initially fixed in 50% ethanol followed by fixation in 10% neutral-buffered formalin (NBF) compared to surgical pathology breast cancer specimens fixed directly in 10% NBF resulted in higher rates of HER2 3+ staining in cytology specimens.^15,22 On the other hand, studies using human breast cancer cell lines comparing 10% NBF fixation to Saccomanno fixative (50% mixture of ethyl, methyl, and isopropyl alcohols; and 2% polyethylene glycol) found that HER2 protein expression assessed via IHC was significantly lower in Saccomanno fixed cells compared to 10% NBF fixed cells, which became more pronounced the longer the specimens were fixed.¹⁴ Therefore, the modality of tissue fixation (i.e. protein precipitation via alcohol fixation versus protein crosslinking via NBF) may impact protein antigenicity and subsequent detection via IHC, which is especially relevant for IHC biomarkers requiring semiquantitative assessment, like HER2. As such, recent guidelines from the College of American Pathologist have emphasized the importance of assay validation for IHC tests: not only using tissues with the same fixation and processing as those that would be tested clinically, but also the need for separate assay validation specifically for cytology specimens that undergo different fixation, such as samples collected in an alcohol-based fixative that then undergo postfixation in NBF.²³

Concerns regarding optimization of preanalytic variables not only impact the cytopathology and immunohistochemistry laboratories, but also the proceduralist at the time of collection. Acquisition of cytology samples in some instances can involve less invasive and more logistically accessible procedures, such as pleural drainage via thoracentesis versus pleural biopsies. In other circumstances acquiring small biopsy specimens in addition to TBNA/FNA specimens requires not only procedural expertise, but also additional sampling equipment and possibly prolonged procedure time (e.g. endobronchial ultrasound TBNA vs. endobronchial ultrasound transbronchial forceps or cryobiopsy). Subjecting patients to procedures that may be less logistically accessible, longer, more invasive, or more costly, may be difficult to rationalize without clear knowledge of the potential downstream benefit of acquiring tissue biopsy specimens for histopathology processing as opposed to cytology specimens alone. Furthermore, in the ever-increasing landscape of biomarkers to guide lung cancer care, the specimen type mandated may be subject to convention and/or unnecessary bias of landmark study methods rather than related to biologic or pathologic necessity. Best practices that are truly necessary therefore require constant reassessment by multi-disciplinary teams including proceduralists as more effective treatments become available. Regarding the findings in our study, much of the benefit to patients for more optimal HER2 assessment via IHC could be attained through submitting TBNA/FNA specimens directly in formalin in addition to Cytolyt, rather than obligatory acquisition of small biopsy specimens. Ultimately, specimen collection and processing entails striking a balance between obtaining optimal cellularity and tumor morphology for diagnosis, proper specimen processing for various ancillary testing techniques including both immunohistochemistry and molecular testing, procedural time and equipment availability, and patient safety during the biopsy procedure.

In summary, our cohort highlights that testing for HER2 IHC on lung cancer specimens is feasible and demonstrates a small but relevant subset of tumors with HER2 IHC 3+, permitting use of T-DXd for advanced disease following prior systemic regimens. Strikingly, there is little concordance between tumors with HER2 overexpression and altered ERBB2 biology at a genetic level, and a significant proportion of samples demonstrated both HER2 IHC 3+ and concomitant canonical oncogenic driver events, the biology behind and clinical ramifications of which are yet poorly understood. Clinical and biological correlations between HER2 overexpression, ERBB2 and non-ERBB2 genetic alterations, and their impact on efficacy of HER2-directed therapies remains largely unknown. Finally, accurate assessment of HER2 overexpression via IHC can be impacted by pre-analytic variables, specifically the type of fixation method used as highlighted in this study. Attention to these pre-analytical variables and proper testing substrate selection (such as testing the primary versus metastatic site) will need further investigation to best identify patients for T-DXd therapy if based solely on HER2 3+ IHC. Implications in disease-specific settings as it relates to tumor and spatial heterogeneity must also be explored. Lung cancer remains a forerunner in the use of molecularly-defined biomarkers to optimally and rationally select best regimens for individual patients and cancer subtypes, and along these lines maximal leverage of this newly approved HER2-directed therapeutic strategy will require careful HER2 IHC testing, with proper control of and consideration for preanalytic variables in all relevant circumstances.

Supplementary Material

Supplementary Figure and Table

Supplemental Table 1. Statistical analysis of associations between clinical/demographic variables and HER2 status (0, 1+, 2+, 3+) across all 166 samples.

Supplemental Table 2. Statistical analysis of associations between genomic alterations/smoking/TPS/TMB and HER2 positivity (3+ vs 0, 1+, 2+) in 110 lung adenocarcinoma samples.

Supplemental Figure 1. Comparison of cytology and surgical specimens within the HER2 IHC testing cohort. A.) Breakdown of cytology and surgical testing substrates by whether they were collected from the primary tumor or a metastasis shows an increase of metastatic site specimens within the cytology cohort compared to the surgical cohort (n=33/47, 70% vs. n=54/119, 45%, p<0.0001). B.) HER2 IHC expression score frequency stratified by both cytology vs. surgical and primary vs. metastasis. When comparing HER2 IHC scoring between the cytology primary vs. cytology metastasis and surgical primary vs. surgical metastasis group, no significant differences were observed (p=0.67 and p=0.74 respectively). When comparing across cytology primary vs. surgical primary and cytology metastasis vs. surgical metastasis, however, the distribution of HER2 scores was significantly different, with a larger proportion of HER2 0 cases in the cytology groups (86% vs. 37%, p=0.01 and 70% vs. 33%, p=0.005, respectively)

Summary Box.

What is already known on this topic

• HER2 immunohistochemical testing has regularly been performed on breast and gastric cancer specimens; however, data on HER2 testing and knowledge of genomic correlates in lung cancer are scarce.

What this study adds

• HER2 IHC 3+ is seen in a small but clinically significant proportion of lung cancer samples, and is associated with a variety of co-occurring non-ERBB2 genomic alterations.

• Preanalytic variables including specimen fixation techniques can significantly impact assessment of HER2 expression via immunohistochemistry.

How this study might affect research, practice, or policy

• For lung cancer patients who may be eligible for treatment with trastuzumab deruxtecan, HER2 assessment via immunohistochemistry should be performed on formalin-fixed, paraffin embedded specimens.