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Diagnostic and Predictive Immunohistochemistry for Non–Small Cell Lung Carcinomas

Hung, Yin P., MD, PhD; Sholl, Lynette M., MD

doi: 10.1097/PAP.0000000000000206
Review Articles

Non–small cell lung carcinoma (NSCLC) accounts for significant morbidity and mortality worldwide, with most patients diagnosed at advanced stages and managed increasingly with targeted therapies and immunotherapy. In this review, we discuss diagnostic and predictive immunohistochemical markers in NSCLC, one of the most common tumors encountered in surgical pathology. We highlight 2 emerging diagnostic markers: nuclear protein in testis (NUT) for NUT carcinoma; SMARCA4 for SMARCA4-deficient thoracic tumors. Given their highly aggressive behavior, proper recognition facilitates optimal management. For patients with advanced NSCLCs, we discuss the utility and limitations of immunohistochemistry (IHC) for the “must-test” predictive biomarkers: anaplastic lymphoma kinase, ROS1, programmed cell death protein 1, and epidermal growth factor receptor. IHC using mutant-specific BRAF V600E, RET, pan-TRK, and LKB1 antibodies can be orthogonal tools for screening or confirmation of molecular events. ERBB2 and MET alterations include both activating mutations and gene amplifications, detection of which relies on molecular methods with a minimal role for IHC in NSCLC. IHC sits at the intersection of an integrated surgical pathology and molecular diagnostic practice, serves as a powerful functional surrogate for molecular testing, and is an indispensable tool of precision medicine in the care of lung cancer patients.

Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA

Present address: Yin P. Hung, MD, PhD, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.

L.M.S. discloses a consulting relationship with Gfk group. Y.P.H. has no funding or conflicts of interest to disclose.

Reprints: Lynette M. Sholl, MD, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115 (e-mail:

All figures can be viewed online in colour at

Non–small cell lung carcinoma (NSCLC) accounts for significant morbidity and mortality worldwide, with most patients diagnosed at advanced stages and managed with systemic therapy and, increasingly, targeted therapy and immunotherapy.1 Approximately 60% of lung adenocarcinomas harbor oncogenic driver alterations, including in KRAS, epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS1, BRAF, RET, MET, ERBB2, and others,2,3 most of which occur in a mutually exclusive pattern.4–6 Lung squamous cell carcinomas generally lack these alterations and instead have a high proportion of TP53 mutations and frequent PI3K pathway activation.7,8 The 2018 clinical testing guideline from the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) defined EGFR, ALK, and ROS1 as the “must-test” biomarkers for advanced lung adenocarcinoma; given established and emerging clinical trial data, an expanded panel should also at a minimum include BRAF, MET, RET, ERBB2, and KRAS.9 Programmed cell death protein 1 (PD-1/PD-L1) immunohistochemistry (IHC) is the dominant predictive biomarker in guiding advanced NSCLC patients to immunotherapy. This large number of diverse clinically relevant targets present a logistical challenge, both in terms of methodology and tissue allocation. With increasing use of minimally invasive techniques in specimen procurement, the available tumor tissue can be scant and, in ~25% of cases, insufficient for molecular testing.10 For optimal management of patients with NSCLC, one of the most common tumors encountered in surgical pathology, effective use of IHC for diagnosis and treatment prediction is crucial.

This review highlights the role for IHC in diagnosis and therapeutic prediction in NSCLC, with an emphasis on molecular correlates. We highlight 2 recently described and possibly underrecognized entities, nuclear protein in testis (NUT) carcinoma and SMARCA4-deficient thoracic tumors, the proper recognition of which facilitates optimal management given their highly aggressive behavior. We review the role of IHC for obligate and emerging molecular biomarkers in lung adenocarcinoma, including EGFR, ALK, ROS1, BRAF, pan-TRK, LKB1, ERBB2/HER2, MET, and PD-L1 (Table 1).



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Diagnostic IHC should be performed judiciously and always in a manner informed by the patient’s the clinical status. It is important to recognize that for some patients with a known diagnosis of advanced NSCLC, a tumor biopsy will be performed to confirm recurrence and evaluate for resistance mechanisms to targeted therapy, such as EGFR T790M mutation or small cell transformation in EGFR-mutant lung adenocarcinoma following treatment with tyrosine kinase inhibitors (TKI).11,12 Diagnostic immunostains may be of marginal value in this situation. Instead, tissue conservation for biomarkers studies is paramount.

At the time of primary diagnosis for a patient with suspected lung cancer, a biopsy showing a NSCLC with no apparent squamous or glandular differentiation should be further evaluated with a focused panel consisting of TTF-1 and p40 (or p63) for adenocarcinoma and squamous cell carcinoma differentiation, respectively.13–15 Of note, multiple commercially available TTF-1 antibody clones differ in their characteristics: 8G7G3/1 (Dako, Carpinteria, CA) is the most specific for primary lung adenocarcinoma, followed by SPT24 (Leica, Newcastle upon Tyne, UK), while SP141 (Ventana, Tucson, AZ) is most sensitive but least specific and often labels bronchial basal epithelial cells.16–20 The p63 antibody clone 4A4 (Abcam, Cambridge, MA) and p40 antibody 5-17 (CalBiochem, Nottingham, UK) are commonly used for squamous cell carcinoma, with p40 being more specific but less sensitive.21–23 If first-round IHC is uninformative (eg, pan-negative or equivocal expression, or only focal p63 staining), one can use second-line markers such as mucicarmine stain and IHC for Napsin-A (for adenocarcinoma) and CK5 (for squamous cell carcinoma). The lung is a common site for metastatic disease. Thus in the appropriate context and especially when the expected primary lung markers are negative, the pathologist should exclude metastases with relevant transcription factor, keratin, and other cytoplasmic protein markers.

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Nuclear Protein in Testis

NUT carcinoma is considered an aggressive type of squamous cell carcinoma defined by rearrangement of NUT, with fusion to BRD4 in most cases,24,25 rarely to BRD3, NSD3,26,27 and a variety of other partners. NUT carcinoma was initially described in midline head and neck and mediastinum in primarily young patients;28 however, both anatomic distribution and age of onset are diverse.29–31 NUT carcinoma is highly aggressive, with a median survival of <5 to 7 months.31,32 Investigational trials using bromodomain inhibitors are ongoing.33,34 Given its distinct clinicopathologic features, NUT carcinoma has been recognized as a separate entity in the 2015 WHO classification of thoracic malignancies.1 In particular, a “poorly differentiated squamous cell carcinoma” in a young nonsmoker should prompt a consideration of NUT carcinoma. Overall, NUT carcinomas are identified in 0.6% to 3.5% of poorly differentiated or previously unclassified thoracic malignancies.30 Histologically, NUT carcinoma is characterized by monomorphic primitive-appearing, basaloid, epithelioid to ovoid cells, with vesicular nuclei and variably prominent nucleoli (Fig. 1A); abrupt keratinization can be a helpful clue, albeit present in only a subset of cases.28 Differential diagnosis of NUT carcinoma includes other carcinomas, melanomas, and round-cell sarcomas. Immunophenotypically, NUT carcinomas show variable expression of keratin, p63, p40, TTF-1, CD34, and CD99,28,30,32,35 compounding diagnostic challenge. Given its rarity, histologic overlap with other more common tumors, and its indistinct immunophenotype, prospective identification of NUT carcinoma can be challenging. However, use of IHC for NUT protein can readily include or exclude the diagnosis. The anti-NUT antibody clone C52B1 (Cell Signaling Technology, Danvers, MA) has reported 87% sensitivity and 100% specificity for NUT carcinoma relative to fluorescence in situ hybridization (FISH) testing; tumor cells show characteristic speckled nuclear immunoreactivity (Fig. 1B).36 NUT IHC positivity is defined as nuclear expression in >50% of tumor cells, and histologic mimics such as germ cell tumors (including >60% of dysgerminoma) may show focal weak NUT expression.36 FISH for NUT rearrangement may be informative in cases where NUT carcinoma is strongly suspected but IHC is negative or equivocal.



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Mutations in SMARCA4, a member of the SWI/SNF chromatin remodeling complex, are present in diverse human tumors,37,38 including ~6% of lung adenocarcinomas2,39 and rare lung squamous cell carcinoma.7 Germline and somatic mutations in SMARCA4 are present in >95% of small cell carcinoma of ovary, hypercalcemic type,40–42 along with a subset of rhabdoid tumors43,44 and epithelioid sarcoma with retained SMARCB1 expression,45 and a subset of undifferentiated to poorly differentiated endometrial,46 gastrointestinal,47 urothelial,48 and sinonasal tumors.49 Loss of SMARCA4 expression correlates with histologic dedifferentiation in endometrioid carcinomas50 and is a diagnostic IHC finding in small cell carcinomas of ovary, hypercalcemic type.51

SMARCA4-deficient thoracic tumors (SMARCA4-deficient thoracic sarcoma and SMARCA4-deficient lung adenocarcinoma) were recently described.52–58 Initially reported in 19 patients with large aggressive mediastinal tumors,54 SMARCA4-deficient thoracic sarcomas show a predominance for male smokers with a wide age distribution, but median age of 30 to 40 years.55,56 Cases presenting as small (<1.5 cm) primary lung tumors56 or in never-smokers54–56 have also been noted. SMARCA4-deficient thoracic sarcoma is genetically characterized by SMARCA4 inactivating mutation, with a transcriptional profile similar to small cell carcinomas of ovary, hypercalcemic type.54 Histologically, SMARCA4-deficient thoracic sarcoma displays discohesive round-to-ovoid cells with abundant eosinophilic cytoplasm and often rhabdoid morphology (Fig. 1C),54–56 features that overlap with SMARCB1-deficient tumors.59 Loss of SMARCA4 expression appears enriched in thoracic tumors with rhabdoid morphology, present in up to 30% in one series.56 Differential diagnosis includes poorly differentiated carcinomas, melanomas, sarcomas, and germ cell tumors. Immunophenotypically, SMARCA4-deficient thoracic sarcoma is characterized by complete loss of SMARCA4 using clones EPNCIR111A (Abcam) or EPR3912 (Abcam) (Fig. 1D), along with SOX2 overexpression and SMARCA2 loss, variable immunoreactivity for keratin, CD34, and SALL4, but lacking NUT, S100, and claudin-4.54–56 SMARCA4-deficient thoracic sarcomas are highly aggressive, with a median survival of 4 to 7 months,54–56 although rare cases with survival up to 9 years have been documented.56 SMARCA4 loss has also been described in ~2% of NSCLC.52,53,57,58 SMARCA4-deficient lung adenocarcinomas, in contrast, may show differentiated histology with conspicuous gland formation, can express CK7 and HepPar-1, but generally lack TTF-1.57 Claudin-4, a gap junction-associated protein, has been suggested as an IHC marker to distinguish between SMARCA4-deficient sarcomas and carcinomas;60 however, claudin-4 loss has also been observed in dedifferentiated lung adenocarcinoma.55 Definitive classification of SMARCA4-deficient thoracic tumors (as sarcomas or carcinomas) thus remain challenging and may be clinically relevant in light of clinical evidence that SMARCA4 loss predicts increased sensitivity to platinum-based chemotherapy in NSCLC61 and preclinical data suggesting activity of EZH2 inhibitors in tumors with BAF-deficiency dependency.62

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Anaplastic Lymphoma Kinase

Since its initial description in anaplastic large cell lymphoma,63 rearrangement of ALK has been described in a subset of diverse epithelial, mesothelial, and mesenchymal neoplasms.64–66 ALK rearrangement is present in ~5% of lung adenocarcinomas, most commonly as EML4-ALK fusion67 and rarely fusions to other partners (KIF5B, TFG, HIP1, KLC1, GCC2, LMO7, and PHACTR1).68–70 In NSCLC, ALK rearrangement is enriched in young nonsmokers and occurs almost exclusively in adenocarcinomas,71,72 often with solid or annular pattern and signet-ring cells.73–76 In the United States, ALK inhibitors crizotinib and alectinib have been approved for first line therapy;75,77–79 a variety of other newer generation ALK inhibitors have shown efficacy including in patients with acquired resistance to first line ALK inhibitors.80 Currently, ALK-targeted therapy is the standard of care in patients with ALK-rearranged lung carcinomas. Given the clinical efficacy of ALK-targeted therapy and the imperfect correlation between ALK rearrangement status and clinicopathologic features, ALK testing is recommended in all patients with advanced lung adenocarcinomas, regardless of age or smoking status.9,81

ALK rearrangements may be detected using break-apart FISH, molecular or sequencing methods, or by IHC. IHC antibody clones historically suitable for diagnosis of anaplastic large cell lymphoma (eg, ALK1) are insufficiently sensitive in NSCLC;82 however, the newer clones D5F3 (Ventana) and 5A4 (Leica) are ≥95% sensitive and specific (Fig. 2A) relative to ALK FISH.9,83,84 Given the accumulation of data supporting the excellent clinical performance characteristics of well-validated ALK IHC, the molecular testing guidelines have evolved from recommending ALK IHC only as a screening tool requiring FISH confirmation81 to a standalone assay for use in selecting lung cancer patients for ALK-targeted therapy.9 Indeed, some studies suggest that ALK IHC better predicts clinical response to crizotinib than does ALK FISH.85 Furthermore, ALK IHC can be used in limited specimens that may be insufficient for FISH scoring. The 5′ fusion partner affects the subcellular localization of the aberrant protein and thus may be inferred from the pattern of ALK IHC staining.70 Finally, ALK IHC can be an orthogonal tool to confirm bona fide ALK alterations in unexpected scenarios, for instance in rare tumors with concomitant drivers such as EGFR mutation.86



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Initially described in a glioblastoma cell line,87 ROS1 rearrangements are present in up to 3% of lung adenocarcinomas,68,88–93 with ROS1 fusions to diverse 5′ partners, such as CD74, TPM3, SDC4, SLC34A2, EZR, LRIG3, and FIG.68,69,89,94 Similar to NSCLC with ALK rearrangements, ROS1-rearranged NSCLC are enriched in young nonsmokers and are exclusively adenocarcinomas,88,91,93,95 characterized by solid, mucinous cribriform, or papillary architecture with signet-ring cells or variable psammomatous calcifications.90,92,96,97 Similar to the treatment of ALK-rearranged NSCLC, crizotinib is efficacious in patients with ROS1-rearranged NSCLC.98 Currently, ROS1 is considered one of “must-test” biomarkers, along with ALK and EGFR, in patients with advanced lung adenocarcinomas regardless of demographics.9

Although ROS1 FISH testing is the most common approach to detection of ROS1 rearrangement,88 molecular and/or IHC approaches may be used. The ROS1 antibody clone D4D6 (Cell Signaling) is highly sensitive (>95% relative to other techniques; Fig. 2B), but immunoreactivity may be diminished in ROS1-rearranged cases with suboptimal tissue fixation.9,89,90,96,97,99–102 As with ALK, the 5′ fusion partner may be inferred from the pattern of staining, with globular staining in CD74-ROS1 tumors or membranous accentuation in EZR-ROS1 tumors.97 The specificity of ROS1 IHC, however, varies among studies depending on the analytic variables and scoring criteria,9,89,90,96,97,99–102 with one study noting up to 8-fold ROS1 IHC-positive cases relative to ROS1 FISH-positive cases.96 The suboptimal specificity of ROS1 IHC results from occasional staining in macrophages, reactive pneumocytes, and low level expression in non ROS1-rearranged tumors.90,96,97,101–103 ROS1 IHC is currently recommended as a screening test in patients with advanced lung adenocarcinomas, with positive results confirmed by a molecular or cytogenetic method. Given the high negative predictive value, a negative ROS1 IHC result is considered reliable to exclude ROS1 rearrangement,9 and ROS1 IHC can also serve as a useful orthogonal tool to validate findings by FISH or molecular methods.

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Programmed Cell Death Protein 1

Immune checkpoint blockade of the PD-1/PD-L1 pathway104 is now an essential part of the armamentarium in oncology. As of spring 2018, the following immune checkpoint inhibitors (against their cognate targets) have been approved for the treatment of advanced NSCLC: nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), and durvalumab (PD-L1). Since these drugs can lead to immune-related adverse events and confer benefit to only a subset of patients, biomarker testing to optimize therapeutic response is crucial, particularly in the setting of first line monotherapy.105 In addition to PD-L1 IHC, efficacy of checkpoint blockade correlates with genomic driver status, mutational burden, smoking signature, neoantigen burden, and mismatch repair status.106–110

Although an imperfect biomarker for predicting immunotherapy response, PD-L1 IHC has been integrated in clinical trials and routine practice. The PD-L1 tumor proportion score (TPS; percentage of tumor cells with membranous PD-L1 immunoreactivity) cutoff of ≥50% was derived from a phase 1 study of pembrolizumab in advanced NSCLC.111 In treatment-naive patients with advanced NSCLC (both squamous and nonsquamous) and PD-L1 TPS≥50%, pembrolizumab monotherapy is superior to chemotherapy with improvement in both progression-free survival and 12-month survival.112 In treatment-naive patients with advanced nonsquamous NSCLC, combined pembrolizumab and platinum chemotherapy shows better overall survival than chemotherapy alone, irrespective of PD-L1 score, albeit greatest when TPS≥50%.113 Another PD-1 inhibitor, nivolumab, shows no survival benefit over chemotherapy in treatment-naive patients with advanced NSCLC,110 but is superior to docetaxel in both squamous and nonsquamous NSCLC in previously treated patients,114,115 a subset of which demonstrates sustained response.116 Second-line atezolizumab is also superior to docetaxel in this setting.117 Second-line pembrolizumab also shows survival benefits over docetaxel for advanced NSCLC in the context of PD-L1 TPS of ≥1%.118 Of note, first line pembrolizumab is not approved for EGFR-mutant or ALK-rearranged tumors, as patients with these alterations tend not to respond to immunotherapy.119,120

In today’s practice, for EGFR-wild type and ALK-wild type tumors, PD-L1 IHC is required to identify advanced NSCLC patients eligible for first line pembrolizumab, with a cutoff of TPS≥50% required for monotherapy. PD-L1 testing is not required for combined therapy for nonsquamous NSCLC. For second-line treatment, PD-L1 IHC with a TPS cutoff of ≥1% is required for pembrolizumab. There is no PD-L1 IHC requirement for nivolumab or atezolizumab, but in general response to these drugs correlates positively with extent of PD-L1 expression.114,117 For early stage resected NSCLC patients, while PD-L1 IHC alone is neither prognostic nor predictive for adjuvant chemotherapy,121 recent promising results on neoadjuvant nivolumab before surgical resection122 suggests some value to evaluating PD-L1, perhaps in conjunction with other biomarkers, in this setting.

Assessment of PD-L1 IHC can be challenging in clinical practice. Multiple commercially available assays are available with different scoring criteria/cutoffs and variable use of antibodies and platform. Scoring reproducibility is adversely affected by both interobserver and intraobserver variability.123 For pembrolizumab, a companion diagnostic platform using clone 22C3 (Dako) pharmDx (Agilent Technologies, Santa Clara, CA) can be used. For nivolumab, atezolizumab, and durvalumab, clones 28-8 (Abcam), SP142 (Ventana), and SP263 (Ventana) can be used as complementary tests, respectively. Laboratory-developed tests have been developed both using clones associated with specific diagnostics, as well as with commercially available antibodies that have not been partnered with a therapeutic such as clone E1L3N (Cell Signaling Technology). For each of these antibodies, PD-L1 immunoreactivity is defined only as membranous staining (Fig. 2C). Although most assays evaluate only the tumor cell PD-L1 status, the complementary diagnostic for atezolizumab in advanced NSCLC evaluates both tumor cells and immune cells; the best clinical response is seen in patients with high tumor cell score (>50%) or immune cell score (>10%).117,124 Multi-institutional studies, including those affiliated with the International Association for Study of Lung Cancer (Blueprint) and the National Comprehensive Cancer Network, have been assessing the performance and comparability among PD-L1 antibodies, staining platforms, and observer interpretations.125,126 In NSCLC, PD-L1 result discordance is attributed to intratumoral heterogeneity and variations both within and among the assays/platforms in a quantitative immunofluorescence analysis.127 Although the PD-L1 epitopes (intracellular vs. extracellular domain) targeted by the antibody clones differ,128 PD-L1 antibodies including E1L3N, 22C3, SP263, E1J2J (Cell Signaling Technology), 9A11 (Cell Signaling Technology), and 28-8 are highly concordant in their performance in a well-controlled laboratory.129,130 SP142 complementary diagnostic, however, shows consistently lower PD-L1 staining.125,126,131 Pathologists are relatively concordant when scoring tumor but not immune cell expression.125 Scoring PD-L1 IHC in tumor cells in cytologic specimens also shows good concordance with surgical specimens.132

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Epidermal Growth Factor Receptor

As the second most common oncogenic driver (after KRAS) in NSCLC, somatic mutations in EGFR are present in ~10% to 20% of patients in North America and Europe but are enriched in nonsmokers and women. In East Asians populations, EGFR mutations are present in about to 50% of lung adenocarcinomas.3,133–138 Among NSCLC, EGFR mutations are present almost exclusively in lung adenocarcinomas, often showing terminal respiratory unit-type histology with lepidic, acinar, or papillary patterns.139,140 A diagnosis of lung squamous cell carcinoma harboring EGFR mutation most likely represents an adenosquamous carcinoma with an unsampled adenocarcinoma component.141,142 Activating EGFR mutations in lung cancers are located in the kinase domain in exons 18 to 21, and >90% of mutations clusters in 2 hot spots: L858R in exon 21 and in-frame deletion of the ELREA motif in exon 19.143 Germline EGFR T790M mutations have also been reported in patients with a family history of lung cancer or in never-smokers with multifocal lung adenocarcinomas.144,145 Sensitizing EGFR mutations are associated with clinical response to TKIs.133,134,137 In the first line treatment of advanced lung adenocarcinomas, EGFR TKI therapy is superior to standard chemotherapy for EGFR-mutant tumors, but not for EGFR-wild type tumors,137 highlighting the importance of evaluating EGFR status before therapy selection. Response and disease control rates using EGFR-targeted therapy in EGFR-mutant NSCLC are 68% and 86%, respectively, as compared with 11% and 47% in EGFR-wild type NSCLC.81 The irreversible EGFR TKI osimertinib is effective in patients with progressive disease following targeted therapy due to acquired EGFR T790M mutation11 and is also superior to first generation EGFR TKIs in terms of progression-free survival in the front-line setting.146,147 Given the efficacy of EGFR-targeted therapy and the imperfect correlation of EGFR status with clinicopathologic features, EGFR testing is recommended in all patients with advanced lung adenocarcinomas, regardless of age, ethnicity, or smoking status.9,81

In selecting patients for EGFR-targeted therapy, EGFR mutation testing should be performed using a sequencing approach.9,81 IHC for total EGFR protein does not predict patient outcome to TKIs148 and has no clinical utility in selecting this therapy. EGFR mutant–specific antibodies for L858R, including clones 43B2 (Cell Signaling; Fig. 2D) and SP125 (Ventana), and for exon 19 E746-A750 deletion, including clones 6B6 (Cell Signaling) and SP111 (Ventana), have been evaluated using molecular testing as the gold standard.149–156 The clinical performance of these antibodies is variable with some reports suggesting sensitivity of only 44% and specificity of 70%.155 False positive IHC results have been documented in tumors with EGFR exon 20 insertion primary resistance mutations.150 Given these performance characteristics and the increasing availability of highly sensitive molecular assays, including plasma circulating tumor DNA testing, use of EGFR mutant–specific IHC as a sole basis for therapy selection is discouraged.9 Nonetheless, EGFR mutant–specific IHC may be considered in limited-resource specimens,81 particularly in regions with high prevalence of EGFR mutations,157 or when validating unexpected sequencing results such as concomitant oncogenic drivers. Given the low negative predictive value, tumors with negative EGFR mutant–specific IHC results should be retested for EGFR mutations using molecular methods.

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IHC may be used for detection of BRAF V600E, as well as alterations in RET, pan-TRK, and LKB1. ERBB2 and MET alterations in NSCLC include both activating mutations and gene amplifications, detection of which relies on molecular methods with a minimal role for IHC.

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Mutations in BRAF are present in 3% to 5% of lung adenocarcinomas; most of these patients are smokers.158–161 BRAF V600E mutation, accounting for ~50% of all BRAF mutations in NSCLC,158–161 is associated with micropapillary histology.159,162 Combined inhibition of BRAF and MEK by trametinib and dabrafenib163,164 has demonstrated efficacy in patients with BRAF V600E-mutant lung adenocarcinomas. The BRAF V600E mutant–specific IHC using the antibody clone VE1 (SpringBio, Pleasanton, CA) has been evaluated in melanoma, papillary thyroid carcinoma, and colorectal adenocarcinoma. Although BRAF V600E IHC is generally concordant with BRAF molecular testing, its clinical utility largely depends on the tumor type.165,166 Two studies using clone VE1 for detecting BRAF V600E mutation in lung adenocarcinomas have found sensitivity and specificity of ≥90% and ≥95%, respectively.167,168 Given the clinical efficacy of BRAF-targeted therapy in NSCLC, more studies are needed to assess the utility of BRAF IHC for guiding therapy.

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Initially described in a subset of papillary thyroid carcinomas,169 rearrangements of RET are present in 1% to 2% of NSCLC, with fusion to KIF5B in most cases69,94,170–173 and rarely to diverse partners including CCDC6, NCOA4, TRIM33, and others.174–176 RET-rearranged NSCLC are enriched in young nonsmokers, though the age distribution can be wide, and nonsmoking association appears to be weaker than for ALK and ROS1 rearrangements.6,9,95,172,174,177 For a subset of patients with RET-rearranged NSCLC, multitargeted kinase inhibition using cabozantinib or vandetanib has demonstrated clinical benefits.177–181 Histologically, RET-rearranged NSCLC are predominantly adenocarcinomas; 2 RET-rearranged adenosquamous carcinomas have been described in the literature.172 RET rearrangement status has been correlated with papillary or acinar growth with conspicuous intranuclear pseudoinclusions and psammomatous calcifications, reminiscent of papillary thyroid carcinoma.94,182 Other series describe tumors with solid or cribriform histology with signet-ring cells, reminiscent of ALK-rearranged or ROS1-rearranged adenocarcinomas.172,182,183 Evaluation for RET rearrangement is primarily through molecular techniques or break-apart FISH; however, for the most common fusion KIF5B-RET, the probe separation resulting from the causative intrachromosomal inversion can be too subtle for reliable detection by FISH.182 As regards RET IHC, while reasonable performance has been reported in one study using the antibody clone EPR2871 (Abcam), with 100% sensitivity and 83% specificity,182 other studies using this and another clone 3F8 (Leica) have found the overall performance disappointing, being neither sensitive nor specific for RET rearrangements.171,174,184,185 More studies are needed to evaluate the potential utility of RET IHC and the optimal diagnostic algorithm in identifying RET rearrangements in NSCLC.

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Rearrangements of neurotrophic tyrosine kinase 1 (NTRK1) have been identified in rare (0.1%) lung adenocarcinomas,186 or ~3% of NSCLC with no known oncogenic drivers.187 Oncogenic NTRK fusion involves diverse 5′ partners, leading to constitutive TRK signaling. TRK-targeted inhibition by larotrectinib has been shown to be efficacious in NTRK-rearranged tumors from diverse origins and histologic types, including 4 lung adenocarcinomas in adults and adolescents.188 Pan-TRK, an antibody clone EPR17341 (Abcam) that recognizes a conserved C-terminal sequence of TRK proteins, has been used to identify and confirm tumors with NTRK fusions, including 2 NTRK1-rearranged lung adenocarcinomas.189 Although clinical and histologic features of NTRK1-rearranged lung adenocarcinoma have not been well described, identification of NTRK rearrangements in lung adenocarcinomas using NGS and/or IHC is increasingly relevant to guide these patients for TRK-targeted therapy.

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Future Directions: STK11/LKB1, ERBB2/HER2, and MET

Mutations in STK11/LKB1 are present in ~30% of lung adenocarcinomas, often co-occur with KRAS mutations in smokers,3,190–192 and are associated with a lack of benefit of immunotherapy in advanced NSCLC patients.107,119 Using LKB1 antibody clone Ley37D/G6 (Abcam), loss of LKB1 expression (Fig. 2E) has been shown to portend aggressive behavior with frequent metastases in KRAS-mutant NSCLC.193 Although earlier studies show good concordance between STK11 biallelic inactivation and LKB1 IHC,194 LKB1 expression can be heterogeneous among cases, from focal to complete loss. Considering the multiple mechanisms for LKB1 inactivation, data on the sensitivity and specificity of LKB1 IHC for STK11 alterations are limited.193

ERBB2/HER2 alterations in NSCLC comprise both activating mutations and gene amplification. Activating exon 20 in-frame insertion mutations195 are reported in 1% to 4% of NSCLC, enriched in nonsmokers and primarily in adenocarcinomas.196–200 Activating ERBB2 transmembrane domain mutations have also been noted, with altered HER2 receptor dimerization and responsiveness to afatinib.201 In contrast, amplification of ERBB2 is present in 1% to 3% of NSCLC, including a subset of EGFR-mutant NSCLC resistant to targeted therapy.202 There is no consistent association between ERBB2 activating mutations and copy number status.198,203 Detection of ERBB2 mutations or amplification in NSCLC relies exclusively on molecular methods, with no established role for IHC. Clinical trials of targeted therapy on ERBB2-mutated NSCLC are ongoing.204,205

MET alterations in NSCLC include both activating mutations and gene amplification. MET amplification is a well-documented mechanism of resistance to targeted therapy in EGFR-mutant NSCLC.11,206 Diverse MET exon 14 splice site mutations have been described, leading to skipping of exon 14 that encodes the juxtamembrane regulatory domain.207 MET exon 14 mutation is present in ~3% of NSCLC,2,208–210 associated with advanced age, stage, and pleomorphic histology (up to ~32% of cases).208–211 A subset of patients with MET exon 14-mutated NSCLC responds to crizotinib208,212 or carbozantinib.213 MET exon 14 mutation and MET amplification each is associated with worse survival.208,210 IHC using the c-Met antibody clone SP44 (SpringBio) has been used in the research setting to demonstrate MET protein overexpression (Fig. 2F) but has no established clinical role. Although MET exon 14-mutated NSCLC shows stage-dependent MET amplification and c-Met overexpression,208 c-Met expression alone does not predict survival and apparently shows poor correlation with MET mutation status.208,210

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IHC is a stalwart in the rapid diagnosis and definitive classification of lung carcinomas. On limited biopsies, a focused IHC panel conserves tissue for predictive biomarker and molecular testing. For patients with advanced lung cancer, predictive IHC for ALK, ROS1, and PD-L1 rapidly provides clinically actionable information for planning therapy. A library of high-quality antibodies correlating with overexpression of oncogenic rearrangements, loss of expression due to inactivating events in tumor suppressor genes, and expression of specific mutated proteins is now available for use in research and, when properly validated, as clinical tools to confirm or supplement molecular testing. IHC is situated at the intersection of surgical pathology and molecular diagnostic in an integrated pathology practice, where it serves as a powerful functional readout in precision medicine and an indispensable tool in the care of lung cancer patients.

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non–small cell lung carcinoma; immunohistochemistry; biomarker; NUT; SMARCA4; ALK; ROS1; PD-L1; BRAF

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