Ken's Notes

CAP / AMP / IASLC Molecular Testing Guidelines for Selection of Lung Cancer Patients

· Molecular Testing Guidelines (2018 Updated, also info from 2013/14)

· Pre-Analytical:

· Pathologists should select samples for lung cancer biomarker testing. (expert consensus opinion)

· A pathologist should assess tumour content of each specimen (expert consensus opinion)

· Pathologists should determine the adequacy of specimens for lung cancer biomarker molecular testing by assessing cancer cell content, tissue preservation, and nucleic acid quantity and quality. (Expert consensus opinion)

· Note also that mucin and necrosis may inhibit amplification

· A pathologist should guide a trained technologist to perform microdissection for tumour cell enrichment as needed

· Microdissection procedures: gross macrodissection from block, coring from block, macro or microdissection from glass slide, flow cytometric sorting, laser capture microdissection (LCM)

· LCM has been shown to have higher failure rates due to low DNA yield

· Testing of multiple areas within the same tumour is not necessary

· Multiple tumours:

· If separate primary lung adenocarcinomas, labs may test each tumour

· Fixation:

· FFPE, fresh, frozen, or alcohol-fixed

· AVOID:

· acidic (including decalcifying solutions)

· heavy metal fixatives

· Cell blocks or other cytologic preparations are suitable (recommendation)

· Recommendation: In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell-free plasma DNA (cfDNA) assay for EGFR.

· Samples for testing [EGFR testing guidelines (CAP/IASLC/AMP/ASCO) (2013/14, draft 2016)]:

· Lung adenocarcinoma, advanced stage (?stage IV) at diagnosis, or at progression in patients who originally presented with lower stage disease but were not previously tested (2016, strong recommendation)

· Physicians may use EGFR and ALK testing in tumors with histologies other than adenocarcinoma when clinical features indicate a higher probability of an oncogenic driver. (2016, strong recommendation)

· irrespective of clinical characteristics

· when adenocarcinoma cannot be excluded

· Including large cell carcinomas with IHC evidence of adenocarcinoma differentiation, or lacking IHC evidence of squamous carcinoma lineage

· TTF-1, p63 (or p40), and other relevant markers should be performed for poorly-differentiated carcinomas

· Lung cancers with mixed histology (ex. adenosquamous, mixed adeno/small cell), or where an adenocarcinoma compomnent cannot be completely excluded (biopsies, cytology) testing may be indicated but clinical criteria (young age, lack of smoking history, documented preceding adenocarcinoma) may be useful to select patients for testing

· Testing for early stage disease is encouraged but decision should be made locally by each lab in collaboration with its oncology team

· In lung adenocarcinoma patients who harbor sensitizing EGFR mutations and have progressed after treatment with an EGFR-targeted tyrosine kinase inhibitor, physicians must use EGFR T790M mutational testing when selecting patients for third generation EGFR-targeted therapy. (2016, strong recommendation)

· Primary tumours or metastatic lesions are equally suitable for testing, EXCEPT for metastasis or relapse after initially successful response to TKI treatment (i.e. acquired resistance)

· Discordances between primary and met mutation status are rare

· minimal sample requirements should be validated by each laboratory

· tumour content (proportion, and number of cancer cells)

· generally 50% for Sanger (25% allele frequency)

· sensitivity studies should be performed with more than 1 specimen to control for the variation in EGFR copies between tumours or cell lines

· tumour enrichment proceducres should also be assessed during test validation

· fixation

· quality

· Each laboratory should establish minimal cellularity requirements (proportion and number of tumor cells) during assay validation. (expert consensus opinion)

· Cytologic specimens are suitable for testing.

· Cell blocks and other cytologic preparations are suitable specimens

· FFPE, fresh, frozen, or alcohol-fixed specimens

· Recommendation: In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell-free plasma DNA (cfDNA) assay for EGFR. (2016)

· Expert Consensus Opinion: Physicians may use cell-free plasma DNA (cfDNA) methods to identify EGFR T790M mutations in lung adenocarcinoma patients with progression or acquired resistance to EGFR-targeted tyrosine kinase inhibitors; testing of the tumor sample is recommended if the plasma result is negative

· certain tissue treatments—such as decalcifying solutions, acidic or heavy metal fixatives —are not suitable for EGFR testing (specimens processed in these ways shoud not be used for EGFR testing)

· Heavy metal fixatives: Zenker, B5, B plus, acid zinc formalin

· Should not be used for EGFR testing

· Acidic solutions: Bouin, bone-decalcifying solutions

· Nonacidic chelating decalcifying solutions may better preserve DNA for molecular testing

· Unbuffered formalin spontaneously oxidizes to formic acid over time

· fixation time 6-48 h should give acceptable results

· Validation should be performed for each specimen type likely to be encountered, and testing should be reported only on validated specimen types.

· Testing is not necessary for patients who are being considered for palliative or hospice care only

· FFPE with low DNA content may lead to false positive results

· Formalin fixation artifact

· Taq DNA polymerase’s normal error frequency

· Duplicate amplifications on FFPE samples can help ensure accurate results

· Same deal if whole-genome amplification is used to increase yield

· Bidirectional sequencing and confirmatory sequencing of independent PCR products should be used if direct DNA sequencing methods are used

· Analytical / Method:

· Laboratories should employ, or have available at an external reference laboratory, clinical lung cancer biomarker molecular testing assays that are able to detect molecular alterations in specimens with as little as 20% cancer cells.

· Expert Consensus Opinion: Multiplexed genetic sequencing panels are preferred over multiple single-gene tests to identify other treatment options beyond EGFR, ALK, and ROS1

· EGFR [EGFR testing guidelines (CAP/IASLC/AMP/ASCO) (2013/14, draft 2016)]:

· Recommendation: Laboratories testing for EGFR T790M mutation in patients with acquired resistance to EGFR-targeted kinase inhibitors should deploy assays capable of detecting EGFR T790M mutations in as little as 4% of viable cells (2% of EGFR alleles). (2016 draft)

· conventional Sanger sequencing, even with microdissection, is considered insufficient for this testing

· sensitivity to detect mutations in samples containing >= 10% tumor cells is strongly encouraged. (expert consensus opinion, 2013)

· Note: ultrasensitive molecular assays (sensitivity below 1%) can be problematic due to false positives:

· Possible sources of false positives:

· Mispriming

· Low cross-contamination

· Very small mutated subclone

· Clinical management implications of AR mechanisms are still evolving without established treatment guidelines

· If a laboratory performs testing on specimens from patients with acquired resistance (AR) to EGFR kinase inhibitors, such tests should be able to detect the secondary EGFR T790M mutation in as few as 5% of cells.

· EGFR testing assays should be able to detect individual EGFR mutations with a reported frequency of 1% of all EGFR mutations. (expert consensus opinion)

· Limiting testing to the 2 major mutations is no longer considered acceptable (expert consensus opinion)

· See Table 12 in Lindeman et al. for a list of these mutations

· Routine EGFR assays for EGFR exon 19 deletions should be designed to detect not just the common 15-bp and 18-bp deletions, but also the less common 9-, 12-, 24-, and 27-bp deletions, as well as the uncommon 15-bp and 18-bp insertions. (expert consensus opinion)

· EGFR exon 18 should be analyzed for E709 and G719 mutations; exon 20 for S768, T790M, and insertions; and exon 21 for L858R, T854, and L861Q mutations. (expert consensus opinion)

· laboratories may consider offering 2 assays: a rapid assay for the most common mutations, which can be reported within a few days in cases of clinical urgency, and a more comprehensive follow-up assay to detect the remaining mutations, which may take longer to report. (expert consensus opinion)

· Physicians must use EGFR T790M mutational testing when selecting patients for third generation EGFR-targeted therapy. (2016, strong recommendation)

· Methods used include:

· Sanger with and without mutated allele enrichment, amplification refractory mutation system (ARMS), length analysis, restriction fragment length polymorphism, real-time PCR, high-resolution metling curve analysis, single-base extension genotyping (including mass spectrometry-based genotyping), and denaturing high-performance liquid chromatography, massively parallel sequencing

· IHC for total EGFR is not recommended for selection of EGFR TKI therapy

· it has been shown to correlate poorly or not at all with the presence of EGFR mutations

· IHC for mutated EGFR:

· If scoring cutoffs are set stringently to ensure a high positive predictive value, IHC with EGFR mutation–specific antibodies (for L858R, clone 43B2, and E746_A750del, clone 6B6) could be used as an initial screen to identify most patients who are candidates for EGFR inhibitors;

· Diffuse mutation-specific protein expression in tumor cells is highly correlated with EGFR mutation and as such predicts response to EGFR tyrosine kinase inhibitors.

· all specimens negative with these 2 mutation-specific monoclonal antibodies, that is, most samples overall, molecular testing is still needed.

· Tumors with faint cytoplasmic labeling should be designated as equivocal. This result can rarely occur both with and without mutation

· because KRAS and EGFR mutations are mutually exclusive, a rapid and inexpensive KRAS assay may be performed initially to exclude KRAS-mutated tumors from EGFR mutation testing as part of an algorithm designed to maximize testing efficiency, provided that the sample is sufficient to perform the KRAS test without sacrificing EGFR and ALK testing, and that the totality of clinically relevant molecular results can be obtained within the target TAT.

· EGFR copy number analysis is not recommended for selection of EGFR TKI therapy

· ROS1 Testing Guidelines (2016 draft):

· Physicians should use ROS1 molecular or cytogenetic testing on all lung adenocarcinoma patients, irrespective of clinical characteristics, when selecting patients for ROS1-targeted therapy. (Recommendation)

· Dr. Phil Cagle confirmed during an information session Nov. 30 that this should be interpreted that ROS1 is standard of care for advanced adenoCA

· Expert Consensus Opinion: Physicians may use ROS1 IHC as a screening test in lung adenocarcinoma patients; however, positive ROS1 IHC results should be confirmed by a molecular or cytogenetic method.

· ROS1 IHC:

· Absence of ROS1 protein expression in cancer cells suggests that this tumor is unlikely to harbor ROS1rearrangement and to respond to treatment with a targeted inhibitor, such as crizotinib.

· ROS1 protein expression in cancer cells is highly sensitive for a rearrangement involving ROS1 but is not entirely specific. Therefore, confirmatory molecular methods should be used when ROS1 protein expression is detected.

· Tumors with faint cytoplasmic labeling should be designated as equivocal. This result can rarely occur both with and without mutation.

· Other Molecular Testing (2016 Draft):

· There is currently insufficient evidence to support a recommendation for or against routine testing for ALK mutational status for lung adenocarcinoma patients with sensitizing ALK mutations who have progressed after treatment with an ALKtargeted tyrosine kinase inhibitor (2016 draft)

· Not sure but sounded like this might be elevated to similar status as T790M

· BRAF molecular testing is currently not indicated as a routine stand-alone assay outside the context of a clinical trial. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include BRAF in the panel done as an initial test or to identify other treatment options. (2016 draft, expert consensus opinion)

· RET molecular testing is not recommended as a routine stand-alone assay outside the context of a clinical trial. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include RET in the panel done as an initial test or to identify other treatment options. (2016 draft, expert consensus opinion)

· ERBB2 (HER2) molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include ERBB2 (HER2) in the panel done as an initial test or to identify other treatment options. (2016 draft, expert consensus opinion)

· No Recommendation: There is currently insufficient evidence to recommend IHC or FISH testing for ERBB2 (HER2) amplification or expression status to guide selection of therapy in lung adenocarcinoma patients.

· Note: There is insufficient evidence to recommend IHC or FISH testing for ERBB2 (HER2) amplification or expression status to guide selection of therapy in lung adenocarcinoma patients

· KRAS molecular testing is not indicated as a routine stand-alone assay as a sole determinant of targeted therapy. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include KRAS in the panel done as an initial test or to identify other treatment options. (2016 draft, expert consensus opinion)

· MET molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. As part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative, it is appropriate to include MET in the panel done as an initial test or to identify other treatment options. (2016 draft, expert consensus opinion)

· Physicians may use molecular biomarker testing in tumors with histologies other than adenocarcinoma when clinical features indicate a higher probability of an oncogenic driver. (2016 draft, expert consensus opinion)

· No Recommendation: There is currently insufficient evidence to support the use of circulating cell-free plasma DNA (cfDNA) molecular methods for the diagnosis of primary lung adenocarcinoma.

· No Recommendation: There is currently insufficient evidence to support the use of circulating tumor cell (CTC) molecular methods for the diagnosis of primary lung adenocarcinoma

· Validation:

· Strong recommendation: Laboratories must use clinically validated lung cancer biomarker testing methods with appropriate performance characteristics, following standardized best practice guidelines for each technology.

· Expert consensus opinion: Laboratories should establish laboratoryspecific requirements for the minimum proportion and number of cancer cells needed for mutation detection during validation

· [EGFR testing guidelines (CAP/IASLC/AMP/ASCO) (2013/14, draft 2016)]

· Technical validation, as required by CLIA

· CAP published recommendations and examples on validation for a variety of assays

· Validation should be performed for each specimen type likely to be encountered, and testing should be reported only on validated specimen types.

· Cell lines may be used, but not to the exclusion of clinical specimens except for rare mutations.

· FFPE cell pellets may be helpful, especially for mutations that are difficult to obtain

· While some rare EGFR mutations may not be obtainable, the common exon 19 deletions, L858R, T790M, G719, and exon 20 insertions are required

· tumour enrichment proceducres should also be assessed during test validation

· specificity of ultrasensitive methods (< 1% allele fraction) must receive additional attention

· multiple negative lung cancer specimens

· multiple no-template controls

· Precision studies should assess the reproducibility of the entire analytic process, beginning with the pathologist’s tumor assessment and enrichment strategies (eg, dissection)

· Comparison with clinical history of treatment response is suboptimal, but may be used as evidence of true positive mutated specimens, in the absence of another accredited laboratory for comparison

· Analytic sensitivity of EGFR testing should be assessed in DNA from mutated specimens with low tumor content, diluted both in water/buffer and in normal DNA, to determine tumor cell content, in terms of both absolute cell count and tumor percentage, at which accuracy and precision (reproducibility) deteriorate

· sensitivity studies should be done with more than 1 specimen, and the least sensitive result should be stated as the overall test sensitivity

· No template controls and very-low-concentration wildtype specimens are essential to establish specificity of ultrasensitive EGFR mutation detection methods.

· Ongoing QC/QA:

· Strong Recommendation: Laboratories should ensure that lung cancer biomarker testing follows similar quality control and quality assurance policies and procedures as for other clinical laboratory assays.

· [EGFR testing guidelines (CAP/IASLC/AMP/ASCO) (2013/14, draft 2016)]

· Controls for ongoing testing:

· Low-positive control specimen (near the lower limit of tumour content of specimens accepted by the laboratory) should be tested in each clinical assay run

· TAT:

· 2 weeks (10 working days) – within 2 weeks of receiving the specimen in the testing lab, results should be available for oncology team review (Expert consensus opinion)

· If > 2 weeks, lab should ensure that a more rapid in-house or reference lab testing option is available for specimens from patients with advanced stage lung cancer (Expert consensus opinion)

· 3 working days to send specimen to the molecular lab - Laboratories should establish processes to ensure that specimens that have a histopathological diagnosis are sent to the molecular pathology laboratory within 3 working days of receiving requests, or 24 hours to send to an internal molecular pathology laboratory (expert consensus opinion)

· Reporting:

· Recommendation: Pathologists and laboratories should ensure that lung cancer biomarker testing reports of all types include both results and interpretation sections readily understandable by clinical oncologists and by non-specialist pathologists

· Expert Consensus Opinion: Laboratories should ensure test results that are unexpected, discordant, equivocal, or otherwise of low confidence are be confirmed or resolved using an alternative method or sample.

· Preclinical:

· Patient

· Specimen

· Diagnosis

· Tumour content (percentage of total nuclei that are malignant)

· Extensive necrosis?

· Atypical specimen processing or fixation?

· Low total number of tumour cells?

· Results:

· Formal HGVS nomenclature

· More commonly used terminologies maybe, as requested by clinical care team

· Incidental findings, variants of uncertain significance, and benign polymorphic variants clearly presented as such

· the detection of ‘‘novel’’ mutations or mutations only reported very rarely should be viewed with great caution and should prompt replicate assays on new DNA extracts to rule out artifactual mutations due to formalin fixation, PCR errors, or whole-genome-amplification errors (if used). (expert consensus opinion)

· Interpretation:

· EGFR:

· Overall statement of the cancer’s likelihood to respond or resist EGFR TKI therapy

· Pretreatment T790M mutations and most exon 20 insertions are associated with lack of response to first-generation EGFR TKIs, and this should be communicated in the report (expert consensus opinion)

· Most studies have only rarely detected T790M in pretreatment samples. When it is detected in the pretreatment setting, it should be confirmed as either somatic or germline by testing of normal DNA from the patient. Germline T790M mutation has been associated with familial lung cancer, and therefore its detection should trigger evaluation of the family history and genetic counseling, keeping in mind that risk estimates and screening recommendations for unaffected T790M carriers remain to be determined

· Tumour content if relevant

· If tumour cellularity is in question, it may be appropriate to add a recommendation for repeated testing on additional material if it becomes available

· Inconclusive results clearly reported as such

· Reason for inconclusive result (as best is known) and suggest requirements for testing a different specimen that would be more likely to yield a successful result

· Technical section:

· Enough information for another labaratorian to understand what was done

· Assay sensitivity

· List of each variant tested (for targeted assays), or each exon sequenced (for sequencing assays)

· Table format ideally for multiplexed assays:

· Listing each clinically significant variant that is assessed

· Adjacent result for each

· Standard language regarding FDA oversight of laboratory-developed tests, as appropriate

· EGFR:

· Sensitizing EGFR mutations with a population frequency of at least 1% should be reported

· Testing algorithms:

· testing for KRAS may be performed initially to exclude KRAS-mutated tumors from EGFR and ALK testing

· If scoring cutoffs are set stringently to ensure a high positive predictive value, IHC with EGFR mutation–specific antibodies could be used as an initial screen to identify most patients who are candidates for EGFR inhibitors; however, for all specimens negative with these 2 mutation-specific monoclonal antibodies, that is, most samples overall, molecular testing is still needed.

· IHC for total EGFR, EGFR copy number analysis, and ALK real-time polymerase chain reaction, are not recommended as predictive assays.

· Stepwise-testing algorithms make more efficient use of resources, but pose a challenge for timely delivery of final results.

· Given this time constraint, we recommend that stepwise-testing algorithms, if used, should nonetheless be completed within 10 working days. (expert consensus opinion)

· ALK Testing guidelines (CAP/IASLC/AMP/ASCO) (2013/14):

· Samples for testing:

· Including large cell carcinoma with IHC evidence of adenocarcinoma differentiation

· Testing for early stage disease is encouraged but decision should be made locally by each lab in collaboration with its oncology team

· irrespective of clinical characteristics

· or when adenocarcinoma cannot be excluded

· FISH testing can be problematic when performed on alcohol-fixed samples (in contrast to EGFR mutation testing)

· FISH testing should ideally be performed on recently cut sections

· Glass slides:

· some slides designed for tissue microarrays have a heavy coating that generates a fluorescent matrix where tumor cells get embedded and cannot be properly treated for FISH-probe penetration.

· Other slides are designed for microdissection and do not hold the tissue adequately during pretreatment for FISH

· Testing method:

· Recommendation: When performing ALK testing, physicians can utilize IHC as an equivalent alternative to FISH. (2016 draft)

· Our opinion is that tumors that are positive for ALK IHC, either weakly or strongly, should still be referred to FISH for confirmation of a rearrangement. (2013)

· A substantial proportion of ALK-rearranged lung adenocarcinomas are not identified by the ALK1 antibody (used for lymphomas)

· ALK IHC:

· Absence of ALK protein expression in cancer cells suggests that this tumor is unlikely to harbor ALK rearrangement and to respond to treatment with a targeted inhibitor, such as crizotinib and ceritinib.

· ALK protein expression in cancer cells (based on platform criteria) predicts the presence of ALK rearrangement and response to therapy with a targeted inhibitor, such as crizotinib and ceritinib.

· Tumors with faint cytoplasmic labeling should be designated as equivocal. This result can rarely occur both with and without mutation.

· ALK FISH assay using dual-labeled break-apart probes

· A commercial assay (Abbott Molecular Probes, Abbott Park, Illinois) is available, and is FDA-approved

· Although FISH assays have been developed by using both break-apart and fusion strategies, the break-apart assay design has shown the best association with clinical outcome.

· If another set of probes or assay design is used, validation studies should demonstrate comparable or superior performance when compared to the commercial probes with regard to signal intensity, magnitude of signal splitting in positive cases, analytic precision, clinical sensitivity, and clinical specificity in accordance with published standards.

· For laboratories that elect to use laboratory-developed probes for ALK FISH testing, attention should also be given to batch variability of clones, DNA-labeling enzymes, and other reagents

· Moreover, any laboratory-developed tests should retain the ability to detect variant fusions of ALK with partners other than EML4.

· A pathologist should participate in the interpretation of ALK FISH slides, either by performing the analysis directly or by reviewing the interpretations of cytogeneticists or technologists with specialized training in solid tumor FISH analysis (expert consensus opinion)

· Typically, areas selected for FISH evaluation will be marked on a hematoxylin-eosin–stained slide that is directly parallel to the section used for FISH.

· Laboratories may follow the standard operating procedures that have proven to be successful for FISH on formalin-fixed, paraffin-embedded tissue sections in their setting

· Use of automated tissue processors and standardized commercial tissue digestion kits can improve consistency and should be considered.

· Experienced scorers who have undergone specific training in FISH in solid tumors should analyze the slides. (expert consensus opinion)

· The scorers should also have had training on the morphologic appearance of lung cancer, and should have easy access to assistance from a pathologist with training in FISH.

· Laboratories with experienced reviewers may use 1 scorer in cases with clearly negative or positive (.50% of cells) cases and a second scorer for less clear cases; otherwise 2 independent reviewers are recommended (expert consensus opinion)

· Interpretation should be performed in areas of the slide with good signal, in which at least 50% of all nuclei are easily analyzable, with minimal background or nuclear fluorescent ‘‘noise.’’

· The FISH signal intensity should be consistently greater than background intensity

· Areas where the borders of individual nuclei are not clearly identifiable and/ or high cell density causes excessive nuclear overlap are easy to misinterpret, and should be avoided.

· RT-PCR is not recommended as an alternative to FISH

· higher failure rate of an RNA-based assay in routine FFPE pathology material

· risk of false negatives, owing to variability in the EML4-ALK fusion structure and the existence of other ALK fusion partners

· Testing for secondary mutations in ALK associated with acquired resistance to ALK inhibitors is not currently required for clinical management.

· Validation:

· ALK-positive cases with split signals and with loss of signals should both be included in validation sets

References:

· Lindeman NI et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. Arch Pathol Lab Med 2018;142:321-346. (Currently just finished Table 3 at top of page 325)

· Lindeman et al. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association of Molecular Pathology. Arch Pathol Lab Med 2013;137:828-860.

· CAP – Template for Reporting Results of Biomarker Testing of specimens from patients with non-small cell carcinoma of the lung (June 2016)