What is the optimal testing algorithm for the assessment of HER2 status in patients with GEA?

In patients with advanced GEA who are potential candidates for HER2-targeted therapy, the treating clinician should request HER2 testing on tumor tissue (Type: evidence based; Quality of evidence: high; Strength of recommendation: strong).

All patients who have documented advanced GEA and who are considered good candidates for combination chemotherapy plus trastuzumab therapy should have their tumor tissue tested for HER2 overexpression and/or amplification. In patients with HER2-positive GEA, the addition of trastuzumab can increase the response rate, prolong progression-free survival, and prolong overall survival. Other than providing guidance regarding the addition of trastuzumab to cytotoxic combination (when the tumor is HER2 positive), HER2 status provides little additional value such as prognostic or predictive information. Currently, there is no evidence of benefit of HER2-directed therapy in patients without advanced GEA.

In the ToGA trial, patients were randomly assigned to receive capecitabine plus cisplatin or fluorouracil plus cisplatin in combination with trastuzumab.^3,8 Of the 3,803 patients originally screened for eligibility, 810 patients had IHC or FISH HER2-positive tumors, but only 594 patients were randomly assigned to treatment. The HER2 positivity rate was 22.1%, with similar rates between European and Asian patients (23.6% v 23.9%). The eligible patients included those with advanced adenocarcinoma of the stomach or gastroesophageal junction, Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2, adequate organ function, and measurable or nonmeasurable disease. Patients were ineligible if they had congestive heart failure, baseline left ventricular ejection fraction less than 50%, transmural myocardial infarction, uncontrolled hypertension (systolic blood pressure > 180 mm Hg or diastolic blood pressure > 100 mm Hg), angina pectoris requiring medication, clinically significant valvular heart disease, high-risk arrhythmias, lack of physical integrity of the upper gastrointestinal tract or malabsorption syndrome, active gastrointestinal bleeding, or evidence of brain metastases. The median overall survival was 13.8 months for patients receiving trastuzumab plus chemotherapy, compared with 11.1 months for those receiving chemotherapy alone (hazard ratio [HR] = 0.74; 95% CI, 0.60 to 0.91; P = .0038). Patients with IHC of 3+ derived more benefit than those with IHC of 2+ (and concurrent HER2 amplification by ISH). However, upon further follow-up of these patients, reanalyses demonstrated considerable reduction in patient benefit from the addition of trastuzumab (HR = 0.8; 95% CI, 0.67 to 0.97; P = .019). The difference in median survival diminished to a mere 1.4 months.²¹

The cardiac adverse event rate was low (6%) and did not differ between the treatment groups. Trastuzumab was generally well tolerated, but the patients assigned to trastuzumab experienced slightly higher rates of diarrhea, stomatitis, anemia, thrombocytopenia, fatigue, and weight loss. However, there was no difference between the groups in adverse effect frequency, or grade 3 or 4 toxicities except for diarrhea.

NCCN Guidelines recommend systemic therapy, clinical trial participation, or palliative management for patients with a Karnofsky performance score ≥ 60%, or an ECOG performance score ≤ 2, and that trastuzumab should be added to active first-line combination chemotherapy for HER2-positive metastatic GEA (although the ToGA trial combined cisplatin and a fluoropyrimidine with trastuzumab).¹¹ Patients with a Karnofsky performance score < 60%, or ECOG performance score ≥ 3, are best treated with best supportive care.

Although the literature regarding HER2 as a prognostic marker is conflicting, some studies^6,22-24 have demonstrated that HER2 amplification or overexpression in GEA may be associated with a worse prognosis and is independent of other prognostic factors including age, sex, location, or stage. We briefly review only two large and representative studies that failed to correlate HER2 status with prognosis. A retrospective study of 1,006 Japanese patients with gastric cancer established HER2 overexpression in 11.7% of cases.²⁵ The HER2 status correlated with age, sex, grade, growth pattern, and nodal status; however, HER2 overexpression did not correlate with disease-specific survival or recurrence-free survival. Likewise, a combined analysis of 924 German and British patients who had undergone surgical resection demonstrated HER2 expression in < 10% of tumor specimens with considerable intratumoral heterogeneity and no relationship between HER2 expression, patient survival, or stage.²⁶

In summary, the evidence does not support the determination of HER2 status in patients who have a surgically resectable GEA, and HER2 status is not useful to prognosticate survival or similar end points. However, for patients with advanced GEA with a good performance status and low cardiac risk who would otherwise be candidates for systemic therapy including trastuzumab, HER2 testing should be performed and patients should be offered trastuzumab if GEA is HER2 positive.

Treating clinicians or pathologists should request HER2 testing on tumor tissue in the biopsy or resection specimens (primary or metastasis), preferably before the initiation of trastuzumab therapy if such specimens are available and adequate. HER2 testing on fine needle aspiration (FNA) specimens (cell blocks) is an acceptable alternative (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: recommendation/moderate).

Treating clinicians should offer combination chemotherapy and HER2-targeted therapy as the initial treatment for appropriate patients with HER2-positive tumors who have advanced GEA (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

HER2-targeted therapy was established in 2010 as a new standard of care for the first-line treatment of patients with advanced GEA with HER2-positive tumors. The results of the ToGA trial (efficacy and safety) have been described above.³ In addition, health-related quality of life (HRQoL) and quality-adjusted time without symptoms of disease or toxicity (Q-TWiST) were improved for patients who received trastuzumab, with a prolonged time to 10% definitive deterioration in all Quality Of Life Questionnaire (QLQ) –C30 and QLQ-STO22 scores and extended Q-TWiST of 2.42 months, compared with chemotherapy alone.⁴³ Thus, trastuzumab achieved a level-1 evidence for overall survival advantage in patients with HER2-positive advanced GEA in the first-line setting.^11,44

In 2010, the US Food and Drug Administration (FDA) and European Medicines Agency approved trastuzumab in combination with cisplatin and a fluoropyrimidine (fluorouracil or capecitabine) for use in patients with HER2-positive GEAs. NCCN Guidelines, however, recommend the addition of trastuzumab to any active chemotherapy combination. In addition to the ToGA trial, smaller trials combining trastuzumab with weekly paclitaxel (in trastuzumab-naive patients) or capecitabine and oxaliplatin have documented some efficacy, but these results are supportive and not definitive.⁴⁵ When adding trastuzumab to a biweekly regimen (eg, oxaliplatin and a fluoropyrimidine), the loading dose should be 8 mg/kg and then 4 mg/kg every 2 weeks.

With the establishment of HER2 testing as a standard of care for patients with advanced GEA, it is important to note that the treating clinician should not offer HER2-targeted therapy until HER2 positivity is confirmed. Because patients with advanced GEA can be symptomatic, it is recommended to start combination cytotoxic therapy as soon as feasible while waiting for the establishment of HER2 status. This statement is based on expert opinion and on the fact that in the ToGA trial, of 810 patients with HER2-positive tumors, 216 became ineligible (mainly due to deterioration of performance status) while waiting for HER2 test results. Once it is determined that GEA is HER2 positive, trastuzumab can be added to the chemotherapy combination. There is no documented benefit for starting HER2-directed treatment in the absence of confirmed HER2 positivity, and there is an added potential for the patient to incur unnecessary adverse effects or costs. It is also recommended that if there is documentation of a HER2-positive result in any specimen (primary or metastatic tumor), the treating clinician does not need to request additional HER2 testing on additional tumor specimens. Conversely, if there is no documentation of an HER2-positive result, and there is no available tumor tissue, an attempt should be made to collect additional neoplastic tissue (primary or metastatic) for HER2 testing. In addition, there is currently no evidence to support repeating HER2 testing after evidence of progression after HER2-directed therapy (trastuzumab) combined with cytotoxic combination, and there is no evidence to support continuation of trastuzumab beyond progression in patients with GEA. In this regard, the TyTAN trial (Lapatinib Plus Paclitaxel Versus Paclitaxel Alone in the Second-Line Treatment of HER2-Amplified Advanced Gastric Cancer in Asian Populations) randomly assigned 262 patients with advanced HER2-amplified gastric adenocarcinomato lapatinib plus paclitaxel or paclitaxel alone, in the second-line setting, and reported no advantage in overall survival for patients randomly assigned to lapatinib.⁴⁶ Additionally, the LOGiC trial (Lapatinib in Combination With Capecitabine Plus Oxaliplatin in Human Epidermal Growth Factor Receptor 2–Positive Advanced or Metastatic Gastric, Esophageal, or Gastroesophageal Adenocarcinoma) that randomly assigned 545 patients with HER2-amplified advanced GEA to lapatinib or placebo plus capecitabine and oxaliplatin, in the first-line setting, demonstrated no overall survival advantage for patients who received lapatinib over those who received placebo.⁴⁷ Therefore, the efficacy of HER2-directed therapy is demonstrated only by trastuzumab restricted to the first-line setting. An algorithm for clinicians for HER2 testing in patients with GEA is presented in Figure 1.

Fig 1. Algorithm for clinicians. FNA, fine needle aspiration; GEA, gastroesophageal adenocarcinoma; HER2, human epidermal growth factor receptor 2.

In summary, randomized clinical data support the use of HER2-targeted therapy in combination with chemotherapy for patients who are fit and able to tolerate treatment. Addition of HER2-targeted therapy in patients with HER2-positive GEA results in improved survival and quality of life. Trastuzumab provides modest overall survival benefit for patients with HER2-positive advanced GEA in the first-line setting in combination with active cytotoxics.

What strategies can help ensure optimal performance, interpretation, and reporting of established assays in patients with GEA?

Laboratories and pathologists must specify the antibodies and probes used for the test and ensure that assays are appropriately validated for HER2 IHC and ISH on GEA specimens (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

Multiple antibodies are available for HER2 IHC (including but not limited to Ventana 4B5 [Tucson, AZ], Thermo Fisher Scientific CB11 [Waltham, MA], Sigma-Aldrich SP3 [St Louis, MO], Dako AO485 and Dako HercepTest [Glostrup, Denmark]). Ventana 4B5, Thermo Fisher Scientific CB11, and Dako HercepTest are FDA approved.⁴⁸ The ToGA trial³ used the HercepTest antibody, and many studies^30,49-53 have used 4B5 or CB11. There is generally moderate to good concordance between various antibodies,^54-58 although several articles have noted stronger staining for 4B5 than for other antibodies. However, no recommendation is made for the use of a specific antibody.

Likewise, multiple methods for ISH have been evaluated for HER2 in GEA. The Dako pharmaDx (Glostrup, Denmark) HER2 FISH kit was used for the ToGA trial,³ and there is considerable experience with FISH in testing for HER2 amplification in breast carcinomas. Development of brightfield ISH technologies has resulted in several other ISH methods, and one kit has obtained FDA approval (Dako HER2 FISH pharmDx). One of these methods is SISH, where either one HER2 slide, or two separate slides, are stained for HER2 and chromosome enumeration probe (CEP) 17, both using silver as the chromogen. The other major brightfield ISH methods are chromogenic in situ hybridization and dual in situ hybridization, where a nonsilver chromogen is used either alone or in combination with a silver chromogen on a separate probe to mark both HER2 and CEP17 on one slide. The authors of multiple studies^52,59-68 agree that these various ISH methods are comparable and effective for GEA HER2 testing. There have been suggestions that brightfield ISH techniques have some advantages over FISH, in that they can often be performed on automated stainers, do not require fluorescence microscopes, and allow for easier identification of tumor nuclei among normal tissues.^61,63 However, no recommendation is made regarding the use of any specific ISH method, as there is no major diagnostic advantage to one method over another.

Although no recommendation regarding which specific antibody/ISH methodology is given, there is a strong recommendation regarding validation. If using a method other than the FDA-approved kit, pathologists and laboratories should carefully validate both IHC and ISH for HER2, and validation should be performed in the laboratory in which the assay will be used. The cases used for validation should be predominantly GEA cases as opposed to other tumors (ie, breast carcinomas) to allow those scoring to develop and maintain expertise with the different GEA tumor types and appearances. CAP and/or Clinical Laboratory Standards Institute guidelines should be followed for assay validation.^69-72

The method of sampling for the validation specimens (ie, from resections or biopsies) should be similar to those expected in future sampling, and should use the same fixative. The CAP Laboratory Accreditation Program (ANP.22978) for HER2 validation for breast carcinomas proposes validation using 20 positive and 20 negative specimens for an FDA-approved test, and 40 positive and 40 negative cases for a laboratory-developed test.⁷³ If using a brightfield ISH assay kit, initial validation should be done by comparison to an FDA-approved FISH assay.¹⁷ Records of validation must be maintained as per the CAP Laboratory Accreditation Program (ANP. 22750, ANP.22978, and ANP.22956).⁷³ Laboratories must also maintain good quality control. When reporting results, the final HER2 test reports should specify the antibody used for IHC and/or the probe used for ISH, along with a brief description of the kit and methodology.

When GEA HER2 status is being evaluated, laboratories and/or pathologists should order IHC testing first followed by ISH when IHC result is 2+ (equivocal). Positive (3+) or negative (0 or 1+) HER2 IHC results do not require further ISH testing (Type: evidence based; Quality of evidence: high; Strength of recommendation: strong).

The ToGA trial demonstrated that the combination of trastuzumab plus chemotherapy significantly improved survival in patients with tumors showing high HER2 expression.³ The latter was defined as HER2 score 3+ by IHC or HER2 score 2+ by IHC and HER2 positivity (amplified) by FISH. HER2-positive results by FISH were observed in 11% of cases with IHC score 0 and 12% of cases with IHC score 1+.³ Similarly, other studies^29,74-77 have shown HER2 positivity by ISH in up to 14% to 24% of tumors with IHC scores of 0 or 1+. These patients did not significantly benefit from the addition of trastuzumab to the chemotherapy regimen in the ToGA trial.³ Similar findings were reported in subsequent studies and reviews, demonstrating that ISH positivity alone does not correlate with response to trastuzumab therapy in GEA.^8,78

NCCN Guidelines recommend that specimens with 2+ expression of HER2 by IHC should be additionally assessed by FISH or another ISH method. Specimens with 3+ overexpression by IHC or FISH positivity (HER2:CEP17 ratio ≥ 2) are considered positive.¹¹ Specimens with an IHC score of 0 or 1+ are considered negative and do not warrant further testing. The concordance between IHC 3+ and ISH positivity was high, with 94% concordance in the ToGA trial and 62% to 100% in the literature, with most studies reporting concordance of 90% or higher.^3,8,29,74-78 Because the benefit from the addition of HER2-directed therapy correlates with HER2 protein expression, initial HER2 testing should be performed by IHC. ISH should be reserved for IHC 2+ cases. In many studies,^{8,29,74,76,77} ISH-positive results have been observed in 30% to 50% of IHC 2+ tumors. Of note, there can be interobserver variation in the interpretation of HER2 IHC, and the reproducibility of 1+ and 2+ scores can be low. If the IHC score is borderline and the distinction between 1+ and 2+ is challenging, HER2 ISH can be considered. However, this approach is not recommended for cases that show an obvious 1+ IHC score. In most cases, ISH is not indicated if IHC scores are 0, 1+, or 3+. An algorithm for pathologists for HER2 testing in patients with GEA is presented in Figure 2.

Fig 2. Algorithm for pathologists. Additional recommendations: Pathologists should ensure that biopsies or resection specimens used for HER2 testing are rapidly placed in fixative, ideally within 1 hour (cold ischemic time) and are fixed in 10% neutral buffered formalin for 6 to 72 hours. Routine histology processing and HER2 testing should be performed according to analytically validated protocols. Pathologists should identify areas of invasive adenocarcinoma and also mark areas with strongest intensity of HER2 expression by IHC in GEA specimen for subsequent scoring when ISH is required. GEA, gastroesophageal adenocarcinoma; IHC, immunohistochemistry: ISH, in situ hybridization. (*) Tumor cell cluster is defined as a cluster of five or more tumor cells.

Pathologists should use the Ruschoff/Hofmann method in scoring HER2 IHC and ISH results for GEA (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

The scoring system (Table 3) used in the ToGA trial and subsequently modified for biopsies has been used in many studies and has shown excellent correlation between IHC and gene amplification methods.^13,42 The IHC is subjectively scored by using the established criteria on a four-tiered scale as 0, 1+, 2+, and 3+, with scores of 0 and 1+ considered negative, 3+ as positive, and 2+ as equivocal. Representative examples of IHC in GEA specimens are shown in Figures 3A through 3D. Similar to breast cancer, only membranous staining, but not cytoplasmic staining, is considered for HER2 scoring, but unlike breast carcinoma, complete membranous staining is not required for positivity. Often the luminal surface of tumor cells fails to stain in HER2-amplified GEA. Only luminal surface staining in the absence of lateral and basal staining is considered negative. Assessment of IHC as weak, moderate, or strong for scoring is similar to that used for breast carcinoma and is subjective, and thus can be a source of intraobserver and interobserver variability. Scoring using automated image analysis or virtual microscopy can be objective but has not been shown to improve reproducibility.⁷⁹ A few studies have shown good concordance of image analysis with a visual method for scoring HER2 IHC results; however, at this time there are limited data to make a specific recommendation for or against using image analysis for scoring HER2 in clinical practice.^34,79,80

Table 3. Scoring Guidelines for Interpretation of HER2 Immunohistochemistry in Gastric Carcinoma

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Fig 3. HER2 immunohistochemistry showing representative cases for scoring. (A) Negative 0: no reactivity, specifically no membranous reactivity is seen in any of the tumor cells. Any cytoplasmic staining is disregarded for scoring purposes. (B) Negative 1+: tumor cells with faint/barely perceptible membranous staining. (C) Equivocal 2+: tumor cells with weak to moderate complete, basolateral and lateral membranous staining. Columnar cells that are sectioned tangentially tend to show a complete membranous staining pattern. (D) Positive 3+: tumor cells with a strong complete, basolateral and lateral membranous reactivity. Also note that cells showing a complete membranous staining pattern are often tangentially sectioned columnar cells. Original magnification × 40 (A, C, D) and × 20 (B).

Various in situ visualization techniques used to evaluate HER2 amplification, include FISH and brightfield ISH using either HER2 probe or dual HER2 and centromere (CEP17) probes, and all are acceptable strategies. At least 20 nonoverlapping nuclei of tumor cells are evaluated for HER2 probe and CEP17 probe signal enumeration (Figs 4A-4C). A ratio of HER2 signal to CEP17 signal of ≥ 2.0 is considered positive, and a ratio of HER2 signal to CEP17 signal < 2.0 is considered negative. To score ISH/FISH results, first scan the stained slide in all areas designated as invasive tumor to identify areas with higher level HER2 amplification. In these areas, score both amplified and adjacent nonamplified cells that have cytomorphology consistent with malignant cells. Proceed to score in areas marked as strongest IHC intensity, if this information is available, as areas of overexpression may signify gene amplification in heterogeneous tumors.^8,42,81-83 Proceed to other invasive tumor areas until at least 20 cells are scored. Extra (three or more) copies of CEP17, on average, were noted in 4.1% of gastric cancers in the ToGA trial. This phenomenon has been referred to as “polysomy” but technically is not polysomy in many cases, as the entire chromosome is not duplicated. Rather, the extra copies of CEP17 are due to an intrachromosomal segmental duplication overlapping the centromere of chromosome 17, typically also involving the HER2 gene.⁸⁴ In such cases, there are often four to six copies of both HER2 and CEP17 signals with a ratio < 2.0.

Fig 4. HER2 and chromosome enumeration probe 17 (CEP 17) fluorescence in situ hybridization show scores of representative cases. (A) Not amplified: ratio 1.0. Mean number of HER2 signals per cell is 1.9; mean number of CEP 17 signals per cell is 1.8. (B) Not amplified: ratio 1.3. Mean number of HER2 signals per cell is 3.4; mean number of CEP 17 signals per cell is 2.7. Segmental duplication (or polysomy) likely accounts for signal numbers > 2 per cell. (C) Amplified: ratio 3.0. Mean number of HER2 signals per cell is 5.2; mean number of CEP 17 signals per cell is 1.7.

If IHC is 2+ and there are three or more CEP17 signals, on average, with a ratio < 2, then presence of more than six HER2 signals, on average, is interpreted as positive for HER2 amplification by ISH/FISH; fewer than four HER2 signals, on average, is interpreted as negative for HER2 amplification; and four to six signals, on average, indicates another 20 cells should be scored in a different target area. If additional scoring does not allow a definitive result to be rendered, then multiple options are feasible: (1) consultation between scorer and pathologist regarding selection of malignant cells or tumor areas for scoring, (2) switching out CEP17 for an alternative chromosome 17 probe in a retest to calculate the ratio with a new probe, (3) selecting a different tumor block for HER2 testing, (4) using genomics or an alternative analytic method to evaluate HER2 amplification.

Of note, there are currently no definitive studies in the literature on interpreting monosomy of CEP17 in GEA. Furthermore, true monosomy for CEP17 is difficult to distinguish from truncated cells in thin sections, and there are no data on how to interpret CEP17 monosomy even if it were confirmed by orthogonal methods. Until further data are available, relying on the ratio of HER2 to CEP17 signals remains a reasonable strategy for analyzing ISH/FISH results.

Each testing laboratory should specify the section thickness required for HER2 ISH/FISH analysis. Section thickness is especially important for single-probe assays in which absolute counts per cell determine scoring, and this is a major reason why single-probe ISH methods are not recommended. In contrast, dual-probe assays are recommended because they rely on a ratio of HER2 to CEP17 signals, which are less affected by section thickness. We recommend 4-µm–thick paraffin sections unless validation studies demonstrate accurate results when using alternative specimen preparation, or if an FDA-approved kit specifies that another thickness be used. Thinner sections can yield greater sampling error, fewer cells that qualify for scoring by virtue of having at least one signal for each of the two probes, and less intense counterstain. Thicker sections can lead to the presence of overlapping nuclei and more difficulty with deparaffinization, protease digestion, and probe or detection reagent dispersion processes.

The exogenous control slide should be scored to ensure that the assay protocol performed as expected. In each patient specimen, ensure adequate staining and counterstaining without background interference, overdigestion, or other artifacts. Failure to detect probe signals in nonmalignant cells (fibroblasts, endothelial cells, inflammatory cells, benign epithelial cells) serves as an indicator of poor-quality hybridization. At least some of these nonmalignant cells are expected to have up to two copies per cell of HER2 and CEP17 discrete signals, serving as a quality check for DNA preservation, reagent perfusion, and sufficient signal-to-noise ratio. In malignant-appearing cells, discrete signals are enumerated, or an estimate of signal number is done when there are numerous overlapping signals (clusters). Correlation of the scored region(s) on the ISH slide with the tumor cell population marked on the IHC slide is essential to ensure that the scored cell population is tumor. In cases where it is difficult to demarcate the tumor cell population on the slide, direct pathologist review of the ISH slide and comparison with the morphology of the tumor on the IHC and hematoxylin-eosin–stained section is often necessary.

Interpret the HER2 test result as indeterminate if technical issues prevent reporting as positive or negative. Examples of technical failures include improper specimen preparation or handling, quality checks outside acceptable limits, or artifact interfering with analysis or microscopy. Several manufacturers market reagents for HER2 ISH, but as of the date of this publication only one manufacturer has FDA approval for GEA (Dako, eg, HER2 IQFISH pharmDx). The HER2 FISH pharmDx test used in the ToGA trial is no longer available. Manufacturer’s instructions are often helpful for guiding analysis and interpretation of results. In the practice-changing ToGA trial, Hofmann et al¹³ recommended modifications to the duration of pepsin and that temperature stability should be achieved during pretreatment.

Pathologists should select the tissue block with the areas of lowest grade tumor morphology in biopsy and resection specimens. More than one tissue block may be selected if different morphologic patterns are present (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: recommendation/moderate).

As mentioned previously, studies show that HER2 overexpression is strongly associated with the intestinal phenotype, and less frequently with the diffuse (signet ring cell) phenotype of GEA. The rates of HER2 positivity vary for intestinal (3% to 23.5%), diffuse (0% to 6%), and mixed histology (0% to 20%) cancers.^36,85-88 GEA has rare morphologic phenotypes that include adenosquamous, papillary, and neuroendocrine carcinomas,⁷⁴ but data regarding HER2 expression in such morphologic variants are limited. Most studies^8-10,57,86 have shown anatomic variation with HER2 expression/amplification being greater at the gastroesophageal junction than in the stomach (32.2% v 21.4%). Correlation of HER2 expression and/or amplification with histologic grade is difficult to ascertain, as studies have used different methods including two- to four-tiered grading systems. Further, most studies do not specify the criteria used for grading, and grading is subjective. The American Joint Committee on Cancer recommends using a three-tiered system of well differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3). Rare undifferentiated carcinomas are classified as G3 in this system. HER2 positivity seems to be more strongly associated with low-grade than high-grade tumors and varies from 15% to 45% for low grade, and 6% to 28% for high grade, in different studies.^36,86,89-91 When choosing a tissue block, selecting one with the lower grade or intestinal morphology appears more likely to yield HER2-positive results and is thus recommended. If the cancer comprises substantially different grades or histologic patterns, it is reasonable to test different areas, which may require selection of more than one block.

Laboratories should report HER2 test results in GEA specimens in accordance with the CAP Template for Reporting Results of HER2 (ERBB2) Biomarker Testing of Specimens From Patients With Adenocarcinoma of the Stomach or Esophagogastric Junction⁹² (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

The synoptic content of this template lists essential reporting elements. Element selection and the manner in which these elements are reported are at the discretion of the medical professional who issues the report. Key elements are listed in Table 4. The report should include a brief Methods section describing the kit or the critical reagents and instruments used. For a gene test, include the correct gene symbol (ERBB2) as approved by the Human Genome Organization Nomenclature Committee, following the colloquial symbol (HER2 [ERBB2]). “Number of observers” refers to the number of laboratory professionals who performed scoring for ISH or the number who further interpreted the results of any automated scoring system. The reporting professional is responsible for ensuring quality of the result via analytic interpretation of raw data and via use of validated protocols for preanalytic and analytic phases of testing.^93,94 Published guidance from the CAP describes general report elements that promote accurate communication of test results.^95,96

Table 4. Key Reporting Elements

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Pathologists should identify areas of invasive adenocarcinoma and also mark areas with strongest intensity of HER2 expression by IHC in GEA specimens for subsequent ISH scoring when required (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

This recommendation is intended to provide guidance on which parts of the slide to prioritize when scoring cells in ISH assays. Accurate ISH results scoring depends on three aspects of preanalytic histopathologic features that help localize regions to score: (1) areas of invasive tumor, (2) areas of intense HER2 overexpression as visualized on IHC, and (3) cytomorphology of the malignancy to help select individual cells for scoring. Areas of invasive carcinoma are identified on hematoxylin-eosin–stained sections adjacent to the unstained section used for hybridization. If there are distinct and separate histologic patterns of malignancy, different areas can be marked for ISH scoring, although there are few data to suggest that outcome is improved by separate scoring of each histologic subtype. More important is that invasive cancer is marked so that the scorer may scan these areas to identify regions enriched for amplification to prioritize for scoring.

“Heterogeneity” typically refers to intratumor variation in genotype or gene expression. In gastric cancers, this term is used when there is focal positivity by IHC or ISH. Ideally, a HER2 IHC stain of the same block used for ISH should be reviewed to find areas of maximum HER2 intensity irrespective of histologic subtype or grade. False positivity can be seen in areas of intestinal metaplasia, adjacent to ulcer sites, or in high-grade dysplasia, and these lesions should be avoided. Crush artifact and necrotic tissue also should be avoided. Areas with strongest IHC intensity may signify gene amplification in heterogeneous tumors.^8,42,81-83

Good communication between the histopathologist and the scorer is critical for resolving difficult interpretations. If the proportion of malignant cells (as a proportion of all nucleated cells in the marked area) is low, the pathologist should communicate this to the ISH laboratory and mention this in the report, as low tumor cell content reduces confidence in the ISH results. The pathologist should also note the pattern of malignant cells (glands v diffuse, sheets of tumor cells v interspersed benign inflammatory/stromal cells) and the shape and relative size of the malignant cell nuclei (round v oval, medium v large) to assist scorers in identifying those malignant cells after hybridization and counterstaining. The goal is to maximize the proportion of malignant cells scored, while minimizing the proportion of nonmalignant cells scored. Morphologic evaluation of ISH stains helps resolve problematic interpretations due to overfixation or underfixation, delayed fixation with or without tissue-drying artifacts, inadequate deparaffinization, or predicting the value of repeating the test using shorter or longer protease digestion duration.

Tissue architecture and cytology are often better visualized in brightfield ISH than FISH, as in brightfield ISH the morphologic features of malignant cells are typically more distinguishable from those of benign cells, potentially improving the signal-to-noise ratio. However, compared with immunostains, brightfield ISH may suffer from less crisp histopathology because of the protease digestion required to promote probe dispersion into nuclei, and because of the near-boiling heat required to achieve DNA denaturation. The FISH signals are often brighter and easier to count than are brightfield ISH signals.⁹⁷ Nevertheless, as stated previously, ISH and FISH results are generally concordant in GEA,^{59,61,65,98,99} and either method is considered acceptable.

Laboratories must incorporate GEA HER2 testing methods into their overall laboratory quality improvement program, establishing appropriate quality improvement monitors as needed to ensure consistent performance in all steps of the testing and reporting process. In particular, laboratories performing GEA HER2 testing should participate in a formal proficiency testing program, if available, or an alternative proficiency assurance activity (Type: evidence based; Quality of evidence: moderate/intermediate; Strength of recommendation: strong).

Although an HER2-expressing breast specimen may be initially used as the positive control,¹⁷ validation of actual GEA specimens is preferred, when such appropriate specimens are available. Gastric cancer cell lines with HER2 expression may be used as the positive control¹⁰⁰ when a sufficient number of actual GEA specimens are unavailable, and the procedure should be specified and documented, since it may differ from those of breast. Checklists for recording positive and negative controls for each test should be incorporated into the laboratory quality improvement program (CAP or other available local programs). Given the heterogeneity of HER2 reactivity in GEA,^{74,75,85,101-104} laboratories may consider tracking their own statistics of HER2 results in GEA, including interobserver reproducibility between pathologists and the histologic subtypes, which may facilitate a better understating of the relevant issues in HER2 testing in GEA.^105-109 Continuing education of pathologists who report on HER2 GEA specimens is important, especially in laboratories performing limited numbers of GEA specimens in comparison to breast specimens.

There is insufficient evidence to recommend for or against genomic testing in GEA patients at this time.

In addition to IHC and ISH, other techniques have been used to determine HER2 status. These technologies include polymerase chain reaction (PCR), single-nucleotide polymorphism chip, comparative genomic hybridization array, gene expression profiling by RNAseq or microarray, targeted/exome/whole genome sequencing, or proteomics.^84,110 Most studies comparing these technologies to standard HER2 test methods have been carried out in breast cancer.¹¹¹ High concordance has been demonstrated for HER2 status in GEA with droplet digital PCR, when compared with IHC and FISH.¹¹² Gene expression profiling using eight transcripts has been shown to predict response to trastuzumab- and docetaxel-based chemotherapy in GEA with HER2 overexpression.¹¹³

Multiplex ligation-dependent probe amplification is a multiplex PCR technique that simultaneously quantifies several gene segments. This technique can be used to interpret whether the HER2 region of the chromosome is amplified compared with control regions of chromosome 17.¹¹⁴ However, the control regions are difficult to select given that segmental amplifications of chromosome 17, or polysomy 17, may or may not be present in a given tumor. Furthermore, when tissue is ground up to carry out nucleic acid extraction, varying proportions of nucleic acid from malignant and benign cells are represented in the assay, in comparison to IHC and ISH, in which cytologic and morphologic features may help limit interpretation of malignant cells. Thus, the criteria for interpreting gene amplification are difficult to set when using genomic technology. Ideally, the criteria for tissue selection for analysis, and for interpretation of genomic test results, would be validated with tissues from drug responders versus nonresponders.

At this time, the main utility for genomic testing is to help classify cases that are uninterpretable with standard IHC or ISH technology, such as in the setting of borderline amplification with or without extra centromere 17 signals by ISH.¹¹⁴ Currently, however, there is insufficient evidence to provide recommendations for or against the routine use of genomic technologies for purposes of qualifying for HER2-targeted therapy.

Pathologists should ensure that biopsy or resection specimens used for HER2 testing are rapidly placed in fixative, ideally within 1 hour (cold ischemic time), and are fixed in 10% neutral buffered formalin for 6 to 72 hours. Routine histology processing, and HER2 testing, should be performed according to analytically validated protocols, and laboratories should establish policies to ensure efficient allocation and utilization of tissue for ancillary testing, particularly in small specimens. Validation studies must address preanalytic factors supporting the stated range of acceptable tissue preparations (eg, 10% neutral buffered formalin, alcohol fixatives, decalcification, air-dried smears, formalin–post fixation). Laboratories should test a sufficient number of GEA cases to ensure that assays consistently achieve expected results.

GEA specimens need prompt fixation for ideal histology, IHC, and ISH testing. Biopsy specimens should be immediately placed into formalin in the endoscopy suite. Pathologists should communicate with gastroenterology colleagues to ensure prompt fixation and documentation. Surgical specimens require prompt specimen transport and opening of the specimen (by pathologist or appropriately trained personnel) to ensure prompt exposure of the tumor to adequate volumes of 10% neutral buffered formalin. Surgical specimens may need to first be inked, the tumor incised, and the specimen pinned on a cork or wax board to facilitate fixation. Pathologists should work with surgeons, nurses, and/or operating room personnel to facilitate recording of surgical specimen ischemic time and appropriate handling.¹¹⁵

Considerations regarding tissue ischemic and fixation time follow from principles of proteolytic degradation and fixation chemistry,^115,116 with data drawn mostly from the breast cancer literature.^{16,17,115,117}

There is a need for direct data regarding the impact of ischemic time (time from specimen removal from the patient to fixation) and fixation time (time tumor is exposed to adequate volumes of formalin) on HER2 testing in GEA. One model using gastric cancer cell lines of known HER2 expression xenografted into mice demonstrated decreased IHC staining with delayed fixation of 6 and 24 hours with the Hercept test, and decreased HER2 to CEP17 FISH ratios, as compared to immediate fixation.¹⁰⁰ This delayed fixation resulted in negative IHC and FISH interpretation for several samples with expected 2+ IHC staining and HER2 to CEP17 ratio of 2.3 (SHC cell line). Unfortunately, ischemic intervals between 0 and 6 hours were not tested.¹⁰⁰ This same study demonstrated no effect of prolonged fixation of 5 and 7 days as compared to 24-hour fixation, but noted diminished IHC staining with 10-day fixation, or use of fixatives other than 10% neutral buffered formalin.¹⁰⁰

Full validation of the HER2 testing protocol should be performed for FFPE specimens, as described previously in Recommendation 4. Discussion of limited available data for alternatively fixed or decalcified specimens is provided below.

Regarding cytologic specimens, we are aware of a single small study (mentioned previously) of HER2 testing in gastric cancer effusion specimens (formalin-fixed plasma thrombin clots), which demonstrated concordance with tissue specimens in all of 18 cases, but acknowledged more granularity of HER2 staining, and difficulty in interpreting membrane staining in discohesive tumor preparations.³⁹ A sampling of studies comparing cytologic cell block preparations with FFPE breast carcinoma specimens evaluated for HER2 by immunohistochemistry demonstrates 87% to 100% positive agreement and 66% to 100% negative agreement (excluding 2+ equivocal scores)^39,118-124; however, one small study exploring ethanol, cytolyte, and formalin-fixed cytologic breast cancer specimens is calculated to have only 14% to 40% positive agreement and 100% negative agreement with matched FFPE breast tissue samples (again excluding 2+ scores).¹²⁵ Several studies^39,120,122 reported false-positive interpretations, some attributed to cytoplasmic background staining.

There are wide differences in the handling and processing of cytologic preparations between studies and between laboratories (eg, proprietary fixative, alcohol-based fixative, alternative fixative followed by formalin fixation, direct formalin fixation),^{39,118-122,125,126} and effects vary by antigen and/or antibody.¹²⁷ This further emphasizes the need for appropriate evaluation of HER2 staining of cytologic specimens in individual laboratories before testing and reporting patient samples. Nonformalin fixatives also have complexities for HER2 ISH testing,^122,128,129 yet several studies^123,124,130 have shown good results with HER2 FISH on cytologic breast cancer specimens.

Diminished IHC staining occurs after decalcification with a variety of antigen-antibody combinations,^127,131 yet studies of HER2 antibodies are lacking. Prolonged hydrochloric acid–based decalcification after formalin fixation was shown to have deleterious effects on the HER2 ISH assay in a breast tumor and xenograft study.¹²⁹ Again, decalcification protocols vary widely among laboratories, reinforcing the need for local assay evaluation. Although it remains impractical to fully validate every specimen variation (cytology, decalcification), laboratories should confirm test performance of HER2 assays on these types of specimens before reporting patient results (with testing paradigm to be determined by the laboratory director, based on local practices).

Laboratories must provide clinically appropriate turnaround times and optimal utilization of tissue specimens by using appropriate techniques (IHC and ISH) for HER2 in GEA. To inform therapeutic decision making, HER2 results should be reported promptly. The panel recommends a benchmark of 90% of reports available within 10 working days from the date of procedure or specimen acquisition. Laboratories that require send-out of tests for HER2 testing in GEA should process and send specimens to reference laboratories in a timely manner. The panel suggests that a benchmark of 90% of specimens be sent to the reference laboratory within 3 working days of tissue processing.

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/site/ifc.

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Consulting or Advisory Role: Genentech/Roche, Sanofi, Bristol-Myers Squibb, Merck Serono, Merck/Schering Plough, Spectrum Pharmaceuticals, Lilly/ImClone, Celgene, Genomic Health, National Cancer Institute, Vicus Therapeutics, Pharmacyclics, Precision Therapeutics, Taiho Pharmaceutical, Bayer, Alchemia, Infinity Pharmaceuticals, Boehringer Ingelheim, Astellas Pharma, EMD Serono, IntegraGen

Research Funding: Genentech (I), Gilead Sciences, Amgen, Astellas Pharma, Advanced Accelerator Applications, Bayer/Onyx, Novartis, Alchemia, AVEO, Infinity Pharmaceuticals, Merck Serono (Inst), EMD Serono (Inst)

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Consulting or Advisory Role: Roche, Bayer, Merck, Sanofi, Celgene, Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme Oncology, Baxalta

Speakers’ Bureau: Bayer, Merck, Celgene, Amgen

Travel, Accommodations, Expenses: Roche, Merck, Celgene

Consulting or Advisory Role: Beacon LBS, McKesson

Research Funding: Illumina

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Honoraria: Ventana Medical Systems

Honoraria: Lilly/ImClone, Bayer, Novartis, Five Prime Therapeutics, Taiho Pharmaceutical, Genentech, Celgene

Research Funding: Novartis, Bristol-Myers Squibb, Taiho Pharmaceutical, Roche/Genentech, MedImmune, Amgen, Lilly/Imclone, Merck, Delta-Fly Pharma, Gilead Sciences, Takeda, Celgene

Patents, Royalties, Other Intellectual Property: I have research funding from: Genentech, Roche, BMS, Taiho, MedImmune, Merck, Amgen, Lilly

Travel, Accommodations, Expenses: Novartis, Bayer, Five Prime Therapeutics