2025 澳大利亚共识建议：肺癌的分子检测

共识肿瘤内科· 肺癌 · 2025

A B S T R A C T

Key words: lung cancer molecular testing biomarkers best practice

Molecular testing plays a critical role in guiding optimal treatment decisions for lung cancer patients across a variety of clinical settings. While guidelines for biomarker testing exist in other jurisdictions, to date no best practice guidelines have been developed for the Australian setting. To address this need, the Royal College of Pathologists of Australasia collaborated with the Thoracic Oncology Group of Australasia to identify state-based pathologists, oncologists and consumer representatives to develop consensus best practice recommendations. Sixteen recommendations were established encompassing appropriate biomarkers, lung cancer subtype, tumour stage, specimen types, assay selection and quality assurance protocols that can inform and standardise best

\* Address for correspondence: Royal Prince Alfred Hospital, Building 12, Missenden Road, Camperdown, NSW 2050, Australia. E-mail address: wendy.cooper@health.nsw.gov.au (W.A. Cooper).

Received 18 December 2024; Received in revised form 7 February 2025; Accepted 9 February 2025 0031-3025/ © 2025 The Author(s). Published by Elsevier B.V. on behalf of Royal College of Pathologists of Australasia. This is an open access article under the CC BYNC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Please cite this article as: Cooper WA et al., Molecular testing of lung cancer in Australia: consensus best practice recommendations from the Royal College of Pathologists of Australasia in collaboration with the Thoracic Oncology Group of Australasia, Pathology, https://doi.org/10.1016/ j.pathol.2025.02.001

practice in molecular testing of lung cancer. These multidisciplinary evidence-based recommendations are designed to standardise and enhance molecular testing practices for lung cancers and should help ensure laboratories provide high-quality molecular testing of lung cancer for all Australians, including those from regional or remote communities.

1. Introduction

In Australia, lung cancer is the ﬁ fth most commonly diagnosed cancer, yet it remains the leading cause of cancer-related deaths each year, with more deaths than prostate and breast cancer combined. 1 In 2024, Cancer Australia, using data from the Australian Institute of Health and Welfare, estimated there would be 15,122 new cases of lung cancer diagnosed in Australia with an estimated 8,918 deaths. 2 Aboriginal and Torres Strait Islander people are twice as likely to be diagnosed and die from lung cancer compared to non-Indigenous Australians. 3 Lung cancer incidence and mortality are also higher for people from lower socioeconomic backgrounds or from remote areas rather than in major cities. 2 The frequency of targetable molecular alterations in lung cancer varies across different patient populations. Australian studies suggest that overall rates are similar to other Western populations; 4 , 5 however, data regarding the rates in different populations, including the Aboriginal and Torres Strait Islander population is lacking.

Recent advances in the treatment of lung cancer, including the use of immunotherapy or targeted therapies in selected patient populations, has led to improvements in patient outcomes over the last decade. 6 Molecular testing is crucial for guiding optimal treatment decisions in early and late-stage lung cancer; however, there is a lack of standardisation and alignment regarding molecular testing of lung cancers in Australia. This can lead to variation in practice, resulting in differences in patient care and outcomes across Australia. Although guidelines have been developed by professional organisations in other countries; these are not necessarily directly applicable to the Australian health setting, and evidence-based recommendations that acknowledge the Australian context are needed to drive best practice for molecular testing for lung cancer.

While the following recommendations are primarily aimed at pathologists in Australia, they are also highly relevant to other healthcare professionals involved in the management of lung cancer. This includes medical oncologists, surgeons, radiation oncologists, respiratory physicians and radiologists, as well as patients and patient advocates.

2. Methods

The Royal College of Pathologists of Australasia (RCPA) initiated a project in 2024 to develop comprehensive, evidence-based, ‘ Best Practice Recommendations for molecular testing of lung cancers in ’ Australia based on expert consensus. The recommendations aim to provide standardisation across Australia on molecular testing methods and protocols (by biomarker, lung cancer subtype and testing type); sample collection, processing and analysis; and quality assurance and reporting.

The project was developed in conjunction with invited state representatives of the Thoracic Oncology Group of Australasia (TOGA) to obtain clinical input into the development of the recommendations.

A multidisciplinary steering committee was convened with expert pathologist and medical oncologist state representatives as well as two consumer representatives, all nominated from RCPA and TOGA. A literature review was conducted to identify existing international guidelines and peer-reviewed publications on molecular testing of lung cancers. From this, a scoping document was developed that included background information pertinent to the Australian context and an overview and comparison of key international guidelines and best practice recommendations from the:

(i) College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC)/Association for Molecular Pathology (AMP); 7 a draft update of the recommendations released for consultation in September 2024 was also considered; (ii) European Society for Medical Oncology (ESMO); 8 (iii) ESMO Pan-Asia; 9 (iv) American Society of Clinical Oncology (ASCO); 10 and (v) National Comprehensive Cancer Network (NCCN). 11

From this, a set of 16 draft key questions was compiled with provisional recommendations derived from the scoping document and existing international guidelines.

The steering committee met in person to review, revise and con ﬁ rm the key questions and evidence-based recommendations. Consensus was reached if at least $80\%$ of the steering committee members agreed with the proposed recommendation. There was unanimous agreement for all proposed recommendations, with only one recommendation (Key Question 8, recommendation 2) requiring a vote and consensus was achieved.

The key questions and draft evidence-based recommendations were also sent for open consultation, and all responses were considered by the steering committee before ﬁ nalising the recommendations. The recommendations were ﬁ nalised virtually by the steering committee in October 2024. Final recommendations were endorsed by January 2025 (see acknowledgements section for a list of endorsing organisations).

3. Results

Sixteen key recommendations were made, as outlined below and shown in Table 1 .

4. Discussion

4.1. Key Question 1: Of the currently identi ﬁ ed biomarkers, which should be routinely tested in advanced stage non-squamous non-small cell lung cancer (NSCLC)?

4.1.1. Recommendations

EGFR, BRAF, KRAS, ERBB2 (HER2), ALK, ROS1, MET, RET , NTRK and PD-L1 should be tested in all advanced stage non-squamous NSCLC. This includes any advanced-stage lung carcinoma with an adenocarcinoma component (e.g., non-small cell carcinoma with squamous and glandular differentiation) or where an adenocarcinoma cannot be excluded (e.g., a small biopsy/cytology diagnosis of NSCLC-NOS). Testing should be performed on re ﬂ ex by pathologists to expedite result turnaround times (TAT).

4.1.2. Discussion

Identifying biomarkers in advanced stage non-squamous NSCLC is a rapidly evolving and important ﬁ eld. Speci ﬁ c activating gene variants, if identi ﬁ ed, allow access for patients to effective targeted systemic treatments and clinical trials. Several landmark studies with small molecule targeted agents against activating variants in EGFR and oncogenic fusions in ALK and ROS1 have demonstrated improvements in survival, with good tolerability and safety pro ﬁ les. 12 – 14 Bene ﬁ ts are also seen for treatments targeting speci ﬁ c oncogenic variants in MET, RET, NTRK, BRAF, KRAS and ERBB2 ( HER2 ). 15

In Australia, there are currently several options for Pharmaceutical Bene ﬁ ts Scheme (PBS)-approved targeted treatments for patients with

Table 1 Summary of key questions and recommendations

|Key Question| Consensus recommendationa| |---|---| |1. Of the currently identified|| | biomarkers, which should be| · EGFR, BRAF, KRAS, ERBB2 (HER2), ALK, ROS1, MET, RET and NTRK and PD-L1 should be tested in all advanced stage non-squamous NSCLC. This includes any advanced-stage lung carcinoma with an adenocarcinoma component. For example, non-small cell carcinoma with squa- | |routinely tested in advanced stage|mous and glandular differentiation or where an adenocarcinoma cannot be excluded (e.g., a smallbiopsy/cytology diagnosis of NSCLC-NOS).| | non-squamous NSCLC?| · EGFR testing should include all clinically relevant oncogenic variants in exons 18, 19, 20 and 21. ● Testing should be performed on reflex by pathologists to expedite TAT. | | 2. Of the currently identified|● At a minimum, EGFR, ALK, ROS1 and PD-L1 testing must be undertaken in stage IB-II non-squamous NSCLC, and non-resectable locally| || advanced non-squamous NSCLC treated with curative intent chemoradiotherapy. ● More comprehensive testing for EGFR, KRAS, BRAF, ERBB2 (HER2), MET, ALK, ROS1, RET and NTRK can be undertaken in the early stage | | biomarkers, which should be (routinely) tested in early stage non- | ● More comprehensive testing for EGFR, KRAS, BRAF, ERBB2 (HER2), MET, ALK, ROS1, RET and NTRK can be undertaken in the early stage setting if practical. ● No molecular testing is required for surgically resected stage IA NSCLC. | | squamous NSCLC?|| | 3. Should tumour biopsy and| · In patients progressing on any targeted therapy, molecular testing for resistance mechanisms should be considered if tissue is available. · If tissue biopsy is not available or feasible, plasma-based ctDNA molecular testing for resistance mechanisms should be considered. | | molecular testing be undertaken on|| | disease progression during|| | treatment with targeted therapy? 4. Is there a role for more extended || || · More comprehensive testing could be considered for patients with advanced stage NSCLC, especially cases lacking any of the oncogenic variants of the genes outlined in KQ1 when clinically relevant. | | biomarker testing? 5. Should patients with squamous cell |● At a minimum, advanced-stage squamous cell carcinoma should undergo MET and PD-L1 testing.| | carcinoma on biopsy undergo|| |molecular testing?| ● Upon request, molecular testing for all markers in KQ1 should be undertaken for patients diagnosed with lung squamous cell carcinoma on small biopsy or cytology that have a clinical profile suspicious for driver mutation (e.g., |

ALK, anaplastic lymphoma kinase; BRAF, V-raf murine sarcoma viral oncogene homolog B; ctDNA, circulating tumour deoxyribonucleic acid; EGFR, epidermal growth factor receptor; ERBB2, v-erb-b2 avian erythroblastic leukaemia viral oncogene homolog 2 (also known as HER2, human epidermal growth factor receptor-2); FFPE, formalin- ﬁ xed paraf ﬁ n-embedded tissue; FNA, ﬁ ne needle aspiration; IHC, immunohistochemistry; KQ, Key Question; KRAS, Kirsten rat sarcoma viral oncogene homolog; MDT, multi-disciplinary team; MET, mesenchymal epithelial transition factor receptor; MSI, microsatellite instability; NGS, next-generation sequencing; NSCLC, nonsmall cell lung carcinoma; NSCLC NOS, NSCLC not otherwise speci ﬁ ed; NTRK, Neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; PD-L1, Programmed death-ligand 1; RET, rearranged during transfection; RNA, ribonucleic acid; ROS1, c-ros oncogene 1; TAT, turnaround time; TMB, tumour mutation burden; TPS, tumour proportion score.

a All unanimous decisions, except 8b ( $.80\%$ consensus).

advanced NSCLC that harbour activating variants in EGFR, ALK, ROS1, MET and NTRK . Although targeted therapies for BRAF V600E, KRAS G12C, ERBB2 ( HER2) and RET are not currently PBS-reimbursed in Australia, results are still useful for clinical decision-making and PBS reimbursement may be available in the future. In 2022, Medicare Bene ﬁ ts Schedule (MBS) reimbursement was approved for next-generation sequencing (NGS) testing of NSCLC, including all of these genes apart from ERBB2 ( HER2) . 16 More recent data suggest activating ERBB2 ( HER2) variants are also important to identify as they predict bene ﬁ t from several therapies. 17 As these oncogenic variants are strongly associated with adenocarcinomas and other NSCLC with a glandular component, biomarker testing is always recommended for these histological subtypes of lung cancer. 15

High expression of programmed death-ligand 1 (PD-L1) is associated with improved survival with pembrolizumab monotherapy and assessment of PD-L1 protein expression is important for immunotherapy treatment decisions. 18 Bene ﬁ t from immune checkpoint inhibitors is seen in wild-type tumours that lack activating EGFR , ALK or ROS1 alterations, (and possibly other oncogenic drivers apart from KRAS) and the status of these biomarkers is essential for treatment decisions. 19

4.2. Key Question 2: Of the currently identi ﬁ ed biomarkers, which should be (routinely) tested in early-stage non-squamous NSCLC?

4.2.1. Recommendations

At a minimum, EGFR, ALK, ROS1 and PD-L1 testing must be undertaken in stage IB-III non-squamous NSCLC, and non-resectable locally advanced non-squamous NSCLC treated with curative intent chemoradiotherapy. More comprehensive testing for EGFR, KRAS, BRAF, ERBB2 (HER2), MET, ALK, ROS1, RET and NTRK can be undertaken in the early stage setting if practical. No molecular testing is required for surgically resected stage IA NSCLC.

4.2.2. Discussion

The use of peri-operative immunotherapy and adjuvant targeted therapy has extended the role of molecular testing from the advanced setting to earlier-stage NSCLC.

There is evidence for the use of PD-1/PD-L1 inhibitors for early-stage resectable NSCLC, either before surgery (neo-adjuvant), after surgery (adjuvant), or both (peri-operative). Currently, nivolumab in combination with chemotherapy is PBS approved as neoadjuvant therapy combined with chemotherapy in patients with resectable stage IB-IIIA NSCLC. Atezolizumab is PBS approved as adjuvant therapy in patients with resected NSCLC, with PD-L1 tumour proportion score (TPS) ${\geq}50\%$ . Importantly, adjuvant/neoadjuvant studies evaluating immunotherapy have generally excluded patients harbouring EGFR sensitising variants and ALK rearrangements given the lack of bene ﬁ t of PD-1 checkpoint inhibition in the metastatic setting. Testing for EGFR variants, ALK and ROS1 rearrangements and PD-L1 must be done on presurgical biopsies where neoadjuvant immunotherapy is being considered by the multidisciplinary team. Patients whose tumours harbour EGFR variants, ALK or ROS1 rearrangements are excluded from this approach as they are not considered to derive bene ﬁ t from chemoimmunotherapy. In other patients, in particular nonsmokers, more extended molecular testing may be considered as other molecular subgroups may also derive less bene ﬁ t from chemoimmunotherapy [e.g., patients with RET , NTRK fusions or ERBB2 ( HER 2) variants].

If not already performed on a pre-operative biopsy specimen, EGFR and ALK testing together with PD-L1 should be undertaken on the resected specimen to facilitate decisions about adjuvant therapy. There is evidence for the use of osimertinib in the adjuvant setting for resected stage IB-III EGFR -positive NSCLC, and for the use of adjuvant alectinib in the adjuvant setting for resected stage IB-IIIA ALK -positive NSCLC. 20

Additional trials are underway evaluating other targeted therapies in the adjuvant setting (e.g., with the RET inhibitor selpercatinib). 21 Given the evidence for the bene ﬁ t of adjuvant atezolizumab in patients with tumours that have high expression of PD-L1 (i.e., $\mathrm{TPS}\ge\!50\%$ ) and pembrolizumab in tumours with PD-L1 ${\geq}1\%$ (not currently on the PBS), PD-L1 status is needed. As PD-L1 scores may differ between biopsy and resection specimens in some cases, testing may be repeated on the resected tumour to guide decisions about adjuvant therapy.

Full oncogene panel testing in early-stage non-squamous NSCLC has the advantage of ensuring results are available if the patient develops recurrent disease to enable commencement of targeted therapy, particularly as adequate tissue may not be readily available on relapse.

4.3. Key Question 3: Should tumour biopsy and molecular testing be undertaken on disease progression during treatment with a targeted therapy?

4.3.1. Recommendations

In patients progressing on any targeted therapy, molecular testing for resistance mechanisms should be considered if tissue is available. If tissue biopsy is not available or feasible, molecular testing of plasma for circulating tumour (ct)DNA for resistance mechanisms should be considered.

4.3.2. Discussion

Tissue biopsy should be considered in patients with oncogene-driven tumours progressing on targeted therapy in order to identify potential acquired resistance mechanisms, including phenotypic transformation (in particular, small cell transformation or squamous differentiation) and to enable molecular testing for on-target and off-target resistance mechanisms that may guide subsequent treatment (for example, ‘ ontarget acquired EGFR kinase domain or ALK kinase domain variants or ’ ‘ off target MET ampli ﬁ cation in EGFR or ALK driven tumours). Given the ’ variety of molecular changes that can be found at progression, broadbased genomic pro ﬁ ling with panels that include multiple genes that also allow assessment of copy number alterations is recommended. While it may be dif ﬁ cult to target identi ﬁ ed ﬁ ndings with currently approved or reimbursed therapies, the information may be helpful to direct patients towards speci ﬁ c clinical trials or access programs.

If re-biopsy and tissue testing are not available or feasible, plasmabased ctDNA molecular testing may be helpful. At relapse, ctDNA molecular testing has the potential advantage over tissue-based biopsies of providing more information on molecular heterogeneity.

In the setting of patients with EGFR sensitising variants, as most patients are now treated upfront with newer generation tyrosine kinase inhibitors (TKIs; e.g., osimertinib), testing for EGFR T790M resistance variant is not speci ﬁ cally required in most patients. Testing for EGFR T790M is only required if the patient has received ﬁ rst-line treatment with a ﬁ rst- or second-generation EGFR TKI following the development of resistance, in order to enable the option of third-generation TKIs effective against EGFR T790M variants. Biopsy may identify other alterations in EGFR or activation of bypass mechanisms, e.g., MET ampli ﬁ cation, which may be amenable to targeted therapy. Similarly, in the context of ALK - driven tumours, secondary variants in ALK may be identi ﬁ ed that in ﬂ uence choice of subsequent ALK TKI.

4.4. Key Question 4: Is there a role for more extended biomarker testing?

4.4.1. Recommendation

More comprehensive testing could be considered for patients with advanced stage NSCLC, especially cases lacking oncogenic variants in the genes outlined in Key Question 1 (KQ1) when clinically relevant.

4.4.2. Discussion

More comprehensive molecular testing in patients with advancedstage NSCLC plays an increasingly crucial role in re ﬁ ning personalised treatment strategies. Beyond the well-established biomarkers EGFR, BRAF, KRAS, ERBB2 (HER2), ALK, ROS1, MET, RET, NTRK, and PD-L1, expanded molecular pro ﬁ ling offers several key bene ﬁ ts. Comprehensive genomic pro ﬁ ling (CGP) can lead to the identi ﬁ cation of less common and novel targets, including actionable variants in lesser-known genes, such as NRG1 , FGFR 1/2/3, MAP2K1 , PIK3CA or RAF . Therapies are being developed or repurposed for these variants or fusions, and more comprehensive testing can direct patients to appropriate clinical trials.

In patients with tumours harbouring oncogene drivers, e.g., sensitising EGFR variants or ALK gene rearrangements, the presence of common co-variants, e.g., TP53 or STK11/KEAP1 variants, may have prognostic signi ﬁ cance and/or be associated with resistance to therapy. For example, variants in TP53 have been shown to confer an adverse prognosis, 22 , 23 or be associated with resistance to treatments such as immunotherapy ( STK11/KEAP1 variants) in KRAS variant tumours. 24 Randomised trials have demonstrated that intensi ﬁ cation of therapy, for example addition of chemotherapy to EGFR-targeted therapy, can improve outcomes over and above targeted therapy alone in EGFRpositive tumours with TP53 variants. 22 Knowledge of the presence of co-variant (e.g., TP53 variants) may help prognostic strati ﬁ cation and for selection of therapy or clinical trials. In addition, identifying multiple concurrent variants or pathways involved in tumourigenesis, CGP can inform the use of combination therapies. For instance, targeting both a driver variant and a concurrent PI3K pathway alteration may enhance ef ﬁ cacy compared to monotherapy.

4.5. Key Question 5: Should patients with squamous cell carcinoma on biopsy undergo molecular testing?

4.5.1. Recommendations

At a minimum, advanced-stage squamous cell carcinoma should undergo MET and PD-L1 testing. Upon request, molecular testing for all markers in KQ1 should be undertaken for patients diagnosed with lung squamous cell carcinoma on small biopsy or cytology that have a clinical pro ﬁ le suspicious for driver mutation (e.g., ${<}50$ years old or no/minimal smoking history).

4.5.2. Discussion

While most targetable biomarkers have been detected in NSCLC of adenocarcinoma subtype, it is possible that they are occasionally found in other subtypes of NSCLC. In particular, MET exon 14 skipping variants are reported in $1.3\%$ of lung squamous cell carcinomas and $5–32\%$ of sarcomatoid carcinomas. 25 – 29 Testing is recommended in these subtypes of NSCLC and is important due to the availability of an effective treatment option (e.g., PBS listed tepotinib).

Pure squamous cell carcinomas, unlike adenocarcinomas (or any NSCLC with a glandular component), rarely harbour other currently targetable oncogenic driver variants, so routine panel testing of this histological subtype of lung cancer is not required. Incidence rates of oncogenic EGFR variants in squamous cell carcinoma (SCC) have been reported in $0–5\%$ of NSCLC patients and KRAS variants in ${<}1{-}6\%$ of patients, 30 – 33 although it is possible that some of these cases represent adenosquamous carcinomas. 32 Furthermore, EGFR variants are more frequent in SCC from non-smokers than in smokers. 34 As small biopsy or cytology specimens may miss the glandular component of a combined adenosquamous carcinoma, and pure SCC are very rare in non-smokers, testing for all oncogenic drivers is recommended in this clinical setting. As pathologists may not be aware of the patient's full clinical details it cannot be presumed that this testing will be undertaken on re ﬂ ex in SCC, and a request may be required from the treating clinician to enable molecular testing to proceed.

4.6. Key Question 6: Should patients with small cell carcinoma undergo molecular testing?

4.6.1. Recommendation

No biomarker testing is currently indicated for pure small cell carcinoma.

4.6.2. Discussion

Atezolizumab and durvalumab are both PBS-listed (in combination with chemotherapy) for the ﬁ rst-line treatment of extensive stage smallcell lung cancer (SCLC). 35 , 36 Recent data have indicated that consolidation durvalumab confers a survival advantage in non-progressive patients with limited stage SCLC after chemoradiation. 37 However, none of these studies selected patients by PD-L1 TPS status.

4.7. Key Question 7: What biomarkers are required for immunotherapy?

4.7.1. Recommendations

PD-L1 immunohistochemistry (IHC) for assessment of tumour proportion score (TPS) should be undertaken in all advanced stage/ metastatic NSCLC and stage IB-III NSCLC, regardless of histology. This should be performed as a re ﬂ ex test. Molecular biomarker status is also required for immunotherapy treatment decisions in non-squamous NSCLC (e.g., EGFR, ALK, ROS1 ). There is currently no routine clinical role for tumour mutation burden (TMB) or microsatellite instability (MSI) testing in NSCLC in Australia.

4.7.2. Discussion

Immune checkpoint inhibitors (ICI) that target the programmed death1/programmed death-ligand 1 (PD-1/PD-L1) pathway are important therapeutic agents in the treatment of NSCLC and SCLC. Therapies approved in Australia for advanced NSCLC include pembrolizumab, nivolumab, atezolizumab and cemiplimab. Anti-CTLA-4 agent ipilimumab in combination with nivolumab is also approved as a ﬁ rst-line therapy option for advanced squamous-type NSCLC. In unresectable stage III NSCLC, durvalumab is approved following de ﬁ nitive chemoradiation.

There is emerging evidence for the ef ﬁ cacy of PD-1/PD-L1 agents in early-stage NSCLC in the neo-adjuvant, adjuvant and peri-operative settings. In Australia, nivolumab in combination with platinum-based chemotherapy is approved in the pre-operative setting, while atezolizumab is available after surgery and adjuvant chemotherapy in resected stage II IIIA NSCLC with PD-L1 expression of ${\geq}50\%$ of tumour cells. –

The magnitude of clinical ef ﬁ cacy of ICIs, speci ﬁ cally anti-PD-(L)1 agents and the level of tumoural PD-L1 expression as evaluated by PDL1 IHC has been demonstrated in multiple clinical trials. 38 PD-L1 IHC for assessment of TPS should be undertaken in all advanced stage/metastatic NSCLC and stage IB III NSCLC, regardless of histology, – to assist in individual treatment decision-making.

TheprevalenceofPD-L1expressioninNSCLCvariesbystage.Specimens from patients with stage III IV NSCLC report PD-L1 TPS scores of ${\geq}1\%$ and ${\geq}50\%$ more often than specimens from patients with stage I – II NSCLC. 39 Higher PD-L1 expression is also associated with higher grade tumours. – 40

Molecular biomarker status is required for immunotherapy treatment decisions in non-squamous NSCLC as use of ﬁ rst-line ICIs for advanced NSCLC is restricted to patients lacking evidence of an activating variant in EGFR, ALK or ROS1 .

Molecular mechanisms associated with some oncogene-addicted tumours may impact the immune tumour microenvironment and impede ef ﬁ cacy of anti-PD-1/PD-L1 therapy. 41 , 42 Patients with NSCLC harbouring variants in BRAF or KRAS and TP53 co-mutations appear to bene ﬁ t the most from ICIs. 43 In contrast, use of ICIs, particularly as single agents in NSCLC harbouring oncogene-driven variants in EGFR or

rearrangements in ALK or ROS1, has shown limited ef ﬁ cacy. 44 Understanding distinct genomic subtypes may improve patient selection for ICI in NSCLC. Co-alterations in STK11/LKB1 in KRAS -mutant NSCLC are associated with inferior outcomes to anti-PD-1 therapy and may predict primary resistance to ICIs. 24 Further prospective data are needed to better understand these observations and their impact on clinical care.

TMB and MSI testing are not recommended as routine biomarkers for lung cancer in Australia. TMB is de ﬁ ned as the number of somatic mutations per megabase of DNA sequenced. 45 Improved response rates with ICIs have been reported in NSCLC with high TMB. 46 MSI-high NSCLC represents a rare subset of lung cancer associated with smoking, TMB and MLH1 inactivation. A broad range of responses has been reported in MSI-high NSCLC. 47 Cost, accessibility and lack of impact on drug access limit the role of TMB and MSI biomarker testing in routine clinical care in Australia.

4.8. Key Question 8: Which molecular tests should be undertaken on re ﬂ ex by pathologists and which should be requested by an oncologist?

4.8.1. Recommendations

All molecular tests recommended for advanced-stage NSCLC should be undertaken on re ﬂ ex by pathologists to expedite TAT of results and preserve tissue. If the pathologist is unaware of the stage of the patient's NSCLC, molecular testing should still be performed on re ﬂ ex. For known early-stage disease, testing can be performed either on re ﬂ ex, or following multi-disciplinary team (MDT) discussion, depending on local preferences.

4.8.2. Discussion

Since a substantial proportion of lung cancer patients present with advanced disease, and treatment often needs to be initiated rapidly, clinician-only test requests can no longer be supported. The CAP/IASLC/ AMP guidelines recognise this and have recommended that pathologistdirected re ﬂ ex testing is an appropriate approach. 7

The clinical need for biomarker status in early and advanced stage disease, combined with the availability of MBS reimbursement for lung cancer panel testing and PD-L1 IHC without stage-based restrictions supports the role of pathologists in conducting re ﬂ ex biomarker testing for all lung cancers, regardless of stage. Re ﬂ ex biomarker testing also offers the advantage of tissue preservation, minimising the risk of tissue depletion from resectioning after primary histology (and any IHC), which is critical for the success of molecular testing. We recommend pathologist re ﬂ ex biomarker testing for all primary diagnoses of advanced stage NSCLC as outlined in KQ1. If the pathologist is unaware of the stage of the patient's NSCLC, the panel consensus is for molecular testing to still be performed re ﬂ exively so that no patient is missed and to avoid delayed results. This is particularly important in patients from rural and regional communities where one-third of all cancer patients reside and where delays are more profound. For known early-stage disease, testing can be performed either re ﬂ exively or following MDT discussion, depending on local preferences, as neoadjuvant or adjuvant treatment would only be considered in a subset of patients.

It is acknowledged that in some instances, re ﬂ ex molecular testing may result in redundant testing due to the pathologist being unaware of prior testing on a previous sample, or the patient never proceeding to treatment options beyond surgery. It is nonetheless considered the preferred pathway to avoid delays to patient treatment and ensure optimum patient outcome. Certain clinical situations will require a speci ﬁ c request for molecular testing by the oncologist, as they will fall outside the standard re ﬂ ex testing algorithms. Patients with SCC that have a clinical pro ﬁ le suspicious for driver variant (e.g., ${<}50$ years old or no/minimal smoking history) should have driver oncogene testing speci ﬁ cally requested by the oncologist (see KQ5). When molecular testing is required on tumour progression, this should also be speci ﬁ cally requested as testing may otherwise not occur on

the grounds that it has already been performed.

Extended biomarker testing that falls outside of the scope of routine recommendations should also be speci ﬁ cally requested by the oncologist or MTB/MDT.

4.9. Key Question 9: Which specimen types are suitable for molecular testing?

4.9.1. Recommendations

Any specimen type with a suf ﬁ cient proportion and number of viable tumour cells is adequate for molecular testing. Formalin- ﬁ xed, paraf ﬁ n-embedded (FFPE) material (small biopsy or cytology cell blocks) is suitable for routine molecular analyses and IHC, except bone biopsies that have undergone acid decalci ﬁ cation. Non-FFPE cytology specimens (e.g., samples from FNA) are also appropriate for molecular testing but are not recommended for IHC. Anatomical pathologists should select the optimal FFPE specimen block (or cytology smear) from the primary tumour or metastasis. Some cytology specimens are generally not adequate for molecular testing (e.g., sputum, bronchial brushings/washings). Blood samples collected appropriately for ctDNA analysis are suitable for molecular testing.

4.9.2. Discussion

Any lung cancer FFPE or cytology specimen can potentially be suitable for molecular testing or IHC. Pathologists should select the optimal specimen available for molecular testing based on the highest total amount and highest proportion of viable tumour cells while avoiding features that can impair the success of molecular testing (e.g., extensive necrosis, acid decalci ﬁ cation or archival specimens ${\operatorname{>}}5{-}10$ years old). 48 , 49 In general, specimen types include small biopsy or cytology specimens, or a surgical resection specimen for patients presenting with early-stage disease. As oncogenic driver mutations are truncal variants that develop early in tumour pathogenesis, selection of a primary tumour or metastatic sample is acceptable for initial testing. However, contemporary specimens are preferred to archival specimens as nucleic acid and protein degradation in older specimens can impair NGS success rates, and blocks ${\leq}1$ year old are preferred for RNA and ${\leq}5$ years for DNA sequencing. 49 For IHC, freshly cut tissue sections ( $\cdot{<}1$ week) should be used. 49

An anatomical pathologist should select the optimal specimen and block for molecular testing based on the greatest amount (area) of tumour and the highest percentage of tumour cells compared with all nucleated cells in the block. 48 Minimum cellular requirements vary between different assays, but in general, small biopsy or cytology cell block specimens with a tumour cell content of at least $10–20\%$ are suitable for NGS testing. For ﬂ uorescence in situ hybridisation (FISH) testing, at least 50 evaluable tumour cells are required, 15 and for PD-L1 IHC, at least 100 viable tumour cells are recommended for reliable assessment with a preference for the most recent sample. 50

The ability to obtain suf ﬁ cient DNA/RNA required for molecular testing can be dif ﬁ cult to predict from a histology (or cytology) specimen. Some have suggested at least 100 400 tumour cells 15 , 51 although lower – numbers can be compensated by cutting additional sections/scrolls if the tumour proportion is adequate. Others have suggested a tumour area of at least $25\;\mathrm{mm}^{2}$ . 52 Macrodissection can be used to enrich the proportion of tumour cells in some cases by marking an area of interest on the haematoxylin and eosin-stained section. 48

Cytology specimens are an important source of material for molecular testing, as minimally invasive procedures may be the only source of diagnostic material for many lung cancer patients, particularly those with advanced stage disease (e.g., FNA or effusions). Specimens including cell blocks, smears and liquid-based cytology (LBC) may be used for molecular testing if there is adequate tumour cell content and the specimen type has been validated for testing by the laboratory. 53 Rapid onsite

evaluation during FNA procedures can help ensure an adequate yield of tumour cells. 54

Cytology cell block preparations can provide cellular material for IHC and molecular testing 55 and have the advantage of being processed similarly to tissue biopsies, although they may initially be exposed to ethanol or other ﬁ xatives so should also be speci ﬁ cally validated for NGS. 15 Although cell blocks are most commonly used, cytology smears with at least 100 tumour cells can also be used for NGS testing (if validated) and may yield better DNA/RNA quality and higher tumour cell content than cell block specimens with the drawback of sacri ﬁ cing the diagnostic material (although this can be digitally imaged prior to cell scraping). 56 Cytology smears should not be used for predictive biomarker IHC unless rigorous validation has been undertaken. 53

LBC specimens have the advantage of improved cell preservation, ease of specimen transportation, and minimal background debris and blood. The preservative is often alcohol-based, which can affect some assays, such as IHC. Specimens can be used either by scraping cells from the LBC slides for DNA extraction, or by extracting directly from the rinse solution if validated for use in molecular testing. 57

In general, exfoliative cytology specimens, including bronchial brushings, bronchial washings and sputum are not suitable for molecular testing due to low tumour cell content. 7

4.10. Key Question 10: What is the optimal specimen handling technique for molecular testing?

4.10.1. Recommendations

Preanalytical factors should be standardised to ensure suf ﬁ cient ﬁ xation of biopsy specimens and optimal preservation of DNA and RNA. Contemporary FFPE tissue blocks are preferred to archival specimens for both NGS and PD-L1 IHC. Minimal immunohistochemical markers should be undertaken in diagnostic specimens to preserve material for molecular testing.

4.10.2. Discussion

Numerous preanalytical factors occurring between specimen procurement and assay performance may impact DNA, RNA and protein integrity and reduce the reliability of molecular results. 49 , 58 Standardised procedures should be developed to optimise nucleic acid and protein preservation during the preanalytical phase. In general, histology specimens should be ﬁ xed in $10\%$ neutral buffered formalin with a high formalin to tissue ratio (10:1) as soon as possible with a cold ischaemia time (time prior to ﬁ xation) of ${<}1\,\mathrm{h}$ . 15 , 49 , 58 , 59 Lung resections should be in ﬂ ated with formalin by infusing via the airways or directly injecting through the pleura. Fixation time prior to processing of blocks should be 6 72 h (ideally 6 48 h) at room temperature with at least 24 h required for large resection specimens. – – 49 , 54 , 59 , 60 Prolonged ﬁ xation leads to fragmentation of DNA and cross-linkage of nucleic acids that can impact molecular testing and reduce the intensity of FISH signals. 49 , 59 Non-FFPE cytology specimens should also be processed as quickly as possible to prevent nucleic acid degradation.

Delays in ﬁ xation lead to reduced PD-L1 expression, while overﬁ xation does not appear to impact staining. 60 – 62 PD-L1 expression may be reduced in archival specimens $^{>1}$ year old, 50 and a recent meta-analysis showed increasing storage time was associated with lower levels of PD-L1 expression, so older specimen samples should be avoided where possible. 63

For biopsies obtained from bone requiring decalci ﬁ cation, acids such as hydrochloric, nitric, formic or phosphoric acid should be avoided as they lead to altered protein and nucleic acid integrity and risks of false negative results or failed assays. Specimens that have undergone ‘ gentle ’ decalci ﬁ cation using chelating agents, such as ethylenediaminetetraacetic acid (EDTA) which causes less DNA damage, can be suitable for molecular testing 49 , 52 , 64 and PD-L1 testing; 50 however, non-decalci ﬁ ed

specimens are preferable if available.

For PD-L1, tissue should be sectioned at $4–5~{\upmu\mathrm{m}}$ and mounted on positively charged slides. 60 Strategies to minimise tumour wastage in small biopsy samples should be implemented, including placing individual biopsies in separate blocks if multiple biopsies are available, pre-cutting unstained sections to avoid tissue loss during refacing and avoiding unnecessary immunohistochemical markers during diagnostic workup. 15 , 65 Procedures should also be in place to minimise the risk of tissue contamination during sectioning for molecular testing, including the use of a new blade. 15

4.11. Key Question 11: What is the role of molecular testing using circulating cell-free DNA?

4.11.1. Recommendations For upfront molecular testing:

Testing of circulating tumour DNA (ctDNA) in plasma specimens can be used if the tissue specimen is inadequate or unavailable. If no variants are found, repeat tissue biopsy should be considered to enable molecular testing if clinically appropriate.

ctDNA testing could also be considered if molecular results are required as a matter of urgency and where ctDNA testing results can be achieved faster than tissue-based testing. If no variants are found, repeat testing should be undertaken on a tissue specimen.

In the setting of disease progression on targeted therapy:

ctDNA molecular testing for resistance mechanisms should be considered if tissue biopsy is not feasible (as per KQ3).

4.11.2. Discussion

Due to the site of the tumour or the performance status of the patient, $10–17\%$ of lung cancer cases in Australia do not get a tissue diagnosis $(\mathord{>}1000$ patients annually). 66 Timely access to biopsy in rural and regional communities is a disproportionate contributor to this number and is likely partly responsible for lower rates of molecular testing and poorer outcomes from these underserved areas.

The advent of highly sensitive molecular methods to detect circulating tumour DNA in plasma offers an alternative to tissue-based molecular testing and detecting minimal residual disease. 67 Tumour cells, like normal cells, release DNA into the bloodstream [cell-free circulating DNA (cfDNA) of which ctDNA is a subset]. Liquid biopsies have been successfully developed to identify abnormal genomic variants, largely testing ctDNA in plasma. There are many obvious advantages that make this approach attractive for patients (and their treating doctors), such as speed, convenience, minimal risk and less expensive collection.

Although the performance characteristics of ctDNA tests are improving, there are still some challenges to overcome. 68 Since the absolute and proportion of ctDNA levels in plasma are exceedingly low, the ctDNA assays need to be highly sensitive but also speci ﬁ c to ensure a negligible false positive rate. If using ctDNA assays, it is important to understand the limitations of the assay for its speci ﬁ c clinical use provided by the laboratory. Most ctDNA assays should be able to identify the high mutant variant allele fraction in upfront molecular pro ﬁ ling in advanced stage disease where the tumour burden is high. However, in earlier-stage disease, the limit of detection of some assays is often higher than the anticipated ctDNA levels, resulting in false negative results. This is the rationale for recommending biopsy testing where there is an accessible lesion in the event of a negative plasma ctDNA result on progression. 69 – 71

In lung cancer, as well as having a role in primary diagnosis and identi ﬁ cation of therapies, ctDNA assays can also be used for identi ﬁ - cation of primary and secondary molecular resistance mechanisms after targeted treatment(s). Indeed, ctDNA testing may be a better method than tissue re-biopsy at disease progression (not always possible), as this

approach may more adequately represent the genetic burden of all lesions, providing more complete and contemporary information than a single tissue biopsy. 72 , 73

Upfront ctDNA testing is recommended for patients who are unable to have a tissue biopsy, when tissue for molecular testing is inadequate or if access, speed or clinical urgency of tissue molecular testing is an issue. ctDNA testing should also be considered to identify molecular mechanisms of resistance in patients who have progressed on targeted therapies. If no variants are found, a tissue biopsy, if possible, should be considered to enable molecular testing if clinically appropriate.

4.12. Key Question 12: What techniques should be used for molecular testing?

4.12.1. Recommendations

NGS panel testing using both DNA and RNA is recommended. If NGS panels using both DNA and RNA are not available, then other multiplex methods can also be used [for example, polymerase chain reaction (PCR) based tests]. Assays should be able to identify variants in specimens with at least ${\geq}10\%$ tumour cell content. For ctDNA testing, higher sensitive assays are required.

4.12.2. Discussion

Molecular testing techniques should be selected to maximise result accuracy whilst ensuring comprehensive marker coverage. This can be achieved most ef ﬁ ciently by simultaneous high-throughput parallel sequencing using NGS. Other techniques used to identify genetic variants such as multiplex PCR-based assays are often less comprehensive than NGS methods in terms of both gene and variant coverage. 15

The clinically relevant variants that should be covered by routine molecular testing in lung cancer now include single nucleotide variants and small insertions/deletions [ EGFR, KRAS, BRAF and ERBB2 (HER2) variants] that may be detected by DNA-based NGS, as well as larger structural changes such as ALK, ROS1, RET and NTRK gene rearrangements. Genes with large intronic regions and repetitive sequences such as NTRK3 and MET, and MET splice variants, are more reliably covered and detected using RNA-based NGS. 15 , 74 Therefore, a combination of DNA and RNA panels is ideal for the reliable and comprehensive assessment of all relevant genes. While DNA and RNA panels are preferably run in parallel, RNA panels can be performed sequentially if no variants are found using a DNA panel; however, this results in longer TATs, so should be avoided.

A number of commercially available targeted NGS panels designed to cover clinically relevant variants with high sensitivity are suitable for clinical practice. Any assay used for genomic molecular testing should cover a comprehensive range of relevant oncogenic variants in each gene of interest (e.g., all common EGFR variants as well as relevant less common variants such as EGFR exon 20 insertions). Pathologists and oncologists should ensure chosen assays are appropriate for clinical use. The technique selected should also take into account TATs, labour and expertise requirements.

Single gene assays should be reserved for specimens with limited material and should target EGFR ﬁ rst. Single gene assays can be considered if results are needed urgently or if limited tissue is available, preventing NGS panel testing, although repeat biopsy or ctDNA testing should also be considered in this setting.

As lung cancer specimens may contain a low proportion of tumour cells, assays should ideally be able to identify variants in specimens with as low as $10\%$ tumour cell content (e.g., minimal variant allele frequency of $5\%$ presuming disomy and heterozygosity). 7 It is acknowledged that CAP/IASLC/AMP guidelines recommend assays that are able to detect molecular alterations in specimens with $20\%$ tumour cell content; however, the steering committee felt this would exclude some specimens for testing, and higher sensitivity assays were more desirable. Dissection

techniques can be used to enrich tumour cell content in some cases. Higher sensitive assays are required for ctDNA testing due to the characteristically low fraction of circulating tumour DNA compared to total circulating free DNA. 75

4.13. Key Question 13: How should molecular testing results be reported – what guidance is needed for clinical interpretation?

4.13.1. Recommendations

Molecular results should be reported by a pathologist and/or scientist with expertise in solid tumours and somatic variants. Evidence-based tiers of clinical relevance should be provided to classify somatic variants based on their level of clinical signi ﬁ cance.

4.13.2. Discussion

Any molecular pathology report for therapeutic decision-making needs to accurately and easily convey the appropriate information required for treatment planning. Preferably, all important information should be at the beginning of the report and conspicuously presented. The report should show results clearly, stating which test was used, the relevant technical speci ﬁ cations and any test limitations together with pre-analytic, analytic, or post-analytic factors that may in ﬂ uence interpretation. Molecular results should be reported by a pathologist and/or scientist with appropriate expertise in an accredited laboratory using a Therapeutic Goods Administration (TGA) class III registered test. Guidelines as to best practice for molecular report content in lung cancer have been proposed. 8 The corollary of a good molecular report is the ability of clinicians to have undergone appropriate training to be able to interpret the results to guide clinical care.

To ensure consistency of molecular pathology results and reduce any variability in clinical interpretation that might adversely impact patient management, there are published guidelines for the clinical interpretation and reporting of somatic variants. 76 – 78 The AMP/ASCO/CAP 2017 Standards and Guidelines for the interpretation and reporting of sequence variants in cancer are those used in Australia. This classi ﬁ cation system is widely accepted, straightforward, and provides a reliable, standardised framework for molecular variant interpretation, promoting consistency and accuracy in reporting.

The AMP/IASLC/CAP consensus guidelines use a four-tiered system based on the use of standard Human Genome Variation Society nomenclature to categorise somatic sequence variations based on their clinical signi ﬁ cance and underlying evidence.

Tier 1: Variants with strong clinical signi ﬁ cance [e.g., those linked to Food and Drug Administration (FDA)-approved therapies or supported by robust clinical evidence]. Tier 2: Variants with potential clinical signi ﬁ cance (e.g., associated with investigational therapies or eligibility for clinical trials). Tier 3: Variants of unknown clinical signi ﬁ cance (e.g., variants with insuf ﬁ cient evidence for clinical impact). Tier 4: Variants deemed benign or likely benign (e.g., those not associated with oncogenic processes).

Nevertheless, other classi ﬁ cations such as the European Society for Medical Oncology Scale of Clinical Actionability of Molecular Targets (ESCAT) from ESMO are also available and are widely used in the respective jurisdictions. 77 The ESCAT framework offers a detailed, ten-tiered system for prioritising genomic alterations based on the strength of clinical evidence. Although ESCAT is not widely used in Australia, it can offer valuable insights for therapeutic prioritisation where clinicians are experienced in its application. Its use should only be considered if both the clinical team and bioinformatics systems are capable of supporting its integration. 78

Cancer genomics is a rapidly evolving ﬁ eld, and therefore the clinical signi ﬁ cance of any variant should be re-evaluated on an ongoing basis. It

is recommended that routine standard of care molecular results be incorporated into lung cancer multidisciplinary team discussions and highlighted in the patient record to optimise patient management. Should more comprehensive molecular pro ﬁ ling be undertaken and reveal unusual or unexpected molecular results, discussion at a molecular tumour board should be considered, especially if the results suggest potential germline implications, which may indicate hereditary cancer risk. If this does arise, reports must clearly differentiate between somatic and germline ﬁ ndings, offering appropriate guidance on the clinical signi ﬁ - cance of each. Germline variant reporting should adhere to established guidelines for hereditary cancer predisposition, ensuring patients receive comprehensive and accurate information.

4.14. Key Question 14: What is an appropriate turnaround time (TAT) for molecular testing in Australia?

4.14.1. Recommendations

To enable management decision-making, molecular testing results should be available as soon as possible, ideally within 5 business days, but no more than 10 business days from receipt of the specimen in the molecular laboratory. Laboratories should ensure laboratory procedures and rapid referral pathways are in place to minimise the time taken for specimens to reach the molecular laboratory.

4.14.2. Discussion

The Optimal Care Pathway (OCP) for people with lung cancer provides a framework for high-quality cancer care in Australia. Within the OCP, it is advised that diagnostic tests for lung cancer are completed within 2 weeks of the ﬁ rst specialist appointment with the initiation of treatment advised within 6 weeks. 79 To meet the OCP standards, molecular testing results should be available as fast as possible, ideally within 5 business days, but no more than 10 business days from receipt of the specimen in the molecular laboratory, in line with CAP/IASLC/AMP Guidelines. 7 This can be challenging for laboratories to achieve in a cost-effective manner when batching of specimens is required for maximal ef ﬁ ciency.

The unique challenges of delivering timely and quality molecular testing in regional and rural Australia are acknowledged, particularly the impact of centralised testing models where most labs send specimens to an external laboratory for molecular testing using established referral pathways. Delays in molecular results contribute to the total TAT for completion of all diagnostic tests and add to the psychological toll on patients waiting to commence treatment. Appropriate funding and increased workforce resourcing are likely needed to meet the demands of delivering best practice care, particularly with the anticipated additional impact of the National Lung Cancer Screening program. 80

4.15. Key Question 15: What IHC testing is appropriate for molecular biomarker assessment in NSCLC?

4.15.1. Recommendations

PD-L1 TPS by IHC using a validated assay and appropriate clone is required. Although upfront ALK IHC is not essential if RNA NGS is used for molecular testing, it still has clinical utility and should be performed if there is adequate tissue available. There is no role for routine IHC for ROS1, BRAF V600E, EGFR L858R/ ex19del or NTRK if RNA NGS panels are used for molecular testing as recommended in KQ12.

4.15.2. Discussion

PD-L1 IHC is an important biomarker for immunotherapy treatment decisions, enriching the likelihood of patient response with higher levels

of expression. Laboratories should use a validated assay with the antibody clone used in the clinical trials or an equivalent clone. 60 Several studies have con ﬁ rmed concordance of the relevant PD-L1 antibody clones 22C3 and 28-8 and to a lesser extent, SP263 for determining the tumour proportion score in NSCLC. 38 , 81 , 82 Commercial assays are recommended using Dako 22C3 or Ventana SP263; laboratory-developed tests ( in vitro diagnostics) can be used but require higher standards of validation. 60 , 83 Pathologists need to determine the percentage of viable tumour cells with any membrane staining to determine the tumour proportion score. 60 Scoring using the TPS has shown good reproducibility amongst pathologists. 38 , 84

Whilst PD-L1 IHC assessment is preferentially performed on tissue specimens, several studies have shown good concordance between PD-L1 assessment on cell blocks and paired tissue biopsy results. 85 , 86 Cytology smears are not recommended for PD-L1 assessment. 87 For all samples but especially cytology, adequate cellularity is recommended, typically 100 tumour cells, as there is a risk of false negative results in samples with low tumour cellularity. 88

ALK IHC using a validated assay with an appropriate clone such as D5F3 or 5A4 has high sensitivity and speci ﬁ city and positive expression is highly predictive of an ALK fusion. 89 , 90 Although upfront ALK IHC is not essential if RNA NGS testing is used, it still has clinical utility due to the rapidity and high reliability of the result and could be performed if there is adequate tissue. In Australia, unlike in some other jurisdictions, molecular testing using FISH or NGS for identi ﬁ cation of an ALK fusion is required, even with a positive ALK IHC result.

ROS1 IHC can be used to screen for ROS1 fusions if RNA NGS is not available. Most studies have used the D4D6 clone, 91 although SP384 and 1A1 clones have shown similar results. 92 Negative ROS1 IHC has a strong negative predictive value, so if NGS is not available, a negative ROS1 IHC is suf ﬁ cient to exclude a ROS1 fusion. 7 However, positive ROS1 IHC has low speci ﬁ city and is not strongly predictive of ROS1 fusion unless the staining is strong and diffuse. As such, ROS1 IHC is not recommended for routine use, and if performed, always requires molecular or cytogenetic testing to con ﬁ rm a positive result. There is no universally accepted scoring system for ROS1 IHC, and although staining in NSCLC harbouring ROS1 rearrangement tumours is usually cytoplasmic and diffuse, it can be granular/globular, or weak membranous, depending on the fusion partner. 93 – 95

Although IHC antibodies are available for pan-TRK, MET, ERBB2 (HER2) and mutation-speci ﬁ c IHC antibodies for BRAF V600E, EGFR L858R and EGFR exon 19 deletion (15 bp deletion only), molecular testing is required for treatment decisions and these IHC are not recommended for routine use due to lack of clinical utility and risk of wasting tissue.

4.16. Key Question 16: What quality assurance (QA) measures are required for biomarker testing?

4.16.1. Recommendations

All assays and all specimen types used for molecular testing should be fully validated by the laboratory. For IHC markers, validation to a standard suitable for predictive IHC biomarkers is required for all predictive IHC. Laboratories performing any biomarker molecular analyses must participate in an external QA program for all tests (molecular and IHC), as well as performing internal quality control (QC) measures. Laboratories should ensure test results that are unexpected or equivocal should be con ﬁ rmed or resolved using an alternative method or sample.

4.16.2. Discussion

Laboratory accreditation, internal QC, internal audits and participation in external quality assessment (EQA) schemes are required for a reliable molecular service. 96 Testing should be undertaken in accredited

laboratories (ISO 15189 or equivalent), and all assays should be validated across the range of specimen types and ﬁ xatives (e.g., FFPE, cytology smears) according to published guidelines. 50 , 96 , 97

During validation of NGS, criteria for specimen acceptance should be established and minimum quality requirements should be determined to ensure accuracy, precision, sensitivity and limits of detection for each type of genetic alteration. 7 , 97 Quality control metrics that are established during validation must be followed in each run, including, but not limited to, nucleic acid quantity and quality, library quali ﬁ cation and quanti ﬁ cation, sequencing depth and uniformity of coverage. Quality and quantity of DNA and RNA should be assessed using ﬂ uorometric-based techniques and minimal nucleic acid inputs must be determined. 97 Each run should include non-template controls to ensure no contamination and procedures should be in place to prevent carryover from other specimens. Data analysis pipelines for NGS should be standardised.

Any test results that are unexpected based on clinicopathological features, discordant with other testing modalities, or equivocal, should be con ﬁ rmed or resolved using an alternative method or sample wherever possible. 7

For predictive IHC biomarkers such as PD-L1, a higher standard of validation is required compared to diagnostic IHC markers. 50 , 98 A minimum validation set of 20 positive and 20 negative samples is required, and results should be compared with a previously validated or veri ﬁ ed assay (e.g., from another laboratory). Selected cases should cover the full expected range of expression levels (i.e., $0–100\%$ for PD-L1), differing specimen types, and assess the accuracy close to clinically relevant thresholds. Analytical validation (or if necessary, indirect clinical validation) is required for all laboratory-developed tests ( in vitro diagnostics) and is also recommended for unmodi ﬁ ed TGA approved predictive assays if no speci ﬁ c instructions for assay veri ﬁ cation are provided by the manufacturer. 98

All pathologists and scienti ﬁ c staff should be fully trained, and the laboratory should participate in EQA programs to assess ongoing technical and interpretation pro ﬁ ciency and to benchmark performance against other laboratories. 96

5. Conclusion

The recommendations for molecular testing in lung cancer provide pathologists and clinicians with best practices guidelines, re ﬂ ecting a collaborative effort between pathologists, oncologists and patient advocates. This multidisciplinary approach has established a robust evidence base for molecular testing of lung cancer across Australia, which should give all patients and communities the opportunity to bene ﬁ t from the latest advancements in healthcare.

The introduction of consensus best practice recommendations can help ensure that all patients have equal access to high-quality diagnostic services. Implementation of the recommendations can provide an evidence base to inform and enable lung cancer care pathways across our population. The recommendations support a shared care approach requiring collaboration between healthcare teams and patients to ensure high quality molecular testing is provided to guide optimal clinical care. By adhering to these recommendations for molecular testing in lung cancer patients, we anticipate that healthcare providers can optimise resource use, minimise the need for multiple invasive biopsies, reduce patient discomfort and hospital stays and help ensure timely initiation of appropriate therapies, which should in turn enhance outcomes and quality of life for all lung cancer patients. It is acknowledged, however, that these recommendations only address a component of optimal patient management and do not address other barriers to optimal care, such as access to primary healthcare providers or lung cancer specialists, including those that provide diagnostic procedural services or surgical services, as well as oncologists.

Further research is needed to fully map the existing molecular testing pathways in each state and territory to identify gaps in service provision

and ways to improve optimal care pathways nationally. In particular, data are lacking on any differences in concerns or access to molecular testing for different patient populations such as the Aboriginal and Torres Strait Islander population or culturally and linguistically diverse populations. Future steps will include monitoring the uptake of these recommendations in the broader context of optimal patient care, as well as reviewing and updating them as the evidence base changes, to inform future best practices that can be applied ef ﬁ ciently, systematically and equitably for all Australians.

Con ﬂ icts of interest and sources of funding

The RCPA acknowledges the ﬁ nancial support provided by AstraZeneca Australia in the development of the RCPA Consensus White Paper for Molecular Testing of Lung Cancers. No authors received funding for their contributions, and AstraZeneca was not involved in any part of the manuscript development.

Benhur Amanuel has acted as a member of the advisory board in several pharmaceutical companies. Wendy Cooper has received support to attend domestic meetings from Daiichi Sankyo and AstraZeneca. WeiSen Lam has received honorarium from BMS, MSD, Roche, Novartis, Roche, Sano ﬁ , Merck. Member of the advisory board for Amgen and Janssen. Trishe Leong has received speaker's honoraria from MSD, Amgen and AstraZeneca. Zarnie Lwin has received speaker's honoraria and/or ﬁ nancial support for conference travel and/or advisory boards and/or educational meetings from Janssen, MSD, Servier, AstraZeneca, Roche, P ﬁ zer and BMS. Nick Pavlakis has served on advisory boards for MSD, Merck, BMS, AstraZeneca, Takeda, P ﬁ zer, Roche, Amgen, Beigene, Gilead, Zymeworks; received speaker's honoraria from Merck, P ﬁ zer, Roche, Takeda, Illumina, Bayer, MSD; and received institutional research funding from Bayer, P ﬁ zer, Roche. Rachel Roberts-Thomson has served on advisory boards for Bristol Myer Squibb, Amgen, Merck, Sharpe and Dohme and received honoraria/speaker's fees from Bristol Myer Squibb, Merck, Sharpe and Dohme, AstraZeneca, Pierre Fabre, Novartis, Roche. Ben Solomon has served on advisory boards and received honoraria from P ﬁ zer, Roche, AstraZeneca, Merck Sharp Dohme, Bristol Myers Squibb, Janssen, Amgen, Eli Lilly, Glaxo Smith Kline, Daiichi Sankyo. All other authors have no ﬁ nancial relationships to declare.

Acknowledgements

The following organisations have provided endorsement of the recommendations.

Royal College of Pathologists of Australasia, Thoracic Oncology Group of Australasia, Lung Foundation Australia and the Thoracic Society of Australia & New Zealand.

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表 1
Key Question	Consensus recommendationa
1. Of the currently identified
biomarkers, which should be	· EGFR, BRAF, KRAS, ERBB2 (HER2), ALK, ROS1, MET, RET and NTRK and PD-L1 should be tested in all advanced stage non-squamous NSCLC. This includes any advanced-stage lung carcinoma with an adenocarcinoma component. For example, non-small cell carcinoma with squa-
routinely tested in advanced stage	mous and glandular differentiation or where an adenocarcinoma cannot be excluded (e.g., a smallbiopsy/cytology diagnosis of NSCLC-NOS).
non-squamous NSCLC?	· EGFR testing should include all clinically relevant oncogenic variants in exons 18, 19, 20 and 21. ● Testing should be performed on reflex by pathologists to expedite TAT.
2. Of the currently identified	● At a minimum, EGFR, ALK, ROS1 and PD-L1 testing must be undertaken in stage IB-II non-squamous NSCLC, and non-resectable locally
	advanced non-squamous NSCLC treated with curative intent chemoradiotherapy. ● More comprehensive testing for EGFR, KRAS, BRAF, ERBB2 (HER2), MET, ALK, ROS1, RET and NTRK can be undertaken in the early stage
biomarkers, which should be (routinely) tested in early stage non-	● More comprehensive testing for EGFR, KRAS, BRAF, ERBB2 (HER2), MET, ALK, ROS1, RET and NTRK can be undertaken in the early stage setting if practical. ● No molecular testing is required for surgically resected stage IA NSCLC.
squamous NSCLC?
3. Should tumour biopsy and	· In patients progressing on any targeted therapy, molecular testing for resistance mechanisms should be considered if tissue is available. · If tissue biopsy is not available or feasible, plasma-based ctDNA molecular testing for resistance mechanisms should be considered.
molecular testing be undertaken on
disease progression during
treatment with targeted therapy? 4. Is there a role for more extended
	· More comprehensive testing could be considered for patients with advanced stage NSCLC, especially cases lacking any of the oncogenic variants of the genes outlined in KQ1 when clinically relevant.
biomarker testing? 5. Should patients with squamous cell	● At a minimum, advanced-stage squamous cell carcinoma should undergo MET and PD-L1 testing.
carcinoma on biopsy undergo
molecular testing?	● Upon request, molecular testing for all markers in KQ1 should be undertaken for patients diagnosed with lung squamous cell carcinoma on small biopsy or cytology that have a clinical profile suspicious for driver mutation (e.g.,

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