1. bony specimen can be used for molecular testing if other sample is not available.

2. For ALK rearrangement, I perfer IHC, it's simple and unexpensive. For tiny tissue or diffcult to obtain tissue, NGS is a good choice.

3. According to ALEX of J-ALEX, Alectinib in first-line therapy is an optimal method for benefit patient's survival, and may reduce the posibility of brain metastasis in the future.

Course Faculty Response

The patient was initially diagnosed with metastatic carcinoma based on a bone biopsy.  In the pathology laboratory, bone specimens must undergo some form of chemical treatment to dissolve calcium salts before the sample is sectioned for histopathologic evaluation.  Many labs have historically relied on the use of strong acids to rapidly decalcify specimens, however these low pH substances (including hydrochloric acid and nitric acid) degrade cellular proteins and nucleic acids and therefore may interfere with immunohistochemical, cytogenetic, and molecular assays.  As a result, molecular testing guidelines discourage the use of decalcification agents for specimens that require biomarker testing and many patients, as in this scenario, may need to undergo a repeat biopsy of a soft tissue or visceral site to obtain adequate tissue for molecular testing (Lindeman, et al). Alternatively, when bone biopsies are obtained for a suspicion of metastatic carcinoma, laboratories may consider used a neutral-pH chelating agent such as EDTA; EDTA-based decalcification solutions are less efficient than those using acids but can effectively remove calcium salts from small core needle biopsies in a relatively short period of time while preserving protein antigenicity and nucleic acid integrity.  Studies of breast cancer samples undergoing neutral decalcification indicate that quantitative immunohistochemistry (IHC) and fluorescence in in situ hybridization (FISH) results are comparable to those for standardly processed formalin-fixed paraffin embedded (FFPE) tissue (Schrijver, et al).  The need for any decalcification can be circumvented entirely by performing cytology-type touch preps or smears of the bone specimen; these can be stained to confirm adequate tumor cellularity and then used directly for nucleic acid extraction or for FISH or IHC. These types of cytology preparations do not require formalin fixation—methanol fixation is commonly used instead—and as a result may actually have superior quality nucleic acids than FFPE samples.  Current molecular testing guidelines advise that such cytology preparations may be used in lieu of other samples such as core biopsies or cell blocks, however individual laboratories should ensure that each sample type accepted for testing is specifically evaluated in the course of assay validation.  In patients with an established diagnosis of lung adenocarcinoma but whose tumor specimens are inadequate for molecular testing, plasma-based next generation sequencing may be considered to determine the status of essential biomarkers, however this is recognized to have inferior sensitivity relative to tumor tissue testing.  

EGFR and BRAF mutation as well as ALK and ROS1 rearrangement testing is advised for all patients with a new diagnosis of advanced non-squamous non-small cell lung carcinoma.  ALK rearrangements are found in about 3-5% of this population; the relative rarity of this event poses some operational challenges for laboratories.  FISH, the historic gold standard test for identification of patients for ALK targeted therapy, is a relatively expensive and labor-intensive technique, and the common EML4-ALK fusion associated with oncogenic activity in lung cancers results from an intrachromosomal inversion event that can be difficult to interpret in clinical practice.  Therefore, alternative approaches such as IHC, PCR, and sequencing have been widely adopted in practice.  Immunohistochemistry, in particular, can be readily implemented in surgical pathology laboratories, is typically less expensive than FISH, and facilitates reflexive testing protocols.  Using established antibodies (5A4 or D5F3) with well-validated protocols, ALK IHC generates a binary readout, with fusion negative cases showing no staining and fusion positive cases typically showing diffuse and often strong cytoplasmic staining.  Pooled analyses of studies examining the performance characteristics of ALK IHC show a sensitivity of 97% and specificity of 99% relative to FISH (Lindeman, et al).  Small studies of IHC-FISH discrepant cases tend to show that FISH+/IHC- tumors do not respond to ALK inhibitor therapy and the FISH results in these cases likely represent nonspecific structural variants that interrupt the FISH probe binding sites (Gao, et al). ALK IHC is, therefore, considered to be robust enough to serve as a standalone predictive marker and indeed one ALK IHC assay has been approved as a companion diagnostic for both crizotinib and alectinib.  Molecular assays, including hybrid capture DNA-based next generation sequencing or RNA-based sequencing, reverse-transcriptase PCR, or anchored-multiplex PCR, also appear to be effective in the detection of ALK rearrangements.  An ALK rearrangement event detected using one of these methods, assuming it shows a predicted functional, in-frame fusion (e.g., retained ALK kinase domain in the same reading frame as the fusion partner), can generally be considered to be a true positive.  Sequencing and PCR-based methods can, however, be falsely negative if the fusion involves unusual gene breakpoints not covered by a targeted assay.  

In general, many molecular diagnostics laboratories require at least 20% tumor cell content in any tissue specimen, however individual labs may develop targeted assays that detect mutations and structural variants in specimens with lower tumor content.  NCCN guidelines recommend up-front testing of any non-squamous non-small cell carcinoma for EGFR, BRAF, ALK, ROS1, and PD-L1; reporting of the PD-L1 tumor proportion score requires at least 100 tumor cells, thus providing a minimal absolute requirement for biomarker testing.

I believe that, whenever possible, rebiopsy at progression should be standard for those progressing on ALK inhibitors. This helps identifying resistant mechanisms and mutations that may aid in selecting the next ALK inhibitor 

Course Faculty Response

Similar to the experience with EGFR TKIs, most patients develop resistance to ALK TKIs, especially crizotinib, after a while. However contrary to the EGFR mutation positive lung cancers, where most common mechanism of resistance is the development of resistance mutations in the EGFR gene, acquired secondary mutations within the ALK tyrosine kinase domain are seen in only about 20% of patients who develop resistance to crizotinib (Gainor, et al). The most common mutation seen after crizotinib is the L1196M mutation, while other common mutations include G1269A, C1156Y and I1171T/N/S. The common mutations seen after ceritinib use are G1202R, F1174C/L and V1180L mutations and deletion of G1202, while S1206Y, E1210K are commonly seen after alectinib use. Lorlatinib, an ALK inhibitor currently under investigation appears to have activity against many of these mutations.  

Currently patients who fail one ALK inhibitor may be treated with another agent and a repeat biopsy at progression is not routinely recommended. However, that may change with increased understanding of the resistance mechanisms. A recent case report described a patient who developed resistance to crizotinib, who did not respond to a second-generation agent, but did respond to lorlatinib (Shaw, et al). When the patient progressed on lorlatinib, the tumor was found to harbor an ALK mutation that predicted for sensitivity to crizotinib. The patient was treated with crizotinib with clinical response.

A retrospective analysis of EGFR-mutated and ALK-positive patients who received PD-1/PD-L1 inhibitors, found a response rate of 3.6% (Gainor, et al). The number of ALK-positive patients included in the large phase III trials of the PD-1/PD-L1 inhibitors are too small to make any specific conclusions regarding the efficacy in this sub-group. While there have been some reports speculating that the lack of response may be due to low PD-L1 expression in these tumors with oncogenic drivers, the data on PD-L1 expression in ALK positive patients are contradictory. A more plausible explanation for the lack of effect may be the presence of a lower tumor mutation burden in ALK-positive lung cancer.

Course Faculty Summary

• All patients with newly diagnosed metastatic lung adenocarcinoma should be tested for actionable mutations.
• Crizotinib, ceritinib and alectinib are all appropriate for initial management of patients with ALK- positive tumors. However, alectinib has shown an improved PFS as compared to crizotinib and seems to have the most favorable side effect profile.
• ALK mutations account for resistance to crizotinib in approximately 20% of patients.
• Biopsy at recurrence is not recommended in the current guidelines, but that may change with improved understanding of resistance pathways.
• Patients who have progressed on one ALK inhibitor may respond to another ALK inhibitor.
• Checkpoint inhibitors do not seem to be efficacious in ALK positive lung cancer.