Best Practices for Molecular Testing to Guide Optimal Treatment Selection for Patients With Lung Cancer

Course Director

Anjali Saqi, MD

Anjali Saqi, MD
Columbia University Medical Center
New York, New York

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Columbia Technology Ventures


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Part 1 of a 2-part series

Dr. Saqi provides expert feedback to the questions submitted by your peers during a recent survey on this topic.

Overview

The ability to detect driver mutations such as EGFR and EML4-ALK in tumor specimens from patients with lung cancer and administer agents targeting specific molecular profiles is revolutionizing the management of lung cancer, particularly non–small-cell lung cancer (NSCLC). Based on timely and accurate molecular testing, the treatment of patients with NSCLC can be individualized, ensuring that the patients receive the most effective therapy and achieve the best possible outcome. A number of guidelines for the optimal assessment and management of lung cancer are available, including the National Comprehensive Cancer Network (NCCN) guidelines for NSCLC1 and the molecular testing guidelines for the selection of patients with lung cancer for EGFR and ALK tyrosine kinase inhibitor (TKI) therapy developed by the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP).2

However, there are still many unanswered questions and significant practice gaps in applying the latest evidence and recommendations to practice persist. Pathologists, oncologists, and other specialists involved in the care of patients with lung cancer must effectively collaborate in order to ensure timely and adequate tissue collection and processing for molecular testing, remain current on the latest testing methodologies, and be able to interpret the results of molecular testing in a meaningful way to guide and tailor the therapy of their patients with lung cancer.


Disclosures

This activity is supported by an educational grant from Genentech
Additional support provided by Penn State College of Medicine and Answers in CME.

Course Director
Anjali Saqi, MD, has no financial interests/relationships or affiliations in relation to this activity.
Medical Director
Kadrin Wilfong, MD
Answers in CME, Inc.
Kadrin Wilfong, MD, has no financial interests/relationships or affiliations in relation to this activity.

Answers in CME staff who may potentially review content for this activity have disclosed no relevant financial relationships.

Penn State College of Medicine staff and faculty involved in the development and review of this activity have disclosed no relevant financial relationships.

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What are the specimen collection and processing requirements in molecular testing for lung cancer, and how can pathologists and other clinicians best work together to ensure that tissue in sufficient quantity and of sufficient quality is collected in an appropriate and timely manner?

Dr. Saqi: Lung cancer is the leading cause of cancer-related deaths worldwide.3 In the recent past, however, significant progress has been made in the field of lung cancer, including in the understanding of underlying pathogenesis, molecular diagnostic tests, and treatment options. In order to maximize the benefits from these advances, we need to optimize the manner in which lung cancer specimens are managed.

For patients who present at an early stage or have a resectable tumor, requirements for specimen collection and processing for molecular testing are relatively straightforward. Typically, the resected cancers provide sufficient tissue to perform the necessary molecular tests.1,2

However, obtaining sufficient tissue for molecular testing from patients who present at an advanced stage is challenging. Often these patients are not surgical candidates, and they typically undergo a minimally invasive procedure, such as a fine-needle aspiration biopsy or a core biopsy, to obtain a tissue sample and determine the appropriate management.1,2 Relative to their surgical-resected counterparts, samples obtained from the fine-needle aspirations and core biopsies are much smaller, and since these smaller samples may be the only tissue obtained from patients, which applies to approximately 70% of cases, it's critical that pathologists and clinicians work together to maximize their yield.1,4
 
Several published studies and personal experience demonstrate that molecular testing is feasible and provides results similar to those obtained from larger resected cancers.5,6 In order to ensure that sufficient quality and quantity of material is obtained, it is necessary for the pathologist to coordinate efforts with clinicians obtaining the samples to ensure appropriate triage and processing.

So there are several steps that can be undertaken to maximize the results. First, let us address the core biopsies. These are often obtained under CT guidance, and given the risk of pneumothorax and hemoptysis, smaller-gauge and not large-bore needles are understandably frequently used.
 
The question is, how many cores are needed to make a histological diagnosis, and, if necessary, immunohistochemical and molecular diagnoses? This really depends on the content of the core. In some institutions, rapid onsite evaluation by a cytopathologist is performed. Touch preparations are performed by a cytologist to make a preliminary assessment. This is somewhat controversial, and not always available.

So the clinician obtaining the biopsy can play a significant role by performing a gross examination of the biopsy. It is important to see if the core represents a solid piece of tan-white tissue, which is typical of carcinomas. Sometimes the cores consist of mostly red-blood-cell clots, mucus, or liquefied necrotic inflammatory tissue. In these instances, additional cores are necessary.

Also, the greater the number of cores, the greater the likelihood of having sufficient tissue for ancillary testing. And currently, there are no guidelines dictating the minimal number of cores. However, if it's feasible and accessible without significant risk to the patient, personal experience has demonstrated that a minimum of three full-length cores of solid tissue are usually sufficient.7,8 But one to four cores have yielded sufficient tissue for mutational analysis in some studies. In the pathology laboratory, separating the cores into multiple blocks and cutting blanks up front minimizes the loss of tissue that may result from trimming through the tissue with each request for additional slides.

So what about the protocol for fine-needle–aspiration specimens? This is slightly different than that of core biopsies. Fine-needle aspirations are performed even more frequently with the introduction of endobronchial ultrasound, or EBUS, which allows for tissue sampling and staging in one step.1

If pathology department resources allow, having rapid onsite evaluation of the aspirate by an experienced cytologist is invaluable.9 The cytologist can provide a diagnosis and request additional samples in cases of scant specimens. But most importantly, either in the presence or absence of a cytopathologist, it is important to allocate material for ancillary testing. This means not making too many or large smears or smears of clots, which leads to poor utilization of the specimen. And when making a smear, small, tiny, tan-white particles should be selected for the smears, and the remainder should be placed in fixative such as formalin or alcohol for cell block preparation. A dedicated pass for cell block also has a greater likelihood of yielding a sufficient specimen. And as with the cores, requesting blank slides up front minimizes loss of tissue from trimming the cell block with subsequent requests for additional slides.

Also, a first-time diagnosis in patients with advanced lung cancer is sometimes rendered on pleural effusions.1 If there is sufficient volume of specimen, a cell block should be made from these samples, as well. Especially in patients who may be unable to tolerate another procedure, cell blocks can provide a source of tissue for necessary ancillary testing.

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What is the ideal testing modality/methodology to determine EGFR and ALK status in patients with NSCLC? Are there new techniques that might replace our current testing and allow for smaller specimens?

Dr. Saqi: The National Comprehensive Cancer Network, or the NCCN, as well as the College of American Pathologists [CAP], the International Association for the Study of Lung Cancer [IASLC], and the Association for Molecular Pathology [AMP] have provided molecular testing guidelines to select patients for EGFR and ALK tyrosine kinase inhibitors [TKIs].1,2

For EGFR testing, there are guidelines for tissue fixation and methodology. EGFR testing can be performed on fresh, frozen, alcohol-fixed or formalin-fixed, and paraffin-embedded tissues. Other fixatives and solutions, including B5, Bouin solutions, and decalcifying solutions, should be avoided because they interfere with the PCR-based molecular testing.2

Ideal fixation times range from six to 12 hours or eight to 18 hours for small specimens and large specimens, respectively. Of all the preparations, using paraffin-embedded tissue, such as from resections, core biopsies, and cell blocks, is advantageous, because the content of the tissue (that is, the presence or absence of tumor), the proportion of tumor cells relative to non-tumor cells, and the amount of tumor can be assessed from the already available H&E [hematoxylin and eosin] slides. These factors are important in avoiding false-negative ancillary test results.2

EGFR mutation testing should be performed using a PCR-based method.1,2 There are many available methods, with some being more sensitive than others. The guidelines recommend that laboratories use a test that detects mutation specimens with at least 50% tumor,2 but one that detects 10% tumor is ideal.

For selecting patients for ALK TKI therapy, ALK FISH testing should be used.1,2 This is performed on slide sections of tumor, and unlike for EGFR mutation testing, some have suggested that alcohol fixation for ALK testing can be problematic.2

Narrator: ALK immunohistochemistry, if carefully validated, may be considered as a screening methodology to select specimens for ALK FISH testing. However, RT-PCR is not recommended as an alternative to FISH for selecting patients for ALK inhibitor therapy.2

Dr. Saqi: Alternative molecular techniques, such as next-generation sequencing, are available for detecting mutations. Next-generation sequencing allows for testing of multiple biomarkers with smaller amounts of tumor DNA. At the moment, this is not standard. But in the future, this method may become the standard and has the potential to overcome the hurdle of molecular testing in small samples, such as core biopsies and fine-needle aspirations.

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How rapidly should the results of molecular testing be available, and how should they be reported and communicated to clinicians and patients?

Dr. Saqi: The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology have also provided guidelines for turnaround time and reporting of results.2 Following the histopathological or cytopathological diagnosis, the ultimate goal is to have molecular test results available within 10 working days of receiving the specimen in the molecular testing laboratory.2

So, prior to molecular testing, a few specimen parameters need to be assessed, including the percent of tumor relative to the total number of nucleated cells. Other features, such as the presence of necrosis or whether or not a specimen was decalcified, need to be addressed, because these factors may affect the outcome of molecular testing.2

The molecular testing report should include two sections, a results and an interpretation, that should be interpretable by oncologists and non-specialist pathologists. For EGFR testing, clinically significant mutations, incidental findings, variants, inconclusive results, methodology and exon sequence should be reported.

Also important in the presence of a detected EGFR mutation is the incorporation of interpretation. That is, the likelihood of response to EGFR TKI therapy. Similarly, in interpretation, either suggesting response or no response to a targeted inhibitor should be incorporated in the presence or absence of an ALK rearrangement.

For ALK FISH testing, the report should include the total number of cells analyzed, as well as the number and percentage of cells with or without rearrangement. For the commercially available FISH assay, a split signal identified in greater than or equal to 15% of 50 nuclei predicts treatment response.2,10

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References

  1. National Comprehensive Cancer Network (NCCN). Non-Small Cell Lung Cancer, V3.2014. Available at: www.nccn.org.
  2. Lindeman NI et al. Arch Pathol Lab Med. 2013;137:828-860. Available at: http://www.cap.org/apps/docs/membership/lc_patient_guide.pdf.
  3. World Health Organization (WHO). Cancer. http://www.who.int/mediacentre/factsheets/fs297/en/. Accessed February 13, 2014.
  4. Wistuba II. 2012 ASCO Educational Book. http://meetinglibrary.asco.org/sites/meetinglibrary.asco.org/files/Educational%20Book/PDF%20Files/2012/zds00112000459.pdf. Accessed February 13, 2014.
  5. Jurado J et al. Ann Thorac Surg. 2013;96:1196-1202.
  6. Heymann JJ et al. Cytojournal. 2013;28:10:4.
  7. Ferretti GR et al. Lung Cancer. 2013;82:69-75.
  8. Hasanovic A et al. Lung Cancer. 2012;77:299-305.
  9. Bulman W et al. Am J Respir Crit Care Med. 2012;185:606-611.
  10. Camidge DR et al. Clin Cancer Res. 2010;16:5581-5590.

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Genentech

This activity is supported by an educational grant from Genentech.
Additional support provided by Penn State College of Medicine and Answers in CME.

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How Can We Achieve Timely and Accurate Molecular Testing in Lung Cancer?

  1. Current Perspectives and Emerging Directions in Molecular Testing for Lung Cancer