Table 1.
Biomarkers included in this study
| Marker/protocol | Clinical relevance | In practice since | Testing methods* and algorithms† | Relevant issues | Why important example of practices |
|---|---|---|---|---|---|
| Breast cancer | |||||
| HER2 | Predicts response to anti-HER2 therapies: trastuzumab (32–34), lapatinib (40,41), pertuzumab (42), ado-trastuzumab emtazine (43) | 1998 | Multiple methods exist: IHC, ISH (FISH, CISH, SISH, DISH) (44–49); other methods are studied (eg, RT-PCR50) Guidelines recommend 2 algorithms: “IHC, reflex‡ IHC2+ to ISH” and “ISH, confirmed by counting additional cells, repeat ISH or reflex to IHC” (14,20) Literature also recommends expanded reflex testing to increase accuracy: reflex IHC0-IHC1+ (“believe the positive”) algorithm (51); reflex IHC3+ (52–54) |
Accuracy and reproducibility of methods, especially FISH vs IHC, are hotly debated (11,55-60) FISH more accurate but more costly, more complex than IHC and other ISH; IHC widespread, cheaper but more prone to quality problems (44,45) >46 studies compare FISH and IHC (61), but no consensus; other methods less studied (45,61) Testing quality problems found in the past (62–64); national efforts to improve quality (17,65); quality is improving but still deficient (66) |
HER2 expanded to gastric cancers; is studied in other cancers (67–69) HER2 challenges are relevant to other markers where multiple testing methods and algorithms are debated (10,59,70) Future new markers may complicate HER2 algorithm and require multibiomarker protocols (11) |
| ER, PgR | Prognosis; stratification; ER guides use of endocrine therapy (71–73) | 1970s | IHC is the only method recommended in guidelines (21) | Testing quality problems found (74) National efforts to improve quality (21) |
IHC is broadly used for other markers; studied as a screening method for molecular testing, eg, for ALK, EGFR (75,76) |
| Multibiomarker protocols | Confirm triple-positive or -negative diagnoses | N/A | Retest HER2 for triple- positive (HER2+, ER+, PgR+) or triple-negative (HER2-, ER-, PgR-) results to ensure quality | Emerging practice to ensure accurate biomarker assessment (77) | Example of a potential emerging practice to improve testing quality for most difficult patient subgroups |
| Gastric and esophageal cancers (GEC) | |||||
| HER2 | Predicts response to trastuzumab in advanced cancers (35) | 2010 | Multiple methods exist: IHC, ISH (FISH, BISH, CISH, dc-SISH, dc-CISH) (19,78,79) Guidelines recommend “IHC, reflex IHC2+ to ISH” (16) Literature also recommends: ISH (68,78,80) concurrent ISH and IHC (80,81); expanding reflex testing: IHC1+ (82), IHC0-IHC1+ (for biopsy samples) (83) |
Lack of detailed guidelines similar to ASCO CAP guidelines in breast HER2 testing (81,83) Testing algorithms differ from BC HER2 (35,84) Relatively small number of studies to inform practices (81,83) Emerging debate on FISH vs IHC and ISH methods (79,85) |
Opportunity to understand how testing practices for one marker expand to additional tumor sites The number of biomarkers in GEC is expected to increase (86,87),requiring future multimarker protocols |
| Non–small cell lung cancer, nonsquamous carcinomas (NSCLC) | |||||
| EGFR | Predicts response to EGFR inhibitors (gefitinib, eroltinib) (36–38) | 2009 | EGFR mutation testing is guideline recommended (15,18,88); multiple methods exist (sequencing, PCR, other) IHC is emerging but controversial (12): IHC proposed as screening, followed by molecular analysis (18,75) EGFR amplification is not guideline recommended (18) |
Guidelines leave room for multiple methods: any validated EGFR mutation test can be used (18) Various methods have pros and cons (89,90) Emerging debate on methods, including IHC algorithms (70,75) |
Example of how HER2 debate on testing methods permeates new biomarker areas (70,75) |
| ALK | Predicts response to ALK inhibitor crizotinib (39) | 2011 | FISH is FDA approved and guideline recommended (15,18); other methods are emerging: IHC (in an algorithm with FISH) (18,91–93), RT-PCR (94,95), CISH (95) | Remarkably rapid time from discovery to approval (23,96) FISH gold standard, but more costly, complex; IHC widespread, easier, cheaper (76,91-93) Debate emerges on which method and algorithm are best (76,92,93) |
Example of a rapidly developed biomarker, requiring expedient implementation (23,96) Example of how HER2 debate on testing methods permeates new biomarker areas (76,91–93) |
| KRAS | Prognosis (15); role as predictive marker for therapy controversial (12,18,97) | 2000s | May be used as screening test to “rule-out” EGFR and ALK mutations (18) Methods used: RT-PCR, pyrosequencing, Sanger sequencing (98) |
Guidelines vary (15,18) | Novel use of a test as a rule-out may be applicable to other cancers where markers are mutually exclusive |
| Multimarker protocols§ | Facilitate testing of multiple biomarkers | N/A | EGFR, ALK, and KRAS mutations are mutually exclusive, allowing a sequential “rule-out” protocol (18,91,99) Literature recommends various sequential protocols (18,91,99) or concurrent testing (96,100) |
Protocols are debated and practices vary (13,18,76,100–102); guidelines recommend 5–10 day result turnaround time (18) Concurrent testing provides rapid turnaround and better sample management (13,96,101) Sequential testing is more economical, but longer total turnaround time (18) |
NSCLC is a paradigm for multimarker testing in cancer (103) |
* Method, type of technology for testing a biomarker. ALK = anaplastic lymphoma kinase; ASCO = American Society of Clinical oncology; BISH = bright-field situ hybridization; CAP = College of American Pathologists; CISH = chromogenic in situ hybridization; dc-CISH = dual color chromogenic in situ hybridization; dc-SISH = dual color silver in situ hybridization; DISH = dual in situ hybridization; EGFR = epidermal growth factor receptor; FDA = Food and Drug Administration; FISH = fluorescence in situ hybridization; HER2 = human epidermal growth factor receptor 2; IHC = immunohistochemistry; ISH = in situ hybridization; KRAS = v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; PgR = progesterone receptor; RT-PCR = reverse transcription polymerase chain reaction; SISH = silver in situ hybridization.
† Algorithm, a sequence of methods for testing one biomarker, when multiple methods are used.
‡ Reflex testing, confirming equivocal or borderline results by another method.
§ Multibiomarker protocol, a sequence of testing multiple biomarkers.