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  • br Table br Assignment of ALK Status NGS Versus

    2022-05-06


    Table 4
    Assignment of ALK Status: NGS Versus IHC.
    NGS+ NGS- Total, n Accordance
    Abbreviations: NGS, Next Generation Sequencing; IHC,Immunohistochemistry.
    Fig. 2. The Venn diagram of ALK positive(A) and ALK negative(B) cases detected by three methodologies in 40 patients who received Crizotinib. All cases have been evaluated by IHC and NGS for ALK status. Among them, 21 cases had assessable FISH results, while 9 cases failed to be judged as positive or negative.
    Further, we explored the dissimilitude of clinical efficacy between ALK + and ALK- patients in each group when accepting Crizotinib and found no significant differences between ALK status and PFS in three groups (FISH, P = 0.75; IHC, P = 0.93; NGS, P = 0.09), as depicted in Fig. 4A, except that NGS-confirmed ALK positive patients tended to have a better PFS than those patients who were NGS negative but considered ALK positive by other methods. A higher ORR and DCR were also displayed in NGS positive cases compared with NGS negative cases (P = 0.07 and P = 0.02), while there were no statistic difference in ORR or DCR in FISH or IHC groups (Fig. 4B).
    3.7. The correlation between the treatment lines and the efficacy of Crizotinib
    3.8. Association between ALK variants and clinical efficacy of Crizotinib
    We underwent next generation sequencing in all 40 cases who re-ceived Crizotinib, and ALK-positive were detected in 36 cases, of which EML4-ALK occupies the dominant, accounting for 80.6% (29 of 36). We also detected 7 cases with 11 rare ALK variants (4 cases had dual ALK fusion sub-types), namely ALK:intron19-GPR39&MIR663B concomitant with ALK:intron19-NFU1:intron1, HIP1-ALK, LHFPL4-ALK, AFAP1L1
    Fig. 3. The comparison of PFS in ALK positive and ALK negative cases receiving Crizotinib defined by three methods. (A) In ALK positive cases defined by each method in three groups, the results demonstrated no significant differ-ence in mPFS. (B) The PFS of ALK negative cases in three groups were compared with re-ference to the triple positive group (FISH
    Abbreviations: Tri_Pos, triple positive.
    Fig. 4. The comparison of clinical efficacy between ALK + and ALK- positive patients in each group. (A) The comparison of PFS between ALK + and ALK- positive patients in each group. (B) The comparison of ORR and DCR between ALK + and ALK- positive patients in each group. Abbreviations: ORR, overall response rate; DCR, disease control rate. * P < 0.05.
    4. Discussion
    Application of reliable screening methods for gene rearrangement detection is vital in selecting patients eligible for tumor targeted 
    therapy. Optimally, the method should be reproducible and reliable, and validated for its clinical and biologic accuracy. In this Vorinostat study, we compared FISH, IHC and NGS for the detection of ALK fusion and ex-plored the predictive value of clinical outcome including ORR, DCR and PFS by three methods in a comparatively large cohort of ALK-positive cases. As a result, IHC presented the uppermost positive rate (94.5%), followed by NGS (92.7%) and FISH (82.4%), among which IHC and NGS had the highest concordance rate of 87.3%. No difference was detected in ORR, DCR and PFS of ALK positive cases defined in three groups. Notably, NGS positive patients tended to have a higher DCR and longer PFS compared to NGS negative cases, while FISH and IHC status were not distinguishing in predicting the outcome of Crizotinib. In particular, TP53 concurrent mutation might reduce responsiveness to Crizotinib and worsen prognosis in ALK-rearranged NSCLC.
    Hitherto, consensus has not been achieved regarding the best de-tection approaches for ALK fusion gene. Detection of ALK rearrange-ment by FISH is currently the gold standard, which has been validated in a series of clinical trials [9,23]. Nevertheless, in our study, 11 of 45 (24.4%) cases evaluated by FISH had inconclusive results for technical reasons like weak or no green signal detected or isolated R signal, im-plying FISH assays could be technically challenging and complicated to
    Fig. 5. (A)The correlation between the treatment line and the efficacy of Crizotinib. The result showed there was a marginal difference in mPFS between the first-line and the second/posterior line treatment. (B) The association between ALK variant sub-types and clinical efficacy of Crizotinib. (C)The comparison of PFS in ALK fusion without TP53 mutation cases to ALK fusion with TP53 mutation cases.
    interpreting, probably due to the subtlety of the emitted signal and the topographic location of these genes in the chromosome [24]. The possibility of signal decay after long-term tissue block storage might account for it [25]. Secondly, patients with advanced NSCLC usually only provide small biopsy. Sometimes it is difficult to ensure that more than 50 lung cancer cells are interpreted. Thirdly, intratumoral het-erogeneity, which was reported to coexist with histologic heterogeneity in both single-driver and ALK/EGFR coaltered LADCs, may result in the discrepance. Altered oncogenic drivers in spatially separated subclones of the same tumor may be different [26]. Among nine FISH-incon-clusive cases using Crizotinib, 9/9 were IHC ALK + and 7/9 were NGS ALK+, and the overwhelming majority (8/9) achieved response to Crizotinib, indicating an omission of potential targeted therapy popu-lation by FISH. While in 21 FISH-defined cases, 3 of 4 FISH negative cases were proved PR to the targeted therapy and 1 was evaluated as SD. It was in part similar to the study of Ali et al., which identified 11 FISH ALK negative cases in 31 NGS ALK positive patients (35%) and found 7 of 11(64%) FISH ALK negative cases sensitive to Crizotinib [27]. Other studies that have applied NGS to confirm inconsistent IHC and FISH results also implied that FISH could deny patients’ access to ALK inhibitors for a significant false-negative rate [28–31]. Hence, the combination of ALK fusion detection method would achieve higher accuracy. The borderline cut off values, as Camidge et al. confirmed that when > 4 fields were counted, the > 15% cell positivity cut point would ensure maximal sensitivity and specificity, affected the detection results from another perspective [32]. The high costs, time consuming, specific equipment, and trained personnel also make it not feasible for routine use.