Scoring system for immunostaining of small biopsy specimens with mutation-specific EGFR antibodies—L858R mutation in exon 21 and 15-bp deletion in exon 19. (a and b) Negative scores. (a) Absence of staining, zero intensity. (b) Faint staining in >10 % of tumor cells, score of 1+: this pattern of immunoreactivity is associated with false-positive results. (c, d) Positive scores: a positive score corresponds to the presence of specific mutation. (c) Moderate but incomplete membranous staining pattern in the majority of tumor cells, scored as 2+. (d) Strong membranous staining pattern in the majority of tumor cells scored as 3+
Jiang et al. reported that mutation detection by molecular techniques correlates with the intensity of immunohistochemical stain . Accordingly, a reaction with a 1+ score is associated with false-positive results [4, 36, 45] with mutations detected by molecular techniques in only 23 % of cases . Therefore, a greater number of false positives will result when a score of 1+ is interpreted as a positive reaction. Application of stringent criteria for the interpretation of a test as positive is thus important and has significant clinical implications. In fact, a large study showed that administration of TKI, instead of chemotherapy, to patients without EGFR-mutated lung adenocarcinomas resulted in more harm than benefit .
On the contrary, scores of 2+ and 3+ with both mutation-specific EGFR antibodies [34, 36] are not associated with false-positive results, correlate with the tumor mutation profile, demonstrate high sensitivity, and have reported specificity and positive predictive values of 100 % [34, 36]. Therefore, a binary system with 2+ and 3+ regarded as positive reactions and 0 and 1+ as negative or inconclusive should be implemented.
The issue remains, however, in cases with negative or inconclusive results for EGFR mutation using this pair of antibodies (scores of 0 and 1+). Jiang et al. observed that EGFR mutations are detected in 7 % of tumors with a score of 0 by immunohistochemistry . The combined sensitivity of these antibodies is approximately 70 % for the specific EGFR mutation that they are designed to detect, which means that there is at least a 30 % chance of missing an activating mutation with a negative reaction by IHC. Therefore, a negative reaction (score of 0 or 1+) should not be assumed to correspond to wild-type EGFR. Cases that are scored as negative for mutation-specific antibodies should still be submitted for molecular diagnostic testing that can detect other mutations in the EGFR gene that are unrecognized by the pair of antibodies but still confer sensitivity to tyrosine kinase inhibitors. A work flow for the use of EGFR mutation-specific antibodies is illustrated in Fig. 7.2.
Algorithm for the interpretation of immunostaining with EGFR mutation-specific antibody. The pair of antibodies is designed for the detection of L858R and 15-bp deletion only. Other potential sensitizing EGFR mutations are not detected by this pair; therefore, negative or inconclusive results (score 1+) do not exclude the possibility of another mutation in the EGFR gene or another class of mutation such as ALK, KRAS, and others. Samples scored as negative or 1+ should be submitted to molecular tests if adequate material exists; otherwise, re-biopsy is recommended for predictive markers
Use of Mutation-Specific Antibodies in Small Biopsy Specimen
There is a great interest in the application of these antibodies in cytology specimens, since cytologic material is the main source of tissue for patients who present with advanced stages of disease and because a small but significant proportion of cytology specimens fail molecular testing. Cytology also offers an array of specimens from aspirations of primary or metastatic sites, ultrasound-guided endobronchial biopsies (EBUS) of the lymph nodes evaluated for staging information, pleural effusions, and other exfoliative samples like bronchial lavages, brushes, washes, and sputum.
A study evaluating the application of EGFR mutant-specific antibodies to clinical specimens, including cytology samples (FNA and effusion fluid), small core biopsies, and bone biopsies showed that the pair of antibodies performs well in cytology (cellblock and ThinPrep) and small biopsy material with comparable sensitivity and specificity as that reported to excisional material  (Fig. 7.3). In another report, Kawahara et al. tested the performance of these two specific EGFR mutation antibodies in a relatively small sample of cytology specimens from effusions and cerebrospinal fluids and reported a sensitivity of 100 % and specificity of 92 % for both mutant-specific antibodies .
Microphotograph of a positive reaction with EGFR mutation-specific antibody on a liquid-based preparation (ThinPrep® slide). This positive result correlated with the detection of 15 bp deletion in exon 19 in another sample of the same tumor
Moreover, Hasanovic et al.  showed that the mutation-specific EGFR antibodies are also useful in the assessment of mutation status in bone biopsies for stage IV patients. The decalcification process for histological evaluation of bone metastasis renders the material unsuitable for molecular testing since it affects the quality of DNA. Application of the pair of mutation-specific EGFR antibodies can provide useful information if a positive reaction is observed.
To summarize, IHC for EGFR mutations is useful in cytology and small biopsy specimens with scant cellularity for molecular analysis. A positive reaction by IHC in a decalcified material or small biopsy with scant cellularity can prevent the need to re-biopsy patients. Also, using a stringent cutoff of >2+ as an indicator of positivity allows for initiation of therapy with TKI in patients with advanced disease. An algorithm for the utilization of EGFR mutation-specific antibodies in small specimens that are unsuitable for molecular tests is illustrated in Fig. 7.4.
Algorithm for tissue utilization of small biopsy material. Samples with inadequate tumor content (scant cellularity) or processed for decalcification can be processed for EGFR mutation-specific antibody. All other biopsies that have adequate material for molecular studies should be processed as such. The CAP/IASLC/AMP recommendations suggest that priority for molecular diagnostics should be a test that can detect all mutations in the EGFR gene
Response to TKI Therapy in EGFR Mutant Tumors Detected by Immunohistochemistry
Most reports that indicate a response to TKI in patients with EGFR mutation originate from studies that applied standard molecular techniques for the detecting mutations. As mutation-specific antibodies became available for the characterization of pulmonary adenocarcinomas, questions emerged among treating physicians as to whether mutation detection by immunohistochemistry was predictive of response to therapy with TKI.
Initial studies showed that although there was good correlation between response to therapy and mutation identified by immunohistochemistry, the best responses were noted in patients with molecular confirmation and not with mutation-specific antibody detection . This discrepancy was a consequence of two factors. First discussed was the relatively low sensitivity of the antibodies; the authors compared in their analysis all EGFR mutations detectable by molecular techniques with the pair of antibodies designed to identify only two mutations (15-bp deletion in exon 19 and L858R point mutation in exon 21). Second, the criteria indicative of a positive reaction were different from those currently accepted. Inclusion of cases with weak positivity (1+) in less than 10 % of tumor cells resulted in poor correlation with mutation status and response to therapy. These conclusions do not come as a surprise given what is now known about these antibodies, including false positivity stemming from overinterpretation of faint or incomplete membranous staining in <10 % tumor cells.
In a more recent study, Kawahara A et al. , using the established cutoff of 2+ and 3+ as positive, demonstrated that all patients with a positive reaction for mutation-specific antibody had a significant progression-free survival compared to those with a negative reaction (0 and 1+). The authors concluded that patients with EGFR mutation detected by immunohistochemistry are good candidates for EGFR-targeted therapy.
These studies are important because they validate the correlation of immunohistochemistry detection of EGFR mutation with response to specific therapy. Considering that the results of immunohistochemistry can be available and reported within a few days, clinicians can initiate treatment of patients in need, safely, while awaiting molecular confirmation of standard molecular techniques, which may take weeks. In cases with only scant available tissue or a decalcified bone biopsy, a positive antibody reaction reduces the need to re-biopsy for further testing (Fig. 7.4).
Specificity of EGFR Mutation-Specific Antibodies as a Marker for Lung Cancer
In the setting of disseminated metastatic disease of unknown primary, it is not uncommon to find tumors that lack expression of tissue specific markers, which makes it very difficult to pin-point the site of origin and therefore selection of appropriate therapy. Mutations in the EGFR gene seem to be specific to pulmonary adenocarcinomas. However, until recently, the specificity of the pair of EGFR mutation-specific antibodies in other carcinomas was unknown.
Wen et al. , with the working hypothesis that mutation-specific antibodies would not cross-react with overexpressed EGFR wild-type on tumors cells, evaluated the specificity of the pair of antibodies in a large series of carcinomas from non-pulmonary sites such as the breast, pancreas, colorectum, and endometrium. The authors showed that of 300 breast carcinomas, including estrogen-positive, Her2/neu-positive, and triple-negative cases, less than 1 % had a positive reaction (2+ intensity) with the anti-L858R antibody. Molecular analysis of the 2+ L858R breast carcinomas showed no mutation, indicating a false-positive result. When present, false-positive results in breast adenocarcinoma were seen only in estrogen-positive tumors and not in triple-negative carcinomas. All breast carcinomas scored 0 with the E746-A750 antibody, and all the colorectal, pancreatic carcinomas and malignant Mullerian tumors of the endometrium were negative (0 intensity) for both antibodies. These results indicate that EGFR mutation-specific antibodies can be incorporated in the diagnostic work-up of patients with disseminated metastatic diseases when pulmonary adenocarcinoma is in the differential diagnosis with a positive result to an EGFR mutation-specific antibody most likely indicating a pulmonary origin in the setting of a tumor of unknown origin. Therefore, in addition to predictive indicators for therapy with EGFR inhibitors, these antibodies are also helpful in providing a site of origin.
In 2007, Soda et al. described that a subset of pulmonary adenocarcinomas showed an inversion within chromosome 2 that resulted in a transforming fusion kinase between echinoderm microtubule-associated protein-like 4 (EML4) in 2p21 and anaplastic lymphoma kinase (ALK) in 2p23.2 . This fusion results in constitutive activation of ALK kinase, and transfection of this molecular alteration into cell lines was also tumorigenic . ALK rearrangement is the second driver oncogene in pulmonary carcinoma that can be targeted by specific therapy.
Rearrangement of the ALK gene is present in approximately 5 % of the patients with pulmonary adenocarcinoma [50–55] and is more common in younger patients who are light or never smokers. In general, these patients present with advanced disease and are often poorly differentiated with solid or cribriform predominant histology with signet ring cell features [52–55] and psammoma bodies . ALK rearrangement is mutually exclusive of EGFR or KRAS mutations  although tumors expressing double mutation with EGFR have been reported .
In 2009, crizotinib (XALKORI, Pfizer, Inc) was approved by the US Food and Drug Administration for the treatment of patients with lung adenocarcinoma harboring ALK rearrangement. The drug, an inhibitor of the tyrosine kinase activity of both ALK and the MET proto-oncogene, is efficacious in treating mutant ALK lung adenocarcinoma [59, 60].
Identification of ALK rearrangement in tumors is standard in the evaluation of patients with pulmonary adenocarcinoma.
Diagnosis of ALK Rearrangement
The area encompassing the EML4-ALK fusion in chromosome 2 is relatively large and therefore suitable for detection by fluorescence in situ hybridization (FISH). In fact, FISH for ALK rearrangement has been licensed by the US Food and Drug Administration as a companion diagnosis for the detection of this mutation.
The Vysis ALK Dual Color, Break Apart Rearrangement Probe FISH Kit (Abbott Molecular) is the gold standard for detection of ALK rearrangement [59, 61, 62]. This test, however, is marred by relative high cost, limited availability, and technical complexity that require trained technicians and pathologists for its interpretation. With the added infrequency of ALK rearrangement in 5 % adenocarcinomas, performing FISH on all specimens is impractical and there is a great interest in developing a robust screening method for detecting this alteration. The CAP/IASLC/AMP recommendations indicate that screening of ALK rearrangement can be conducted with immunohistochemistry, following strict validation of the antibody in the user laboratory .
Diagnosis of ALK Rearrangement by Immunohistochemistry
Initial analysis using the ALK-1 clone, the same antibody used to detect ALK in T-cell lymphomas, showed variable results [49, 63]. Recently, two commercially available antibodies show higher specificity and sensitivity to ALK-rearranged lung adenocarcinoma when compared to the ALK-1 clone. Mino-Kenudson et al. , using a novel antibody (clone D5F3, Cell Signaling Technology) for the detection of ALK-rearranged lung adenocarcinomas, showed 100 % sensitivity and 99 % specificity with excellent interobserver agreement between pathologists.
In a study by Minca EC et al. , D5F3 clone showed 100 % sensitivity and 100 % specificity for the detection on ALK fusion protein in paraffin-embedded tissue and cytology material, and in instances of inconclusive FISH results, negative IHC was helpful in preventing false-positive FISH. The authors concluded that the high concordance rate between FISH and IHC for ALK supports the use of IHC as a screening method for ALK status determination.
Another clone, 5A4 (Novocastra, Leica Biosystems), is commercially available for the detection of ALK-rearranged adenocarcinoma. Sakairi et al. correlated the results of IHC using this clone with standard FISH in 109 adenocarcinoma specimens obtained by endobronchial ultrasound-guided transbronchial needle aspiration biopsy . A good correlation between the two tests was observed without any reported false-positive or false-negative cases by IHC. Savic S et al. showed that immunohistochemistry for the detection of ALK rearrangement using clone 5A4 is feasible in cytologic material with a reported sensitivity and specificity of 93.3 and 96 %, respectively . The authors used cellblock material, direct smears, and liquid-based cytology in their study and reported that ALK testing with IHC is feasible on all cytology preparations.
Both antibody clones, D5F3 and 5A4, have been compared. Selinger CI  showed that both identified ALK rearrangement detected by FISH. IHC with D5F3 clone and 5A4 clone has been reported by several investigators as having sensitivity of 93 to 100 % and specificity of 96 to 100 %, when compared to FISH [64, 65, 67, 68].
A recent publication by the IASLC, dedicated exclusively to ALK testing in lung carcinomas, emphasized the need to validate IHC tests for ALK, since there are several available antibodies with different detection systems . The report suggested the need to standardize the procedure, if results are to be comparable from different laboratories.
Scoring of ALK Immunohistochemistry
The ALK staining pattern is predominantly cytoplasmic with a granular quality and has membranous accentuation in cases with high-intensity stain. This is in contrast to the pattern seen with EGFR mutation-specific antibodies that show predominantly membranous staining.
In most studies, ALK staining pattern is scored from 0 (zero) to 3+: zero (no stain), 1+ (weak cytoplasmic stain, best visualized by 40× objective), 2+ (moderate cytoplasmic stain best visualized using 10× to 20× objectives), and 3+ (strong stain, seen at 2× to 4× objective) (Fig. 7.5). The definition of positivity among studies is variable, though [64–68]. Most consider 2+ and 3+ positive demonstrating good correlation with FISH tests.
Scoring system for immunostaining of small biopsy specimens with antibodies that recognized rearranged ALK clone D5F3. (a) Absence of staining with the antibody. This has been shown to correlate lack of ALK rearrangement. (b) Score 1+, weak cytoplasmic staining in all tumor cells. This pattern is considered equivocal. Some authors reporting the presence of rearrangement confirmed by FISH but not others. (c) 2+ intensity score: this is considered a positive reaction and correlates with a positive ALK-FISH test. (d) 3+ intensity score: this is considered positive and correlates with a positive ALK-FISH test
As the staining intensity plays a major factor in the decision for screening algorithms, there is still considerable amount of variability when a staining pattern of 1+ is encountered, with some regarding it positive [64, 65, 68] and others considering it inconclusive with FISH follow-up [62, 69]. Some authors suggest that a diffuse reactivity in all tumor cells with an intensity of 1+ is indicative of ALK rearrangement [64, 65, 68], whereas others have reported that heterogeneous staining patterns is common in ALK-rearranged tumors . The issue of a weak reactivity is more likely the result of pre-analytical problems such as fixation and antigen retrieval, as well as the clone and detection system used, rather than different levels of the fusion protein expression. Therefore, before a screening algorithm for ALK rearrangement detection can be established, a standardized procedure including pre- and post-analytical parameters needs to be outlined by competent regulatory bodies. At the time of writing this chapter, the recommendation of the CAP/IASLC/AMP is that before a screening algorithm for ALK is established, each laboratory should validate its own test with strong internal positive and negative controls.
More importantly, what appears clear from most reports on the subject is that a negative stain by IHC with either clone (D5F3 or 5A4) is indicative of negative FISH rearrangement (negative predictive value of 100 %). Therefore, in most screening algorithms that have been established outside of the United States, a negative (zero intensity) stain by IHC indicates absence of ALK rearrangement and no further testing is required. An example of a possible screening algorithm is illustrated in Fig. 7.6.
Algorithm for ALK screening using ALK antibody either clone D5F3 or 5A4. Since ALK FISH is the companion diagnostic test for ALK rearrangement, all positive cases should be sent for FISH test for confirmation of the arrangement. This is also true to cases scored as 1+ (equivocal). Most studies using IHC for ALK rearrangement screening suggest that a negative reaction (no staining) is indicative of native ALK. The CAP/IASLC/AMP guidelines suggest that an ALK screening algorithm using IHC should be strongly validated in each laboratory before implementation of the test. There is no agreed-upon screening algorithm for ALK rearrangement
At least in the United States, all cases considered positive for ALK by IHC need to have confirmation of ALK rearrangement by FISH (companion diagnostic) test.
Other Mutation-Specific Antibodies
ROS1 is a proto-oncogene translocation identified in approximately 1–2 % of pulmonary adenocarcinomas  and is responsive to treatment with crizotinib, an ALK/MET inhibitor. Similar to ALK-rearranged tumors, most adenocarcinomas with this translocation are poorly differentiated and have a predominant solid, micropapillary, or cribriform growth patterns with signet ring cells and microcalcifications [72–74]. The translocation can be detected by a specific FISH probe. Due to the rarity of this mutation, most testing is conducted in tumors that are triple negative for EGFR, ALK, and KRAS mutations.
Recently, an antibody that detects ROS1 has become commercially available. The D4D6 clone [73, 74] has a sensitivity of 100 % and specificity of 97 % in two studies [73, 74] for the detection of ROS1 when compared to standard FISH test.
A study from Mescam-Mancini L. et al. reports good correlation between IHC and FISH test when 2 + and 3+ are considered positive reaction by IHC. ROS1 antibody shows cytoplasmic staining, similar to ALK antibodies. Interestingly, the same authors reported that the antibody cross-reacts with HER/2neu mutant lung adenocarcinomas .
BRAF mutations are detected in approximately 1 to 2 % of pulmonary adenocarcinomas; the most common mutation is V600E . BRAF mutations can also be targeted by specific therapy . An antibody that recognizes V600E mutation is commercially available, with clone VE1 having good sensitivity and specificity for detecting mutations in pulmonary carcinomas, thyroid carcinomas, and melanoma [77–79].
Other Uses of Mutation-Specific Antibodies
Synchronous and metachronous adenocarcinomas of the lung pose a significant challenge for diagnosis and staging of patients with lung cancer, since prognosis and clinical management are highly dependent of pathological staging.
Although pulmonary adenocarcinomas are histologically heterogeneous and therefore morphological comparison of two tumors may be sufficient for determining whether they represent metastases or separate primary tumors , there are still cases in which this differentiation cannot be achieved with certainty by morphology alone. At the same time, it is not uncommon to encounter a clinical situation of metachronous tumors with different histological patterns that are favored to represent separate primaries, but the clinical presentation is suggestive of metastatic disease. In these situations, most pathologists rely upon the molecular characteristics of the tumors for further differentiation.
D’Angelo SP et al.  evaluated 1831 resected lung adenocarcinomas from clinical stages I–III and suggested that information generated by mutational profiling following tumor excision permitted distinction between multiple primary tumors and metastases and assignment to expected directed therapy and specific clinical trials. In their evaluation of 82 multifocal adenocarcinomas of the lung, Takamochi et al.  showed that EGFR and KRAS mutations occurred randomly, thus suggesting that in synchronous or metachronous carcinomas, the identification of the same mutation in more than one tumor supported the concept of clonality therefore determining metastatic disease. Different mutational profiles are consistent with separate clones, therefore separate primary tumors.