Background
Nowadays, non-small-cell lung cancer (NSCLC) might be considered as a universe of different diseases. Particularly in the context of adenocarcinoma, reliable evidence is available suggesting that cancer development and progression might be led by the addiction from aberrant pathways triggered by genetic abnormalities acting as oncogenic
drivers (such as the activating mutation of EGFR or the translocation of ALK). In this setting, the inhibition of these
drivers with selective agents has radically changed the natural history of the disease [
1‐
7]. Unfortunately, only a limited subpopulation of lung cancer patients might benefit from this personalized treatment. Hence, the importance of identifying and validating new molecular alterations with prognostic and predictive significance in order to extend the proportion of lung cancer patients who might benefit from targeted drugs.
ALK is a versatile oncogene whose role has been recognized in a large variety of tumors through different activation mechanisms, mainly the chromosomal rearrangement with different fusion partners (as the microtubule associated protein EML4 in NSCLC or the nucleophosmin NPM1 in anaplastic large cell lymphoma). In NSCLC, the successful history of ALK inhibitors started with crizotinib and is still ongoing [
8]. Crizotinib is an orally available tyrosine kinase inhibitor (TKI) originally designed to target the mesenchymal to epithelial transition process, but it also potently inhibits ALK and ROS1 phosphorylation and signaling [
9]. Based on encouraging preclinical data, impressive preliminary results were published from an expansion cohort of a phase I trial [
10] and from the PROFILE 1005 phase II trial [
11]. In the second-line trial PROFILE 1007, 347 patients with ALK-rearranged NSCLC pretreated with a platinum doublet received either crizotinib or second-line chemotherapy with docetaxel or pemetrexed. Patients benefited from crizotinib in both terms of overall response rate (ORR) (65 % versus 20 %;
p < 0.001) and median progression-free survival (PFS) (7.7 versus 3.0 months; HR 0.49;
p < 0.001) [
6]. Moreover, in the first-line phase III trial PROFILE 1014, 343 treatment-naive patients with ALK-rearranged NSCLC were randomized to receive either crizotinib or standard platinum-based plus pemetrexed first-line chemotherapy. Also in this setting, crizotinib met not only the primary end point, achieving a significantly longer median PFS (10.9 versus 7.0 months; HR 0.45;
p < 0.001), but also a significantly higher ORR (74 % versus 45 %;
p < 0.001). The more frequent adverse events associated with the administration of crizotinib were vision disorders, diarrhea, edema, increased aminotransferase levels and neutropenia [
7]. The main limitations of the crizotinib efficacy are represented by its low brain penetrance and its inactivity against secondary mutations of the ALK gene. Therefore, clinical trials evaluating next generation ALK inhibitors are currently ongoing. Promising response rates and PFS have been reported particularly in crizotinib-refractory ALK-rearranged NSCLC patients treated with ceritinib [
12‐
14], alectinib [
15,
16], brigatinib [
17] and lorlatinib [
18]. Recently, the pre-planned interim analysis of the J-ALEX trial, demonstrated the superiority of alectinib to crizotinib in untreated ALK-rearranged NSCLC patients (median PFS not reached versus 10.2 months; HR 0.34;
p < 0.0001) [
19].
Another ALK aberration, the gene copy number gain (CNG), has been identified in several tumor types and significantly correlated with poor prognosis and/or advanced disease status.
In this regard, ALK-CNG has been reported in the 10 % of renal cell carcinoma patients and the presence of more than 5 copies of the ALK gene was significantly associated with high tumor size, nuclear grade and worse 10-years survival rate [
20]. In colorectal cancer patients, ALK-CNG was found in the 3.4 % of non-molecularly selected patients and in the 37 % of RAS-BRAF-PI3KCA wild-type patients [
21,
22]. In both studies, the ALK gene copy number increase was significantly associated with poor prognosis. Moreover, in
triple wild-type patients, the response rate to cetuximab or panitumumab was significantly higher in the subgroup of disomic ALK (70 %) as compared with ALK-CNG subgroup (32 %), with similar results in term of PFS and overall survival (OS), raising the hypothesis of a possible role of ALK-CNG in resistance to anti-EGFR therapy [
21,
22]. ALK-CNG was further identified in the 13 % of patients affected by hepatocellular carcinoma, negative for serum hepatitis B virus DNA, with a significant correlation among ALK-CNG (≥4 copies versus < 4 copies), 3-year PFS rate (27 % versus 42 %) and 3-year OS rate (18 % versus 47 %) [
23]. In rhabdomyosarcoma (RMS), ALK-CNG was detected in the 88 % of alveolar RMS and in the 52 % of embryonal RMS (ERMS). In ERMS, specific ALK gain in the primary tumor correlated with metastatic disease and poor 5-year disease-specific OS (62 versus 82 %) [
24]. Moreover, ALK-CNG was retrospectively detected in the 47.2 % of patients with inflammatory breast cancer and significantly correlated with worse overall survival (24.9 versus 38.1 months) and recurrence free survival (RFS) after curative mastectomy (12.7 versus 43.3 months) compared to ALK-CNG negative patients [
25]. Finally, an aberrant activation of ALK, both by activating mutation and amplification, might drive the tumorigenesis of neuroblastoma (NBL) [
26]. In a retrospective analysis of pediatric patients with NBL, ALK amplification due to polyploidy (ALK/CEP2 ratio > 4) represented a negative prognostic factor for OS [
27].
Regarding lung cancer, the presence and potential prognostic role of the different ALK gene aberrations (translocation and gene copy number gains) have been widely investigated with debatable results (as more extensively revised in the discussion paragraph). Our study, retrospectively conducted in a large series of NSCLC patients, aimed to investigate the potential correlation between ALK-CNG and clinical features, exploring in particular the prognostic implications of this genetic abnormality in resected and advanced NSCLC patients.
Discussion
Our analysis aimed to investigate the potential correlation between ALK-CNG and clinical features, exploring the prognostic implications of ALK-CNG in a retrospective cohort of 167 NSCLC patients. We decided to exclude from the analysis patients harboring EGFR mutant and ALK rearranged NSCLC in order to avoid additional confounding factors reducing the reliability of the prognostic analysis. In fact, to evaluate the prognostic effect of ALK and EGFR alterations, patients should not be treated with the selective inhibitors commonly used in clinical practice, because the benefit deriving from these agents might mask the prognostic value of the biomarker in-study. Overall, ALK-CNG was observed in the 34.1 % of cases. The 25.1 % of the samples showed ALK fluorescent signal ranging from 3 to 7 and, for all of them, the ALK-CNG was due to polyploidy (ALK/CEPs ratio < 2), whereas only 9 % of tumors showed ALK fluorescent signal > 7. Therefore, similarly to what demonstrated by our group in the study of the 3q chromosomal amplification in squamous cell lung carcinoma [
29], ALK-CNG seems mostly related to polyploidy (whole DNA reduplication) rather than locus specific gene amplification.
As reported in other retrospective studies [
24], our results suggest a correlation between ALK-CNG and early stage disease (the majority of T1 tumors clusterize in pattern B, none in pattern C). Moreover, smokers and former smokers showed an increased probability to harbor an ALK-CNG compared to never smokers, which are in the 80 % of cases identified in pattern A. These findings support the hypothesis that ALK-CNG might represent a marker of chromosomal instability appearing early in NSCLC carcinogenesis and potentially triggered by cigarette smoking.
Although no definitive conclusions can be drawn from the results of our prognostic analysis, some interesting hypotheses emerged regarding the intrinsic biological features of the three ALK-CNG patterns, potentially reflecting different levels of chromosomal instability. In fact, while for ALK-CNG pattern B a greater 1-year DFS (80.0 %) and 2-year OS (69.5 %) clearly emerged in resected tumors, conversely, for pattern C, the DFS rate was very low (26.7 %) and in the metastatic setting none of these patients survives at 2 year time-point. Regarding the PFS, the greater 1-year PFS observed in patients with ALK-CNG pattern C (38.9 % versus 19.6 % for pattern A and 15.8 % for pattern B) might reflect the higher chemo-sensitivity of these tumors featured by a strong chromosomal instability. Although in our work we calculate a 2-year survival rate, future studies should be performed with long-term survival outcomes in order to reliably evaluate the prognostic role of a biomarker for clinical setting.
Similarly to our analysis, some other studies evaluating the incidence and potential prognostic implications of ALK gene aberrations have been performed in lung cancer with debatable results. In the context of a retrospective analysis of 107 NSCLC cases, ALK amplification (>5 copies of ALK per cell in 10 % of analyzed cells) and ALK-CNG (mean copy number of 3–5 in 10 % of cells) were identified in the 10 % and 63 % of NSCLC patients, respectively. Although no significant correlation between ALK and clinico-pathological features or prognosis emerged, a significant association between ALK amplification and early stage has been reported, supporting the hypothesis that ALK amplification might represent an early genetic event in NSCLC and potential marker of genomic instability [
30]. In a retrospective analysis of 20 pulmonary sarcomatoid carcinomas (PSC), the frequency of ALK-CNG was significantly higher compared to NSCLC with adenocarcinoma histology (22 % versus 0.02 %) with a mean copy number gain of 7 and a significant association with chromosome 7 (EGFR) and 17 (HER2) polysomy. This finding suggests the implication of ALK-CNG as an oncogenic event in PSC, potentially correlated with epithelial-mesenchymal transition and sarcomatoid differentiation [
31]. Future confirmation of the oncogenic role of ALK-CNG could support the design of selective studies exploring the potential activity of the ALK inhibition in PSC. Another study conducted in patients with NSCLC and brain metastases reported ALK-CNG in the 11 % of cases with an interesting increased ALK-CNG in brain metastases compared to primary tumors, supporting the rationale of ALK-CNG as a genetic aberration connected to aggressiveness and metastatic behavior [
32]. Moreover, in a large retrospective analysis of 1500 NSCLC patients, ALK-CNG (mean native copy number ranged from 2 to 7) was reported in the 80 % of cases and was significantly more common in ALK non-rearranged tumors compared to the translocated ones (62 % versus 19 %). Furthermore, as observed in our analysis, ALK-CNG was mostly related to polysomy, whereas focal amplification was a rare event (<2 % in ALK non-rearranged tumors) [
33]. Regarding ALK inhibitors, ALK-CNG, besides representing a marker of insensitivity to crizotinib, was reported to be a mechanism of resistance to crizotinib in ALK-translocated NSCLC both in vitro [
34] and in patients progressing during crizotinib treatment [
35]. To summarize, our analysis supports the fact that, although ALK pattern B seems to appear early in the tumorigenesis of NSCLC and might mirror an early genomic instability, an high ALK gene copy number (>7, pattern C) related to polyploidy, could be considered as a cut off to discriminate genomically unstable and smoking-related NSCLC featured by an aggressive biological behavior, frequently in advanced stage and with a poor awaited prognostic outcome.
Conclusion
Although limited by the retrospective nature, the results of our analysis are able to generate interesting hypotheses regarding the biological behavior and the potential therapeutic implication of ALK genetic aberrations, ALK-CNG in particular. As observed in other studies, our analysis confirms that a high ALK gene copy number gain is not a driver genetic event in lung cancer tumorigenesis but it might represent a marker of chromosome instability, correlated with an aggressive metastatic behavior. In this regard, early pathogenic events probably induce different ALK aberrant NSCLC. The ALK non-translocated tumors are frequently associated with chromosomal instability and ALK-CNG, whereas the ALK-translocated NSCLC presents a low native ALK copy number and the increase in ALK-CNG emerges as a mechanism of resistance to crizotinib treatment. Unfortunately, conversely to ALK and ROS1 translocations that are widely recognized oncogenic drivers in a small subset of NSCLC able to predict sensitivity to specific inhibitors, ALK-CNG represents a candidate mechanism of resistance to this target therapy. Therefore, it is mandatory to clarify the role and mechanism of this genetic aberration in order to identify and validate effective therapeutic approaches in this subpopulation of lung cancer patients harboring ALK-CNG. In this context, our analysis is conceived and designed as a further step towards a more biologically rationale approach to treat NSCLC patients. The implications for prognosis should be prospectively investigated and validated in larger patients’ series.