Squamous cell carcinoma differentiation at progression as a resistance mechanism in metastatic non-small cell lung carcinoma harbouring a targetable mutation

Abstract

Non-small cell lung cancer (NSCLC) comprises approximately 85% of patients diagnosed with metastatic disease,1Testa U. Castelli G. Pelosi E. Lung cancers: molecular characterization, clonal heterogeneity and evolution, and cancer stem cells.Cancers (Basel). 2018; 10: 248Crossref PubMed Scopus (183) Google Scholar of which the major histological subtypes are adenocarcinoma (ADC) and squamous cell carcinoma (SCC).2WHO Classification of Tumours Editorial BoardThoracic tumours.in: Borczuk A.C. Cooper W. Dacic S. WHO Classification of Tumours. 5th ed. IARC, Lyon2021Google Scholar These tumour types are thought to originate from different cell types, typically arise in different locations within the lung, express differing molecular alteration profiles and are variably associated with tobacco exposure. Lung ADC are thought to arise from alveolar type II cells, bronchoalveolar stem cells, or club cells,3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar are more often located distally, and have been identified to harbour a variety of therapeutically targetable oncogenic molecular alterations involving genes such as EGFR, ALK, ROS1, RET, BRAF, MET, NTRK, TP53 and HER2. In contrast, SCCs are typically more centrally located, arising from basal cells in bronchiolar epithelium,3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar and harbour mutations in genes such as TP53, CDKN2A, PTEN, PIK3CA, KEAP1, MLL2, and NFE2L2.1Testa U. Castelli G. Pelosi E. Lung cancers: molecular characterization, clonal heterogeneity and evolution, and cancer stem cells.Cancers (Basel). 2018; 10: 248Crossref PubMed Scopus (183) Google Scholar Most patients with SCC will have a prior smoking history, whereas a subset of patients with ADC have minimal or no cigarette exposure and these adenocarcinomas are associated with mutations of EGFR (approximately 47% in Asia-Pacific NSCLC/ADC cohorts4Midha A. Dearden S. McCormack R. EGFR mutation incidence in non-small-cell lung cancer of adenocarcinoma histology: a systematic review and global map by ethnicity (mutMapII).Am J Cancer Res. 2015; 5: 2892-2911PubMed Google Scholar and 15% in Western cohorts).5Mead S. Lucas M. Pang J.-M. et al.EGFR mutation profile in Australian patients with non-small cell lung cancer.Pathology. 2021; 53: 933-936Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Adenosquamous carcinomas (ADSC) are rare NSCLCs (2–3% of lung cancers) which by definition include at least 10% of both ADC and SCC in resection specimens.2WHO Classification of Tumours Editorial BoardThoracic tumours.in: Borczuk A.C. Cooper W. Dacic S. WHO Classification of Tumours. 5th ed. IARC, Lyon2021Google Scholar They may occur in non-smokers and may harbour similar oncogenic driver mutations to lung ADC2 but can be difficult to diagnose in small biopsy samples and sampling bias may lead to misclassification as either ADC or SCC. Amongst EGFR-mutated lung ADC treated with EGFR tyrosine kinase inhibitors (TKIs), transformation to small cell carcinoma is a recognised mechanism of treatment resistance.1Testa U. Castelli G. Pelosi E. Lung cancers: molecular characterization, clonal heterogeneity and evolution, and cancer stem cells.Cancers (Basel). 2018; 10: 248Crossref PubMed Scopus (183) Google Scholar Emerging within the literature is the recognition that squamous differentiation/transformation may also be a potential mechanism of treatment resistance; either due to true ADC to SCC transition (AST),3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar or clonal selection that has occurred in the context of initial ADSC histology. We describe two intriguing cases from our institution, with initial ADC and ADSC histology harbouring an oncogenic driver mutation and subsequent development of predominant SCC differentiation on re-biopsy at progression. Case 1 was a 55-year-old life-long never-smoker of South-East Asian ethnicity, who was diagnosed with de novo metastatic NSCLC in June 2018 with radiological evidence of a lung primary, lymphangitis carcinomatosis, bilateral thoracic node involvement and a single 4 mm brain metastasis. At diagnosis, fine needle biopsy of a supraclavicular fossa lymph node demonstrated metastatic adenocarcinoma histology (TTF1+, p40–), a programmed death ligand-1 (PDL-1) tumour proportion score (TPS) of 10%, with an ALK translocation confirmed by fluorescence in situ hybridisation (Fig. 1A–C). Following stereotactic radiosurgery to the brain metastasis she commenced treatment with the ALK TKI crizotinib via a clinical trial. In December 2018, at intra-cranial progression, she proceeded to second-line alectinib, an ALK directed TKI. In July 2021 she underwent biopsy of a newly enlarged axillary lymph node, which revealed a keratinising SCC with occasional cells demonstrating intracytoplasmic mucin, and immunohistochemistry (IHC) profile consisting of p40+, TTF1– (Fig. 1D–H), and preserved ALK translocation on IHC. Subsequently, a progressing isolated brain metastasis was resected in December 2021, with evidence of adenocarcinoma and squamous components and ALK alteration preserved. Case 2 was a 52-year-old life-long never-smoker of Caucasian ethnicity, who was diagnosed with de novo metastatic NSCLC in December 2018, with baseline imaging demonstrating a lung primary, regional lymphadenopathy and bilateral lung and skeletal metastases. Surgical biopsy of a femoral lesion during orthopaedic intervention revealed metastatic carcinoma demonstrating predominant ADC (acinar and cribriform architecture, TTF1+) with a small focus (approximately 5–10%) of squamous morphology (p40+, cytokeratin 5/6+), raising the possibility of ADSC (Fig. 2A–F). An EGFR exon 19 deletion was detected and PDL-1 TPS was 3%. First-line systemic therapy was commenced with erlotinib, a first generation EGFR TKI. In September 2019 at progression a liver biopsy demonstrated TTF1+ adenocarcinoma harbouring an EGFR T790M resistance mutation. Second-line osimertinib, a third generation anti-EGFR TKI, was commenced until progression in July 2020. A further biopsy of a liver lesion demonstrated metastatic adenocarcinoma, TTF1+. The patient commenced third-line treatment with platinum-doublet chemotherapy (carboplatin/pemetrexed) in combination with doublet immunotherapy [durvalumab, a PDL-1 inhibitor; and tremelimumab, a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) inhibitor] via clinical trial until progression in July 2021 (ILLUMINATE trial NCT03994393). Hip lesion core biopsies demonstrated metastatic keratinising SCC (p40+/TTF1–) with no evidence of glandular differentiation. Repeat mutation testing revealed preservation of the original EGFR exon 19 deletion with no T790M mutation detected. Subsequent treatment response with trial anti-EGFR therapy was noted (Fig. 2G,H). Whilst development of small cell lung cancer histology is a well described but uncommon mechanism of TKI resistance in metastatic lung ADC, morphological change involving subsequent development of SCC histology has been rarely reported.3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar Amongst the just over 20 cases described to date, the majority have been recognised to harbour an EGFR mutation at initial diagnosis, which have been preserved at re-biopsy with some harbouring a T790M mutation.3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar To our knowledge, only five cases of ALK-translocated cases have been reported with subsequent SCC differentiation: one after progression on crizotinib;6Wang F. Qin J. Xie F. et al.Transformation of EML4-ALK fusion-positive adenocarcinoma into squamous cell carcinoma in association with acquired resistance to crizotinib.Lung Cancer. 2020; 140: 118-120Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar one after exposure to crizotinib, ceritinib and alectinib;7Gong J. Gregg J.P. Ma W. et al.Squamous cell transformation of primary lung adenocarcinoma in a patient with EML4-ALK fusion variant 5 refractory to ALK inhibitors.J Natl Compr Canc Netw. 2019; 17: 297-301Crossref PubMed Scopus (13) Google Scholar one after exposure to alectinib;8Kaiho T. Nakajima T. Iwasawa S. et al.ALK rearrangement adenocarcinoma with histological transformation to squamous cell carcinoma resistant to alectinib and ceritinib.Onco Targets Ther. 2020; 13: 1557-1560Crossref PubMed Scopus (9) Google Scholar one after alectinib and crizotinib;9Park S. Han J. Sun J.M. Histologic transformation of ALK-rearranged adenocarcinoma to squamous cell carcinoma after treatment with ALK inhibitor.Lung Cancer. 2019; 127: 66-68Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar and one heavily pre-treated patient with prior exposure to cytotoxic therapy, multiple ALK-directed TKIs including most recently lorlatinib.10Ueda S. Shukuya T. Hayashi T. et al.Transformation from adenocarcinoma to squamous cell lung carcinoma with MET amplification after lorlatinib resistance: a case report.Thorac Cancer. 2021; 12: 715-719Crossref PubMed Scopus (5) Google Scholar Whether the phenomenon is more likely to occur in patients with EGFR-mutated NSCLC compared to ALK-translocated NSCLC is difficult to surmise given the relatively lower incidence of ALK-translocations (approximately 3–7% of patients diagnosed with metastatic NSCLC),11Itchins M. Chia P.L. Hayes S.A. et al.Treatment of ALK-rearranged non-small cell lung cancer: A review of the landscape and approach to emerging patterns of treatment resistance in the Australian context.Asia Pac J Clin Oncol. 2017; 13: 3-13Crossref PubMed Scopus (9) Google Scholar and re-biopsy at progression may be less common than for EGFR-mutated cases. The fact that EGFR and ALK alterations are preserved despite morphological change from initial ADC to SCC in cases illustrating this phenomenon in the literature, suggests a phenomenon of ADC to SCC transition (AST). In ADSC cohorts, the identification of oncogenic drivers in both the ADC and SCC components has likewise been suggested to support the notion of AST.1Testa U. Castelli G. Pelosi E. Lung cancers: molecular characterization, clonal heterogeneity and evolution, and cancer stem cells.Cancers (Basel). 2018; 10: 248Crossref PubMed Scopus (183) Google Scholar Observational data from case reports for AST is further supported by Chen et al., who described loss of the LKB1 gene in genetically engineered mouse models harbouring KRAS G12D mutations as being associated with development of AST.3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar The authors also note the role of YAP, a transcriptional co-factor, which acts as an oncogenic driver in the case of lung ADC, whereas it exerts a tumour suppressor function in SCC, suggesting that additional understanding of the function of YAP may contribute to greater understanding of AST.3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar It is unclear whether the cases presented represent true AST or clonal selection in the context of initial ADSC histology, such as for Case 2, and the implications for subsequent clinical management. Whether future treatment should be guided by SCC rather than ADC histology is unclear. In both cases, SCC differentiation has been demonstrated upon disease progression, and for Case 1 was indicative of TKI resistance. In similar cases, development of SCC differentiation has in some cases been described alongside acquisition of known resistance mechanisms to first or second generation EGFR-TKIs given at the time of re-biopsy (eg., T790M, PIK3CA).3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar Likewise one of the ALK-translocated cases was noted to have MET amplification at re-biopsy.10Ueda S. Shukuya T. Hayashi T. et al.Transformation from adenocarcinoma to squamous cell lung carcinoma with MET amplification after lorlatinib resistance: a case report.Thorac Cancer. 2021; 12: 715-719Crossref PubMed Scopus (5) Google Scholar Whether development of SCC differentiation is a resistance mechanism particular to patients with oncogene-addicted NSCLC following TKI exposure is unclear; however, we are only aware of two reported cases with wild-type for EGFR and ALK mutations.3Chen Y. Tang W.Y. Tong X. Ji H. Pathological transition as the arising mechanism for drug resistance in lung cancer.Cancer Commun (Lond). 2019; 39: 53Crossref PubMed Scopus (17) Google Scholar Hence, there may be several potential explanations for apparent development of SCC histology in both cases: (1) the cases were both ADSC initially (with failure to detect SCC component in Case 1 due to sampling bias) and subsequent TKI therapy led to clonal selection of the resistant SCC component; (2) true AST occurred (e.g., Case 1) through acquisition of additional resistance mechanisms; (3) both cases were true ADSC with random sampling of ADC and/or SCC components at different time points and apparent squamous transformation unrelated to the development of resistance. Whilst in both cases described above SCC differentiation was noted on tissue biopsy, it is noteworthy that there is growing momentum for a ‘plasma-first’ approach to re-biopsy of patients during the course of metastatic NSCLC to guide therapeutic decision making. Although there may be advantages to liquid biopsies with respect to accessibility and safety, the described cases illustrate the importance of tissue biopsy to aid morphological pathological assessment to identify resistance mechanisms, such as transition to SCC or small cell carcinoma.12Clery E. Pisapia P. Feliciano S. et al.There is still a role for cytology in the 'liquid biopsy' era. A lesson from a TKI-treated patient showing adenocarcinoma to squamous cell carcinoma transition during disease progression.J Clin Pathol. 2017; 70: 798-802Crossref PubMed Scopus (15) Google Scholar At this stage, given the paucity of reported cases, it is unclear whether subsequent management should account for histological transformation to SCC; however, identification of subsequent small cell histological transformation enables use of platinum/etoposide chemotherapy and informs prognostication. Additional challenges, especially for cases of ADSC (illustrated by Case 2), include sampling bias where a single site of tissue biopsy may fail to capture both the SCC and ADC components within a patient’s tumour. In the case of an ADC component not being appreciated, this is further problematic given testing for oncogenic driver mutations does not occur routinely for cases of metastatic SCC. As such, clinician suspicion in the event of an apparent new diagnosis of SCC in a never-smoker is important to raise the question as to whether the patient may instead have ADSC and hence warrant mutation testing.