Acinetobacter baumannii has become a major threat in health care institutions and the food industry due to multiple antibiotic resistances and its ability to quickly adapt to very different ecological niches. Here, we report an additional, novel survival strategy of A. baumannii. Upon prolonged incubation in high-salt media, cells became unculturable. However, LIVE/DEAD staining followed by flow cytometry, respiratory activity assays, and resuscitation experiments revealed that these cells were viable but non-culturable. VBNC cells underwent large morphological changes. Entry into the VBNC state was also induced by pH and temperature stress, as well as by desiccation and anaerobiosis. The VBNC state was found in several strains of A. baumannii. Genome-wide expression profiling revealed a plethora of genes differentially regulated upon entry into the VBNC state. In summary, this study presents unequivocal evidence for a dormancy state in A. baumannii that has important consequences for detection of this pathogen and recurrent outbreaks.The viable but non-culturable (VBNC) state is a persistence strategy adopted by bacteria to withstand long-lasting periods of unfavorable conditions. VBNC cells evade classical detection methods and are therefore easily transmitted in the hospital causing relapsing infections. The opportunistic human pathogen A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various  Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that has become a major threat in healthcare institutions worldwide. Due to its broad repertoire of antibiotic resistance mechanisms, the World Health Organization has put A. baumannii first place on the list of bacteria for which new therapeutics are urgently needed . Actively respiring cells will import CTC and reduce it to insoluble formazan, which can be detected . Cells were again grown with high salt, and their viability was addressed on 4 consecutive days PSP, while cells grown in low-salt medium served as control . Corresp6 CFU/mL before single samples failed to regain culturability. After 7 months, the resuscitation efficiency started to fluctuate strongly between 2.1 × 106 and 1.7 × 103 CFU/mL with a success rate of only 30–50%. Eleven months after entry into the VBNC state, none of the samples could be resuscitated after repeated attempts of stress removal by dilution in PBS. The addition of succinate to PBS did also not lead to successful resuscitation. However, when resuscitation was performed in LB medium, cells started to grow after approximately 48 hours (3.1 × 108 CFU/mL), indicating the presence of a component in LB that supports resuscitation.To address the timeframe in which cells can be revived from the VBNC state, a VBNC culture was further incubated for 11 months PSP and the resuscitation assay was repeated monthly . Within A. baumannii ATCC 19606T grown at low salt exhibited a nearly rod shape structure with a length of 0.85 × 0.94 µm , including tricarboxylic acid cycle (TCA) genes suggesting a slowdown of the entire metabolic machinery. This is in line with the observation that many genes coding for enzymes that catalyze critical steps of different degradation/catabolic pathways were downregulated . In contrast, expression of genes of the respiratory chain was only slightly affected. In general, expression levels of transport systems such as the MFS transporter for uptake of nutrients/carbon and energy sources were extremely high . These analyses are in line with the hypothesis that VBNC cells are energetically compromised.A. baumannii AB5075 and named DtpA for its desiccation tolerance-promoting property . All cultures became unculturable, and the timeframe in which culturable cells could still be detected  ranged from 7 to 63 days depending on the carbon source used. Cells could be resuscitated by stress removal, indicating VBNC-state entry under all conditions tested .+-gluconate, 600 mM sucrose, or 600 mM glucose induced the VBNC state, demonstrating high osmolarity in general as inducer of the VBNC state .Since succinate oxidation increases the medium pH up to pH 9.5, we addressed the role of pH in VBNC-state induction and its carbon-source dependence. First, we checked the pH of the medium after growth with the carbon sources tested above, and strikingly, the corresponding cultivation intervals correlated strongly with the pH: the more alkaline the medium, the earlier the VBNC state was induced (Table S2). When cells were grown on arabinose (no pH change during growth) in the presence of 300 mM NaCl and pH 10, cells became unculturable within 3 days of incubation, similar to cells grown on succinate in the presence of high salt . Not only high-NaCl concentrations but also 300 mM KCl, 300 mM NaA. baumannii ATCC 19606T could be regained in each case, underlining that VBNC-state entry is a common mechanism for coping with prolonged stress adaptation. However, it is noteworthy that the resuscitation efficiency varied strongly. Desiccated cells lost major parts of their population, such as only 2.9 × 104 CFU/mL were detected after resuscitation, whereas after heat and cold stress 4.6 × 106 and 3 × 106 CFU/mL were detected after resuscitation, respectively. Highest numbers of resuscitated cells were observed after anaerobiosis (5.9 × 108 CFU/mL).Next, we addressed whether the entry into the VBNC state is also induced by other stressors. Indeed, desiccation, anaerobiosis, cold (4°C), and heat (42°C) stress also induced the entry into the VBNC state . HoweverAfter having seen the accelerating effect of additional pH stress on the timepoint of VBNC-state entry, we addressed the influence of different temperatures on the VBNC-state induction of previously stressed cells. Therefore, cells were grown on succinate in the presence of high sodium chloride before they were exposed to 4, 37, and 42°C, respectively . As observed before, salt-stressed cells at 37°C became unculturable within 4 days PSP, but at 4°C, no decline in the culturable cell count could be observed 4 days PSP. In contrast, cells exposed to 42°C were already unculturable after 2 days PSP. These results demonstrate that the entry into the VBNC state can be induced by two different stressors but the response can be amplified or reduced.6 CFU/mL; Fig. S9A and S9B). Viability analyses by flow cytometry provided clear evidence that also strain AB5075 enters the VBNC state in the presence of prolonged salt stress . The same was observed for the strains AYE-T, ACICU, CS36, CS121, and SC1946 (T being the least (4 days of culturability) and AYE-T being the most resilient . Under desiccation conditions, ATCC 19606T lost culturability after 63 days , while the more virulent reference strain ACICU was culturable for at least 147 days. The other strains were culturable throughout the whole incubation time . In fact, their culturable cell count declined very slowly and reached a final cell count of 2.79 × 106 to 5.7 × 107 CFU/mL by the end of the experiment.The more virulent strain AB5075 also became unculturable in high-NaCl medium within 6 days PSP and also could be resuscitated . Cold sA. baumannii strains became unculturable after a few days under high-salt conditions and succinate as carbon source. LIVE/DEAD staining, CTC measurements, and successful resuscitation analyses provide unequivocal evidence that A. baumannii enters the VBNC state. Successful resuscitation of non-culturable cells is seen as the most vital proof for the VBNC state; however, the specific conditions needed to regain culturability depend on the species under study, the applied stress condition, and the resuscitation time window  and of the respiratory chain  are repressed, indicating a slowdown of metabolic activity as it is also described for VBNC Vibrio species   , 20 and NC state , the recA. baumannii ATCC 19606T presented here may provide suitable candidates. One promising target may be iron siderophore biosynthesis, which not only is highly upregulated in A. baumannii but was also among the top-ranked DEGs observed in other bacteria in the VBNC state, such as Pseudomonas syringae and V. cholerae  and incubated for 2 days on a rotary shaker at 37°C. Samples were plated on LB agar plates immediately after dilution in PBS and also after incubation for 2 days in PBS. Plates were incubated overnight and the colony-forming units were determined.As soon as the CFU/mL of a cell suspension dropped below 100, the culture was addressed as non-culturable. At this point, the resuscitation assay was performed as described . BrieflyThe viability of a cell culture was determined using the LIVE/DEAD BacLight Bacterial Viability Kit . Staining was performed according to the manufacturer’s instructions using the fluorescent stains Syto9 and propidium iodide. For quantification purposes, flow cytometry analyses were conducted using the Flow Cytometer BD FACSVerse . SYTO 9 and propidium iodide were both excited by a laser at 488 nm, and fluorescence was collected by a 527/32-nm (FITC-A cytogram) or 700/54-nm (PerCP-Cy5.5-A cytogram) bandpass filter, respectively. Populations were set  according to the populations received with the culturable-  and dead-control (isopropanol-inactivated) cells. Viable cells (populations 6 and 7), and total cells (populations 5 to 8), were quantified using the kit’s microsphere standard. In addition to the quantitative flow cytometry analysis, stained cells were further qualitatively assessed by fluorescence microscopy (Zeiss Axio Imager M1) using a 63× objective.g); each pellet was resuspended in 30 µL Ampuwa , negatively stained with 0.5% phosphotungstic acid  according to the manufacturer’s instructions. Actively respiring cells metabolize CTC to formazan which emits a purple fluorescence. Therefore, dead cells will appear green and viable cells purple/magenta. Fluorescence was visualized using confocal laser scanning microscopy (CLSM). After staining, samples were fixed in 2% fresh paraformaldehyde. Subsequently, all samples were counterstained with DAPI and mounted on glass slides and at least three representative spots with standardized areas were imaged with the CLSM  using the Plan-Apochromat 63×/1.4 Oil DIC objective lens. All bacteria and bacteria with CTC staining were counted afterward using ImageJ  and percentages calculated.A. baumanni ATCC 19606T-specific probe to reduce the amount of rRNA-derived sequences. Sequencing of samples was performed on the NovaSeq 6000 instrument (Illumina) with the NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles), and the NovaSeq XP 2-Lane Kit v1.5 was used in the paired-end mode and 2 × 61 cycles. For quality filtering and removing of the remaining adaptor sequences, trimmomatic-0.39 (A. baumannii ATCC 19606T (Ref: NZ_CP058289.1) was performed with Salmon (v1.9.0)  . As mapp(v1.9.0) . DeSeq2 (v1.9.0)  was used(v1.9.0)  and the (v1.9.0) . Genes wt-test with GraphPad Prism Software. Data differences were considered significant if P ≤ 0.05.Error bars denote the standard deviation of the mean derived from at least three independent biological replicates. Standard deviations were calculated and analyzed with Student’s


MET) polysomy in previous studies. We included three non–small cell lung cancer (NSCLC) cohorts in this retrospective study to establish new criteria for detecting MET polysomy and to explore the clinical relevance of MET polysomy. Cohort 1 included 53 patients whose tissues were available for both FISH and NGS assays. Paired plasma and tissue samples were obtained from 261 patients with NSCLC as cohort 2. Cohort 3 included 46 patients with metastatic NSCLC, who presented with MET copy-number gain assessed by NGS. ROC analysis demonstrated that a cut-off point of 2.3 copies achieved the maximum Youden index in discriminating polysomy from normal copy number. Compared with the FISH test for MET polysomy, the sensitivity, specificity, and agreement of NGS were 90%, 90%, and 96.2%, respectively. Following optimization using maximum somatic allele frequency, the sensitivity and specificity of NGS for defining polysomy using plasma samples according to different circulating tumor DNA mutation frequencies were 42% and 63%. The concordance rate between tissue and plasma samples for detecting polysomy was 85%. Regarding the response to MET inhibitor, the median progression-free survival (PFS) of the MET amplification group was significantly higher than that of the polysomy group. The median PFS was similar between the polysomy and normal groups. Our results indicated that NGS may serve as an alternative method for detecting MET polysomy in NSCLC tissues. Moreover, patients with MET polysomy may not benefit from MET inhibitors.Next-generation sequencing (NGS) has failed to detect mesenchymal epithelial transition factor gene (MET polysomy from amplification, for the former may dilute the clinical benefit of MET inhibitors.In this study, we established a methodology to differentiate polysomy from normal copy numbers and amplification using NGS. Moreover, this study suggests that it is critical to discriminate  MET), such as exon 14 skipping mutation and amplification, have been regarded as driver oncogenic mutations and are potentially targetable (MET copy-number gain (CNG) has been described as one of the mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors  ratios  ratios . In contMET CNG is actually harboring polysomy rather than amplification  and progressed after at least one line of therapy. In total, 46 patients were included in this retrospective study from June 2018 to November 2021.Cohort 3 was used to determine the correlation between the response to Supplementary Figure  shows a MET Spectrum Red FISH Probe Kit and Vysis CEP7 Spectrum Green FISH probe kit. According to the present criteria, MET amplification was defined as gene copy number ≥5 and/or MET/CEP7 ratio > 2.0.FISH was performed using the Vysis DNA was extracted from fresh-frozen tissues or formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens using the QIAamp DNA Mini Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega). DNA was extracted from plasma specimens using the QIAamp Circulating Nucleic Acid Kit (Qiagen). All samples were obtained from patients after signing informed consent. Prior to pooling, the samples’ concentrations were quantified by Qbit. A minimum of 15 ng of cell-free DNA was required for NGS library construction. Next, the DNA was sheared into fragments at a 200–250 bp peak using a Covaris S2 ultrasonicator , and indexed NGS libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB). Finally, the 1,021 panel , covering approximately 1.5 Mbp of the genome and targeting 1,021 cancer-related genes, was used for hybridization enrichment. The indexed libraries were sequenced using a 100-bp paired-end configuration on a DNBSEQ-T7RS sequencer (MGI Tech) or Gene+Seq-2000 sequencing system (GenePlus-Suzhou), producing 3 Gb of data for fresh specimens/FFPE.Standard quality control was performed on all sequencing data for all samples. A coverage depth of not less than 100X in more than 98% of exons, sequencing depth of at least 200X, and mapping rate of not less than 95% must be achieved.https://github.com/etal/cnvkit). Genomic DNA extracted from peripheral blood leukocytes in the same batch was used as a background control. On average, we set a bin size of 120 bp for the capture regions and 150 kb for the non-capture regions. Default settings were used for all remaining parameters.Variant calling and copy-number variation analysis of each capture and non-capture regions were performed using the cnvkit  The proportion of chromosomal bands without significant difference in copy number between smaller and larger regions was higher than 50%. (ii) The larger region contained more than seven chromosomal bands, and the proportion of chromosomal bands without significant difference was higher than 30% in smaller and larger regions. The mean copy number of each region was calculated. CNG was defined as a mean copy number of the region higher than 2.3. The length of the amplified region was estimated as a percentage of the total length of chromosome 7q.First, the MET copy number was increased and the copy number of the region including 7q32.1 did not increase or the region with increased copy number accounted for <10% of the length of 7q, the MET gene was defined as amplified. If the copy number of the region including 7q32.1 was increased and the region with increased copy number accounted for >10% of the length of 7q, it may be defined as MET gene amplification or polysomy, that is, pan-MET gene amplification. If the copy number of the region including 7q32.1 was increased and the region with increased copy number accounted for >80% of the length of 7q, it was defined as polysomy. The detection process is provided in the Supplementary Data  was analyzed using the Kaplan–Meier method, and patients were censored if their status was not available at the last follow-up.To differentiate polysomy from normal cases by copy number using NGS, the Youden index was calculated, and the point at the maximum Youden index was considered as the cut-off value. In addition, ROC analysis demonstrated that a cut-off value of 2.3 copies achieved the maximum Youden index in differentiating polysomy from normal copy number.MET status using NGS.On the basis of the established cut-off value, the kappa coefficient was calculated for concordance between tissue and plasma assessments of P < 0.05. All analyses were performed using the IBM SPSS Statistics 24.0 and R 4.2.1.Statistical significance was set at https://ngdc.cncb.ac.cn/gvm). Other data generated in this study are available within the article and its Supplementary Data. All data generated and analyzed are available from the corresponding author upon reasonable request.Variant Call Format files supporting the findings of this study have been deposited in the Genome Variation Map (GVM) database of National Genomics Data Center under accession number GVM000490  were male, and the rest  were female. The median age of the patients was 57 years, ranging from 31 to 75 years. The predominant histology type was adenocarcinoma , while 5 patients had adenosquamous carcinoma. Most of the patients were diagnosed with stage IIIA or IIIB , and the remaining patients had stage II  and stage IV . Among the 53 patients with paired tissue FISH and NGS results, 29 cases were classified as normal copy number and 10 cases were classified as polysomy according to FISH status. The distribution of  0.0001] .MET amplification, 10 cases of polysomy, and 29 cases with normal copy numbers. Using FISH as the gold standard, NGS correctly identified 13 cases with MET amplification, nine cases with polysomy, and 28 cases with normal copy numbers. Compared with the FISH test for MET amplification, the sensitivity, specificity, and agreement of NGS were 92.9%, 100%, and 98.1%, respectively. Compared with the FISH test for MET polysomy, the sensitivity, specificity, and agreement of NGS were 90%, 90%, and 96.2%, respectively. A kappa value of 0.886 also revealed high congruence.In total, 53 patients whose tissues were available for both FISH and NGS were enrolled. The FISH and NGS results are presented in MET-FISH–positive tumor was identified as negative by NGS. The mean MET/CEP7 ratio (10.5/3.9) was 2.7. The copy number was 2.26 copies and no MET amplification signal was observed in the CNV map (MET-FISH polysomy tumor was classified as negative using the NGS assay. The MET/CEP7 ratio (5.1/2.7) was 1.9, while the copy number derived from NGS was 2.24 (MET-FISH–negative patient was classified as having MET polysomy by NGS. The MET/CEP7 ratio (3.6/2.2) was 1.6, and copy number was 2.5596  and tissue samples was 96.6%. For polysomy detection using NGS, sensitivity and specificity were 12.5% and 50%, respectively. The concordance rate between the two sample types was 90.7%.The plasma and tissue NGS results were compared for 261 patients who underwent concurrent testing, and the details are listed in y number . The senEGFR mutation rate was higher than 5% in 45 patients. Among the 45 patients, MET amplification was identified by NGS in the tissues of 6 patients. Among the 6 patients, 3 presented with MET amplification in plasma ctDNA. When limited to 10 patients with a MSAF higher than 5%, MET amplification was detected in plasma ctDNA from 7 patients. Comparison of ctDNA with tissue in detecting MET amplification using NGS identified a 70% positive percent agreement (PPA) and 95% negative percent agreement (NPA) after optimization.On the basis of these results, we calculated the detection rates of liquid biopsies in selected patients. First, the EGFR in ctDNA was found to be mutated at a frequency higher than 0.5% in 8 patients. Among the 8 patients, only 1 presented with polysomy in plasma ctDNA. Among the 12 patients with MSAF > 5%, polysomy was detected in plasma ctDNA from 5 patients. Following our optimization, the sensitivity and specificity of NGS for defining polysomy with plasma according to different ctDNA mutation frequencies were 42% and 63%, respectively. The concordance rate between the two sample types for detecting polysomy was 85% after optimization.Of the 48 patients in whom polysomy was identified by NGS in tissue, MET copy number assessed by NGS were enrolled in the study. Detailed characteristics of the recruited patients are listed in Supplementary Materials and Methods  rate was 35% and the median PFS was 2.6 months. The PR rate was significantly higher in the MET amplification group than in the polysomy group , whereas there was no significant difference in the PR rate between the polysomy and normal groups . The median PFS was 5.9 months in the MET amplification group, which was significantly higher than that in the polysomy group . The median PFS was similar between the polysomy and normal groups , positive predictive value , and negative predictive value  in detecting polysomy than those reported in the TATTON study, demonstrating that the proposed approach has better detection performance (MET status were in good concordance with each other, which lay the foundation for NGS as routine method to detect MET aberrant molecular alterations. What's more, NGS based on liquid biopsy may be an alternative when tissue biopsy is not available.FISH testing for tissue is commonly used in the detection of malities . Both MEformance . In geneMET abnormalities in patients with NSCLC. The results showed that liquid biopsy had a high NPV and high specificity for detecting MET amplification. NPV and specificity were lower for polysomy detection. In agreement with previous studies, liquid biopsy has a moderate PPV for detecting polysomy (MET amplification and polysomy were higher than those in unselected patients.Liquid biopsy can overcome many limitations of conventional solid biopsy and has become a valid alternative to tissue biopsy . Liquid polysomy . In patiMET in tissues and higher ctDNA content can increase the detection rate of polysomy in plasma. In addition, we suppose that the degree of intratumoral heterogeneity can also influence the detection rate of polysomy. Therefore, the generally good concordance between tissue and plasma for testing MET amplification based on NGS in this study highlights the feasibility of liquid biopsy as an alternative to tissue testing for MET detection.Following a case-by-case analysis, we found that a higher copy number of MET activity, may determine which type of testing is the most relevant in predicting the response to MET inhibition (MET status acts as a tumor driver gene. According to our study, we found significant survival benefits among patients with MET amplification detected using NGS with a MET inhibitor; however, there was no significant benefit in both polysomy and normal copy groups with similar PR rates and PFS. This finding demonstrates that polysomy may not be a therapeutic target for MET inhibitors. A few oncogene overlaps were observed in the MET amplification group, whereas gene mutation profiles were comparable between the polysomy and negative groups. This finding is consistent with polysomy, which does not act as an oncogenic driver. Multivariate analysis revealed that PFS was associated only with MET status, other than MET copy number, which is consistent with a previous study (It has been previously suggested that the response to crizotinib, an inhibitor of c-hibition . For relus study .MET aberrations. Our results indicate that NGS may serve as an alternative method for detecting MET amplification or polysomy in NSCLC tissues. Liquid biopsy could serve as a substitute for tissue biopsy for detecting MET amplification using NGS. It appears that polysomy does not act as a true oncogenic driver, even though it has a high MET copy number. These findings guaranteed further testing and validation in cohort with enlarged sample size.In summary, the reasonable cut-off value to distinguish polysomy and normal copy number may be 2.3 copies. Identification of amplification and polysomy depends on the different proportion of the amplified region on the chromosome 7q. MET status using NGS. However, this study has some limitations. First, it had a relatively small sample size. Second, its retrospective nature and the fact that the results were from one institution without an external validation group are obvious limitations. Third, most MET inhibitors used in our cohort were crizotinib, and whether the poor response in patients with MET polysomy might be related to drug accessibility needs to be explored.As the first exploratory study to establish a methodology for detecting polysomy using NGS, our results lay the foundation for future studies on the same topic. Furthermore, our study also demonstrated the feasibility of detecting Table TS1Clinicopathologic characteristics of patients in cohort 3.Click here for additional data file.Figure S1Flowchart of the study designClick here for additional data file.Table TS2Summary of MET status tested by NGS, MET inhibitors with clinical outcome and dynamic changes in gene alterations in cohort 3Click here for additional data file.Figure S2The detection process using NGS of the study.Click here for additional data file.Figure S3One MET-FISH-positive tumor was identified as negative by NGSClick here for additional data file.Figure S4One MET-FISH polysomy tumor was classified as negative using the NGS assay.Click here for additional data file.Figure S5One MET-FISH-negative patient was classified as having MET polysomy by NGSClick here for additional data file.


However, it may also occur de novo in 2–8% of EGFRm+ NSCLC cases as a potential mechanism of intrinsic resistance. These patients represent a group with unmet needs, since there is no standard therapy currently approved. Several new MET inhibitors are being investigated in clinical trials, but the results are awaited. Meanwhile, as an alternative strategy, combinations of EGFR-TKIs with the MET/ALK/ROS1-TKI Crizotinib may be used in this setting, despite this use is principally off-label. Thus, we studied five of these MET amplified cases receiving EGFR-TKI and Crizotinib doublet after progression on EGFR-TKI treatment to assess the benefits and challenges related to this combination and the possible occurrence of genomic and phenotypic co-alterations. Furthermore, we compared our cases with other real-world reports on Crizotinib/EGFR-TKI combinations, which appeared effective, especially in patients with high-level MET amplification. Yet, we observed that the co-occurrence of other genomic and phenotypical alterations may affect the response to combined EGFR-TKI and Crizotinib. Finally, given the heterogeneity of MET amplification, the diagnostic methods for assessing it may be discrepant. In this respect, we observed that for optimal detection, immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing should be used together, as these methods possess different sensitivities and complement each other in characterizing MET amplification. Additionally, we addressed the issue of managing EGFR-mutated NSCLC patients with de novo MET amplification causing primary EGFR-TKI resistance. We conclude that, while data from clinical trials with new MET inhibitors are still pending, adding Crizotinib to EGFR-TKI in NSCLC patients acquiring MET amplification at progression on EGFR-TKI monotherapy is a reasonable approach, with a progression-free survival of 3–19 months.Amplification of the  EGFR-mutated (EGFRm+) NSCLC treated with EGFR-TKIs, there is no specific recommendation at the time of progression. Several clinical practices, including chemotherapy alone or in combination with an angiogenesis inhibitor (Bevacizumab) and single-agent immunotherapy or in combination with chemotherapy, result in a low objective response rate (ORR) and a short progression-free survival (PFS) [For patients with al (PFS) ,2,3,4. Tal (PFS) . EGFRm+ NSCLC, MET signaling deregulation due to MET gene amplification is the most frequent off-target mechanism of acquired resistance during first-line treatment with Osimertinib. It occurs with a frequency ranging between 7% and 20% of cases in different studies with various numbers of investigated patients, types of specimens, and utilized methods for MET amplification detection [MET amplification during treatment [MET amplification during first-line Osimertinib treatment is even higher than otherwise reported. In any case, comparable frequencies of acquired MET amplification have been observed after treatment with first- and second-generation EGFR-TKIs and during second- or next-line treatment with Osimertinib [MET exon 14 skipping mutations (METex14) [MET amplification. The FDA, on the basis of results from the PROFILE 1001 trial, has also granted Crizotinib, a type 1a MET-TKI, which also inhibits the ROS1 and ALK kinases, a breakthrough therapy designation for the treatment of patients with metastatic NSCLC with METex14 alterations progressing after platinum-based chemotherapy [In etection ,7,8,9. Hreatment , which mmertinib ,12. Two METex14) ,12, but otherapy .MET-amplified NSCLC to Crizotinib of approximately 30% and limited median PFS (max 5 months) and overall survival (OS) (max 7.7 months) [EGFR and high-level MET amplification may achieve a median OS of >11 months and that Crizotinib is more effective than chemotherapy or immunotherapy as first-line therapy in this setting [EGFR and METex14 with a median OS of 22.8 months than in those with MET amplification (median OS 5.4 months) [MET amplification as a driver can also be found in patients with squamous or sarcomatoid phenotypes of NSCLC, in whom response to Crizotinib may be poorer [MET amplified patients progressing on first-line platin-based chemotherapy [Initial phase II trials showed an objective response rate (ORR) of  months) ,15. Yet  setting ,17. In t months) . MET ampe poorer ,20. Furtotherapy .MET amplification as a mechanism of resistance to EGFR-TKIs is clinically actionable in a combination approach attempting concomitant inhibition of EGFR and MET signaling [EGFR T790M-negative cell lines with acquired Osimertinib resistance due to MET amplification indicated that Osimertinib combined with Crizotinib or other investigational MET-TKIs may circumvent resistance to EGFR-TKIs in vivo and in vitro [EGFRm+ NSCLC patients with acquired MET amplification [Given the availability of the above-mentioned anti-MET drugs and their proven effect in preclinical models and phase I–II trials, ignaling ,23,24,25ignaling . Preclinin vitro . Despitefication ,28,29,30EGFRm+ NSCLC patients with acquired MET amplification as a resistance mechanism at the first, second, or later progression on EGFR-TKIs, who were treated by combining these drugs with Crizotinib. The cases also serve to illustrate that because of tumor heterogeneity, only certain tumor clones may harbor MET amplification in NSCLC, thereby making their detection challenging in small tumor biopsies [MET amplification in the context of different co-existing genomic alterations, challenging diagnostic methods, and efficacy of combined treatment with EGFR-TKIs and Crizotinib. We present five different cases of biopsies ,31,32. CEGFRm+ NSCLC acquiring MET amplification during EGFR-TKI treatment. In all these cases a combination of EGFR-TKI and Crizotinib was administered in different treatment lines and with variable outcomes. Furthermore, these cases illustrate the challenges in the diagnostics of MET amplification (including discrepancies between IHC or FISH and NGS).Five real-world cases are presented, reflecting the heterogeneous configuration of EGFR ex19del (p.E746_A750del). This variant was also detected in baseline plasma cfDNA, with a variant allele frequency (VAF) of 0.33%. The patient achieved a complete objective response (OR), as evaluated by positron-emission tomography (PET)/computed tomography (CT) , and 18F-Fluoro-deoxy-glucose (FDG)-PET/CT revealed progressing lesions in the right pleura and pancreas . NGS analysis detected, in addition to the founder EGFR ex19del, mutations of TP53 (p.Q331*), FGFR2 (p.R201L), and SMAD4 (p.L540R) together with JAK3 amplification (7 copies). However, MET amplification was not captured by NGS, possibly reflecting the above-mentioned clonal heterogeneity of MET amplification. A 53-year-old, North-African male, never-smoker, with a performance status of (PS) 1, was diagnosed by tissue biopsy with T4N2M1a lung adenocarcinoma (LAC) a harboriphy (CT) b, and shpancreas c. RebiopThe patient started a standard dose of Crizotinib (250 mg BD) and continued Osimertinib 80 mg QD. The treatment was feasible, and only manageable diarrhea grade 1–2 was observed. EGFR ex19del with a VAF increasing to 3.68%, a finding that can predict worse survival and impending progression [EGFR ex19del in the cfDNA. Next FDG-PET/CT performed nine months after combination treatment showed intrathoracic progression , JAK3 amplification, and TP53, FGFR2, and SMAD4 mutation. All these co-alterations could represent mechanisms of resistance to the Osimertinib–Crizotinib combination, yet they were not obvious druggable targets. Thus, Crizotinib was discontinued after 12 months, and the patient began chemotherapy with Carboplatin and Pemetrexed, while continuing Osimertinib. After four cycles, clinical and radiographic regression was observed, but the newly sampled cfDNA displayed persistence of EGFR ex19del at a low VAF (0.28%). The patient continued maintenance Pemetrexed together with Osimertinib; however, further progression of the T-site and remaining circulating EGFR ex19del (AF 0.18%) were observed after two months. Due to increasing dyspnea, the patient received palliative radiation against the progressing lung tumor (3 Gy × 10 fr) and, given the persistent and increasing circulating EGFR ex19del (VAF 1.7%), he continued Osimertinib. The patient’s condition deteriorated quickly, and he died after having reached an OS of 34 months. A schematic time course of this case is shown in The patient was treated for 5 months, achieving complete clinical and radiographic response, despite the significant genomic co-alterations. However, a cfDNA control sample showed persistent gression . Accordigression d, but thEGFR ex19del (p.E746_T751delinsVA). The patient’s NSCLC progressed after six months of successful treatment with Osimertinib. Analysis of plasma cfDNA did not display any variants among the investigated genes. Rebiopsy from progressive metastasis in the left lower lung lobe showed histology and immune profile of squamous cell carcinoma (SCC) , in PS 1, was diagnosed with T3N2M1c NSCLC, adenocarcinoma type, harboring ma (SCC) , which sMET p.H1112Y variant, MET receptor upregulation (MET-IHC 3+ in 70% and 2+ in 30% of tumor cells), and low-level MET amplification  as well as TP53 mutation (p.K320*). No MET amplification was observed in the NGS analysis. The patient was initiated on Crizotinib 250 mg BD, while continuing Osimertinib 80 mg QD. Significant clinical improvement was quickly obtained, and no adverse events were observed. After four months of this combination treatment, growth of a pericardial and a left adrenal metastasis was observed, while other lesions were stable. A second rebiopsy from the left adrenal gland displayed the original founder EGFR ex19del and the TP53 mutation (p.K320*) as well as three acquired potential mechanisms of TKI-resistance mechanisms: mutation of the NF2 tumor-suppressor gene (p.S87*) and high-level amplification of EGFR and MET genes (both >10 copies by NGS and 100% of tumor cells with MET gene “clusters” of >15 copies as assessed by FISH and resulting in MET-IHC 3+ in 100% of tumor cells). Therefore, the patient received four cycles of chemotherapy with Carboplatin/Vinorelbine, while continuing Crizotinib and Osimertinib. Radiographic evaluation showed a partial response (PR); however, due to cumulative toxicity of the two TKIs, the patient continued the treatment with only Osimertinib. After two months, two symptomatic progressive sites  were observed, and local radiation therapy was administered with a good palliative effect, while Osimertinib was continued beyond progression, reaching an OS of 22 months. Yet, the original EGFR ex19delins together with new mutations in KRAS (p.G13D) and TP53 (p.R273H) were detected in the last cfDNA alongside further deterioration of the patient’s condition. A schematic time course of this case is shown in We identified additional potential mechanisms of acquired Osimertinib resistance in the tumor rebiopsy, such as the EGFR p.L858R and TP53 p.C277F co-mutations. The patient progressed after two months on first-line Osimertinib. Since tissue rebiopsy was unfeasible, plasma cfDNA was analyzed and exhibited no EGFR or TP53 mutations but the presence of a KRAS mutation . The patient was unfit for platin-based doublet chemotherapy but was offered Pemetrexed, continuing Osimertinib in standard doses. After three cycles, we observed radiographic tumor regression and disappearance of the KRAS variant from the corresponding cfDNA. However, the treatment was temporarily paused because of toxicity. During the treatment break, cfDNA analysis unveiled the reappearance of the original EGFR p.L858R (VAF 0.11%) together with the TP53 p.C277F (VAF 0.20%) and KRAS p.G12R (VAF 0.10%) mutations. The patient was re-challenged with Osimertinib and Pemetrexed in reduced doses. Unfortunately, radiographic progression occurred three months later. Despite the absence of circulating EGFR/KRAS/TP53 mutations, new cfDNA analysis detected the acquired pathogenic gain-of-function ALK p.R1275Q mutation (VAF 0.15%) in the ALK-TK domain. This variant is characteristic of neuroblastomas and supposed to be Crizotinib resistant in both neuroblastomas and NSCLC [EGFRm+ NSCLC becoming resistant to Osimertinib through acquired ALK fusions.The patient was an 80-year-old female, smoker (53 py), with reduced lung capacity due to chronic obstructive pulmonary disease, and a previous history of breast cancer . She had a PS of 2 and was diagnosed with T3bN1M0 LAC harboring nd NSCLC ,36,37,38ALK, indicating that the ALK p.R1275Q variant may represent a mechanism of Osimertinib resistance that may be effectively counteracted by Alectinib [MET amplification  and overexpression (MET-IHC 3+ in 60% and 2+ in 40% of tumor cells) together with high-level amplification of three other genes (detected by NGS): PDGFR-A (26 copies) on chromosome 4, MYC (26 copies) on chromosome 8, and CDK4 (25 copies) on chromosome 12. Furthermore, a new mutation in the tumor suppressor and DNA repair gene FANCA (p.Y998*) was observed, together with both the original EGFR p.L858R and TP53 p.C277F co-mutations. Corresponding analysis of cfDNA also identified the EGFR p.L858R (VAF 4.1%) and TP53 p.C277F (VAF 6.8%) variants, but not the ALK p.R1275Q variant. The latter was not detected in the rebiopsy either. Thus, the patient discontinued Alectinib and initiated Crizotinib 250 mg QD (due to her fragile status) while continuing Osimertinib 40 mg QD. The first evaluation after three months revealed improvement in symptoms, and CT scan showed reduction of the axillary lymph nodal conglomerate and primary tumor response , and the patient progressed intracranially with three brain metastases, which were subsequently treated with stereotactic radiosurgery (SRS). The patient received the fifth line combination treatment for 11 months, after which her condition deteriorated rapidly, and she died shortly thereafter, reaching an OS of 34 months. A schematic time course of this case is shown in Thereafter, new NGS analysis of plasma cfDNA showed reappearance of the circulating EGFR p.L858R mutation. Following initial Gefitinib (250 mg QD) in first-line treatment (36 months) and Afatinib (20 mg QD) in second-line treatment (five months), supplemented along with palliative radiation therapy against solitary thoracic lesions, the patient received third-line Osimertinib (80 mg QD) and progressed after four months. Rebiopsy from the relapsed tumor in the right pulmonary upper lobe showed an acquired TP53 mutation (p.L265P) and high-level MET amplification , with significant comorbidities , was diagnosed in 2016 with right malignant pleural effusion containing metastatic LAC cells with EGFR p.L858R and the previously identified TP53 p.L265P variants, loss of MET amplification and a newly acquired EGFR p.T790M mutation, as well as PMS2 amplification (five copies). Based on these findings, Gefitinib and Crizotinib were discontinued and a re-challenge with Osimertinib in reduced dose of 40 mg QD was initiated. However, the patient’s PS quickly deteriorated, and he deceased after four months. The patient reached an OS of 71 months. A schematic time course of this case is shown in Inclusion of the patient in the SAVANNAH trial with the MET-TKI Savolitinib was considered, but the patient declined this possibility. Thus, he was treated with immunotherapy (three cycles of Atezolizumab stretched over four months), resulting in further progression. The combination of EGFR- and MET-TKI was reconsidered. As the patient had experienced gastrointestinal toxicity with Osimertinib, Gefitinib rechallenge together with Crizotinib was initiated. The patient responded for 18 months with an excellent quality of life and no evident adverse events. Thereafter, new progression occurred, and a new rebiopsy from a metastatic lesion in the chest wall revealed, in addition to the founder EGFR ex19del (p.E746_A750del) and TP53  co-mutations. A baseline liquid biopsy (plasma cfDNA) showed shedding of the EGFR ex19del. The patient was neurologically stable on steroids (Prednisolone 12.5 mg QD) and no additional radiation therapy was needed. She initiated first-line Osimertinib (80 mg QD), and first assessment following three months of treatment showed subtotal regression of cerebral metastases and extracranial PR together with disappearance of the EGFR ex19del from the cfDNA. After 18 months of remission, an isolated progression of the T-site was observed, and the patient received palliative local radiation therapy (3 Gy × 10 fractions) while continuing Osimertinib. At the 24-month assessment, growth of a single brain metastasis was observed, and the patient received supplementary SRS radiotherapy (18 Gy × 1 fraction). At the 26-month assessment, oligoprogression was detected intrathoracically (EGFR ex19del (VAF 7.82%). The patient continued Osimertinib beyond progression. Rebiopsy from new metastases in thoracic lymph nodal stations 7 and 11R revealed acquired MET overexpression (MET-IHC with 3+ in 80% and 2+ in 20% of tumor cells) associated with high-level MET amplification identified by FISH , whereas only 5 MET gene copies were detected by NGS. This was accompanied by co-amplification of the BRAF (five copies), SMO (five copies), and PMS2 (five copies) genes on chromosome 7 (like the MET gene), as well as amplification of the CCNE1 gene (seven copies) on chromosome 19. Furthermore, the original TP53 mutation was also found, whereas neither phenotypical transformation nor druggable fusions were detected. The patient initiated Crizotinib (250 mg QD) while continuing Osimertinib (40 mg QD), and after three months of this combination treatment, FDG-PET/CT revealed PR , in a PS of 2, with well-treated hypertension, was diagnosed with LAC in the right lung’s lower lobe with multiple brain and bone metastases and acically a. cfDNA ealed PR b. The trEGFRm+ NSCLC, the 5-year survival remains low and achievable only for ~24% of patients, with a median OS of 36.8 months [MET amplification is the most frequent off-target mechanism of acquired resistance to Osimertinib, an EGFR-TKI widely preferred in first-line settings [MET dysregulation may be further druggable [MET gene status in tumor rebiopsies from progressive lesions by thoroughly assessing the possible occurrence of its amplification or mutation as well as the overexpression of the MET receptor protein, since these dysregulations may occur as concurrent subclonal events or in a temporal sequence. Each of these dysregulations—and the extent of their changes—may influence the response to MET targeted therapy. Furthermore, the complexity of acquired resistance to EGFR-TKIs, as also illustrated in the presented cases, with the co-occurrence of different molecular and phenotypical changes, makes it difficult to find a treatment capable of counteracting multiple simultaneous resistance mechanisms. Nevertheless, MET amplification is now well proven to function as an oncogenic driver with the ability to significantly impair the response to EGFR-TKIs by causing persistent reactivation of the RAS-RAF-MAPK, PI3K-AKT, and STAT signaling pathways downstream of EGFR [EGFRm+ NSCLCs with co-amplification of the MET gene [Despite the advances of the last decade in the treatment of metastatic 8 months . MET ampsettings ,41,42. Mruggable ,12,43. T of EGFR ,12,44. TMET gene ,12,44.EGFRm+ tumors with acquired MET amplification may develop dependence on MET activation alone as a result of an entire switch of oncogenic addiction. In such a subset of tumors, a single-agent MET-TKI, rather than the currently recognized treatment regimen of EGFR-TKIs combined with MET-TKIs, might be sufficient to control the growth [However, preliminary results from cell lines suggest that some rare cases of e growth . MET amplification is also one of the most frequent off-target mechanisms of acquired resistance to inhibitors of the ALK, ROS1, RET and TRK kinases as well as the KRAS GTPase, thus justifying the usage of combinations with MET-TKIs also in these settings [Notably, settings ,44.MET amplification after EGFR-TKI therapy, Crizotinib both in monotherapy and in combination with an EGFR-TKI provided promising outcomes with median PFS (mPFS) of 6.0 and 12.6 months, respectively [EGFRm+ NSCLC patients, who acquired MET amplification during EGFR-TKI treatment and exhibited mPFS of only 1.4 months [MET amplification after EGFR-TKI therapy, inhibition of both EGFR and MET seemed to be a more effective therapeutic strategy [p = 0.010), however without OS being significantly different . Another report provided clinical evidence for the efficacy of a combination regimen with either first- or third-generation EGFR-TKI together with Crizotinib after the emergence of MET amplification-mediated resistance to EGFR-TKIs [MET amplification was 19 months [EGFRm+ NSCLC with acquired MET amplification detected by liquid biopsy of plasma cfDNA and treated with combined Osimertinib–Crizotinib have been reported with a PFS between three and four months [In a small group of Asian NSCLC patients (n = 14) with acquired ectively . Yet, re4 months . A simil4 months . In a lastrategy . In thatGFR-TKIs . One of 9 months . However9 months ,52. Comb9 months . Single r months ,54. MET amplification detected by analysis of plasma cfDNA, the response to the combination of Crizotinib and Erlotinib was reportedly nine weeks due to rapid emergence of other resistance mechanisms [MET amplification was also reported in a NSCLC patient harboring two synchronous uncommon EGFR mutations (exon 18 p.G719S and exon 19 p.L747S) and exhibiting sarcoma-like (spindle and/or giant cell) features [In another case of acquired chanisms . Finallyfeatures . MET dysregulation has been reported to have a longer effect on PFS when using the combination of EGFR-TKI + Crizotinib, rather than Crizotinib alone or chemotherapy, as the next line treatment upon progression on EGFR-TKIs. This may be explained by the fact that EGFRm+ NSCLCs progressing on first-line EGFR-TKI therapy usually remain dependent on EGFR-signaling, so that combining MET inhibition with continued EGFR-TKI treatment is more likely to be more effective than switching from EGFR to MET inhibition alone [As displayed in on alone ,57.EGFRm+ NSCLC patients progressing on first-line Osimertinib, when their second-line treatment was adjusted based on identified mechanisms of resistance at progression using tissue-based genomic analysis [As presented in our cases, we recommend performing tumor rebiopsies in patients progressing on EGFR-TKIs, if feasible, to enable the next treatment based on a biomarker-matched approach. Indeed, recently published data support this approach by showing improved survival of analysis .MET amplification by FISH and NGS, as previously described [MET amplification this gene alteration results in overexpression, auto-aggregation, and ligand-independent activation of the MET receptor protein [MET amplification, which subsequently needs to be verified by FISH/NGS. It may also be utilized to verify that in tumor cells with MET amplification, the encoded MET protein is overexpressed, as this is ultimately the target for MET-TKI treatment. Conversely, IHC-detected MET overexpression does not necessarily mean that the MET gene is amplified [MET amplification assessed by FISH or by NGS. Moreover, there is no complete concordance between MET amplification detected by FISH and by NGS [MET gene amplification or mutation, MET overexpression in NSCLC and other cancer types may also be caused by transcriptional/post-transcriptional/post-translational mechanisms [MET amplification [In all our five cases, tumor rebiopsies were taken at progression and tested for MET overexpression by IHC and for escribed ,59, sinc protein ,32,44. Tmplified ,32,57. Id by NGS ,43,44,57chanisms ,61. Consfication ,60,61,62EGFRm+ NSCLC with IHC-assessed MET overexpression not linked to MET amplification or mutation are resistant to EGFR-TKIs. In this situation, MET upregulation is not an optimal predictor of response to MET-TKIs either, as these drugs appear less effective in NSCLC patients with MET overexpression in the absence of MET mutation or amplification [EGFRm+ NSCLC patients with acquired MET amplification/overexpression [It is unclear whether cases of fication ,57. One fication . This mification ,57,62,64pression ,62. MET amplification, clinical studies have not entirely clarified the concordance between MET overexpression and MET mutation/amplification as predictive biomarkers and indicators of NSCLC dependence on MET signaling [MET-GCN/MET amplification alone or combined with IHC-assessed MET overexpression is currently preferred for predicting responses to TKIs and rating MET addiction of tumors [Even though IHC-assessed MET overexpression may be utilized as a surrogate marker to screen for ignaling ,60,64,65f tumors ,57. MET amplification in all our five cases. Moreover, strong concordance and comparable ORR were observed between MET-IHC positivity and MET-FISH positivity in the TATTON trial combining Osimertinib with Savolitinib for treating EGFRm+ patients with acquired MET amplification/overexpression [Nonetheless, IHC showed MET overexpression correlating with FISH-detected pression . These rMET amplification in clinical NSCLC tissue samples with high sensitivity, minimal false negative rate, and the possibility of distinguishing MET-GCNG due to focal amplification of the MET genomic region on chromosome 7 from GCNG due to polysomy. Thereby, FISH-determined MET amplification remains the optimal biomarker to identify suitable candidates for MET-TKI therapy [In any case, in keeping with our current data, FISH, despite being relatively laborious and observer-dependent , remains therapy ,63,64. MET-GCNG due to duplication of the whole chromosome 7 or parts of it larger than the MET region; (2) the subclonal nature of MET-amplified tumor cells in NSCLC, which can be captured by direct morphological assessment using FISH but may be “diluted” by DNA of other cells. Consequently, the subclonal MET amplification may be below the detection level in the bulky genomic DNA analyzed by NGS, which does not allow any morphological correlation [MET amplification in clinical settings [MET amplification and its various levels by NGS (or qRT-PCR) analysis on cfDNA may be particularly challenging and result in a high rate of false negatives, as it is dependent on the level of obtained ctDNA [MET-GCNG in both tissues and peripheral blood samples, which could be worth future clinical consideration, not least for cases where FISH is not applicable, given the very high concordance with FISH in detecting MET amplification [In comparison, amplification identified by NGS does not seem as robust as a predictive biomarker . The rearelation ,67. Simisettings ,64. Accoed ctDNA . In thisfication ,68,69,70MET amplification, which was, however, identified by FISH. Moreover, in case 2 NGS did not detect the low-level MET amplification revealed by FISH at first progression on Osimertinib , whereas in case 5, NGS detected a MET-GCNG of 5 instead of the MET-GCN >8 uncovered by FISH. Our data are therefore consistent with other real-world studies showing that NGS can identify cases with high-level MET amplification (samples with GCN ≥10) and those that are also FISH-negative, but is not reliable for assessing the various levels of MET amplification, nor for distinguishing MET-GCNG due to true gene amplification from that caused by polysomy [MET amplification than between NGS and FISH [MET amplification and overexpression assessed by FISH and IHC, respectively, have been shown to bear a very important clinical impact in the SAVANNAH study [MET amplification  and MET overexpression  was improved in terms of ORR 49% and mPFS 7.1 months, as compared with the overall population (with IHC 3+ in ≥50% and/or MET-GCN ≥5 by FISH) showing an ORR of 32% and mPFS 5.3 months. These findings were also the reason for redefining the inclusion criteria for enriching the patient population most likely to derive clinical benefit in the current version of the study (NCT03778229).In our cases 1, 3, and 4, NGS failed to detect the acquired polysomy ,62,67. Oand FISH . FurtherAH study ,70. IndeMET amplification and prediction of response to TKI treatment. As elegantly reviewed elsewhere, the most relevant of these challenges with NGS are the lack of consensus on cut-off values for detecting MET amplification; the overall amount, quality, and composition of tumor material (risk of insufficient tumor cell content and purity for sensitive detection of MET amplification); tumor heterogeneity; difficult distinction of MET-GCNG caused by focal gene amplification from that due to polysomy; difficult quantification of tumor clones with different amplification levels as compared to FISH, which results in poor concordance between NGS and FISH detection (discrepancy with FISH in different studies ranging between 37% and 75%); and NGS often unable to detect MET amplifications otherwise revealed by FISH [Thus, despite the successful implementation of targeted NGS panels in routine clinical diagnostics of NSCLC , this me by FISH ,64,67. MET amplification in NSCLC in routine clinical practice, it is not sufficiently standardized to allow ideal comparisons of different studies [MET-GCNG required to induce clinically significant MET overexpression and MET-signaling deregulation as well as the threshold for which each anti-MET treatment has an effect remain uncertain [MET amplification in certain clinical studies, while others also include an intermediate-level, and even a top-level amplification, based on various MET/centromere 7(CEN7) ratios and/or average MET-GCN/cell that slightly differ from study to study [MET/CEN7 ratio is considered by many as a parameter reflecting true gene amplification, with a ratio ≥2 used for defining amplification in general, but also utilized by several studies to classify high-level amplification, whereas others have referred to a ratio of 1.8≤ MET/CEN7 ≤2.2 for low-level amplification, 2.2< MET/CEN7 <5 for intermediate-level amplification, and MET/CEN7 ≥5 for high-level amplification. Instead, increased MET-GCN/cell can be determined either by true amplification of the gene  or by polysomy [Although FISH is currently the preferred method for determining  studies ,62,64. Encertain ,44,57,71to study ,73,74. Ipolysomy ,64,72.MET amplification, a TP53 mutation. In two of these patients, this mutation was already present as a de novo alteration at baseline . This is consistent with the high rate of concomitant TP53 mutations in MET amplified NSCLCs [EGFRm+ NSCLC patients with MET amplification and/or MET overexpression also harbored a TP53 mutation [MET amplification promotes proliferation and survival of TKI-treated EGFRm+ NSCLC cells by co-activating MAPK and PI3K/AKT signaling and by inhibiting the proapoptotic proteins BIM and APAF-1 [MET amplified tumor cells by concomitant disruption of the p53 tumor-suppressive function, which relies on p53-mediated cell-cycle arrest or apoptosis in response to not only DNA damage and hypoxia, but also mitogenic oncogenes (such as mutant EGFR or amplified MET) [All five of our patients progressing on Osimertinib carried, as the most frequent co-alteration accompanying d NSCLCs ,12 and wmutation . These dd APAF-1 ,76,77. Tied MET) ,79,80,81MET amplification are likely due to clonal selection of preexisting MET amplified cells during TKI treatment [MET amplification, which is observable by FISH and may be missed by NGS analysis [MET amplified cell subpopulations, representing <1% of cells in a tumor, have reportedly been uncovered in treatment-naïve cases of EGFRm+ NSCLC that subsequently progressed on EGFR-TKIs with MET amplification as the main mechanism of resistance [MET amplified clones may emerge from very low-frequent pre-existing cells under the selective pressure imposed by EGFR-TKIs.In the clinical setting, cases with acquired resistance to EGFR-TKIs linked to reatment ,57,77,82analysis ,43,57, asistance . CollectMET alterations in intrinsic TKI resistance  is litiation . ConcurrGFR-TKIs ,85,86,87 therapy ,66,85,87 therapy . Moreoveatio >15 . Similarof NSCLC  and in afication . MET amplification as a mechanism of primary resistance to EGFR-TKIs, a retrospective Japanese study identified MET amplification at baseline in 11 of 35 EGFRm+ LAC patients treated with Gefitinib and showed that it predicted a higher risk of tumor progression and death [EGFRm+ NSCLCs showed that concomitant MET amplification at baseline correlated with a shorter time to progression on first-line EGFR-TKI with a HR of 3.7 [EGFRm+ NSCLC patients not responding to first-generation EGFR-TKIs [EGFR p.T790M mutation or MET amplification [Yet, in keeping with concurrent de novo nd death . SimilarR of 3.7 . The impGFR-TKIs . In thisfication .EGFRm+ NSCLC is an event that may cause intrinsic resistance to EGFR-TKIs, but at the same time, it may also represent a potential target for first-line combination therapy aimed at disabling the inherent resistance to EGFR inhibition. The results from the ongoing FLOWERS study (NCT05163249) dedicated to EGFRm+ NSCLC patients with de novo MET amplification or overexpression will be important for finding the best clinical approach for such patients [Taken together, the reported results indicate that co-activation of MET signaling at baseline in patients .MET alterations  already present at baseline may or may not respond to Erlotinib alone [EGFRm+ NSCLCs, four cases harboring de novo MET co-mutations yet responding to first-generation EGFR-TKIs were described [Nonetheless, we previously reported cases in which ib alone ,32. Simiescribed .MET amplification at baseline. In this regard, co-alterations in other oncogenic drivers, such as EGFR, ALK, ROS1, KRAS, BRAF, ERBB2, and RET, have been reported more frequently in NSCLCs with low-/intermediate-level MET amplification than in cases with high-level amplification, suggesting that MET is the main driver in the latter tumors [EGFRm+ NSCLCs with concomitant de novo high-level MET amplification at baseline [EGFR or amplified MET as a driver [EGFRm+, FISH analysis revealed concomitant MET-GCNG at baseline in 52 of them, which was due to polysomy in 46 patients and true MET amplification (as MET/CEN7 >2) in the other six [MET-GCNG displayed by the 46 patients with polysomy did not correlate with the response to EGFR-TKIs, while five out of the six patients with a de novo true MET amplification displayed considerably worse response to these drugs, with the two cases exhibiting the highest MET/CEN7 ratio progressing already within the first month of treatment [EGFRm+ NSCLC with true high-level MET amplification at baseline may harbor clones of MET-driven tumor cells that may very rapidly take over when EGFR signaling is inhibited and may cause intrinsic resistance to EGFR-TKIs, resulting in very rapid cancer progression [These variations in response to EGFR-TKIs could be explained by the level of concomitant r tumors ,72,73. Ybaseline ,91,92 maa driver . Supportreatment . Thus, pEGFRm+ cases with de novo co-amplification of MET are imputable to the lack of standardized methods for determining MET amplification. Hence, NSCLC cells with de novo deregulated MET signaling may already be present at baseline; however, their clinical significance in intrinsic resistance seems heterogeneous, possibly because of the polyclonality of resistance mechanisms and different levels of MET signaling deregulation or tumor cell addiction to this signaling. Accordingly, the role of these cells in intrinsic TKI resistance requires further investigation in larger cohorts. These studies may also elucidate to which extent the above-mentioned discrepancies regarding response to TKIs in EGFRm+ NSCLC patients with both de novo and acquired MET amplification, who cannot be included in these trials, may be offered a combination of EGFR-TKI and Crizotinib, which is a feasible and reasonable approach. Notably, the longest PFS in the real-world cases reported so far ; thus, combinations of MET inhibitors with EGFR-TKIs that are less CNS penetrable than Osimertinib are being investigated too [While we are expecting conclusive data from the clinical trials currently investigating new MET inhibitors in the setting of acquired resistance to EGFR-TKIs in ,12,67,96ated too ,96.MET amplification as both de novo and acquired mechanisms of resistance to EGFR-TKI treated with Crizotinib was searched in the following databases: PubMed, Cochrane, and Medline.Available literature on The immunostaining for membranous and cytoplasmic expression of MET receptor was performed as previously described ,59. Briei.e., 3+ (strong intensity in ≥50% of cells), 2+ (moderate intensity in ≥50% of cells), 1+ (weak intensity in ≥50% of tumor cells), and 0 (no staining or <50% of tumor cells stained). Both 2+ and 3+ are considered as indicative of MET upregulation (“MET-positive”) as opposed to no upregulation (“MET-negative”), as previously described [MET protein expression was scored in a blinded manner (before knowing the FISH results) by one observer (E.S.-R.), assessing staining intensity  and the percentage of stained cells, thereby defining four diagnostic “immunoscores”, escribed ,33. Endoescribed ,33. ImagMET gene and the centromeric portion of the MET-harboring chromosome 7 (CEN7), as previously described [MET signals) with the ×100 objective, counting MET (green) and CEN7 (orange) signals. Representative images were acquired using a 19 mm sCMOS Leica DFC9000 camera incorporated with the microscope after identification of representative areas with the Leica LAS X Navigator Software, v.3.6.0 Widefield . FISH was assessed by two readers (E.S.-R. and a trained and experienced laboratory technician). The tumor samples were classified into the following four groups of MET amplification status [MET gene copy number gain/gene amplification (GCNG/GA) = MET/centromere 7 (CEN7) ratio ≥2.0 or an average MET-GCN/cell ≥6.0 or ≥10% of tumor cells with ≥15 MET signals (“clusters”); (B) Intermediate-level MET-GCNG/GA =≥50% of tumor cells with ≥5 MET signals; (C) Low-level MET-GCNG/GA =≥40% of tumor cells with ≥4 MET signals; and (D) No MET-GCNG/GA = none of the above criteria fulfilled.FISH was performed with the Zyto-Light SPEC MET/CEN7 dual-color probe  that detects the escribed ,33. Brien status ,33: (A) MET amplification were assessed as described above.To identify TKI-resistance mechanisms during treatment, baseline biopsies and longitudinal rebiopsies from new consecutive metastatic lesions emerging during tumor progression were analyzed histologically and by IHC with specific markers  for possible phenotypic transformation to small-cell carcinoma or squamous carcinoma and for epithelial-mesenchymal transition (EMT), as previously described ,98. ImmuEGFR and 21 other lung cancer-relevant genes was determined using the AmpliSeq Colon and Lung Research Panel v.2 on the Genexus™ system . Rebiopsies were investigated for single nucleotide variants (SNVs), short indels, and copy number variations (CNVs) across 161 unique cancer-associated genes using the Oncomine Comprehensive Assay v.3 according to the manufacturer’s instructions , as previously reported [For targeted next-generation sequencing (NGS) analysis, genomic DNA was purified using the Maxwell RSC Blood DNA kit  for cytological samples and an in-house crude DNA extraction method for FFPE material (protocol available upon request). The genomic DNA was quantified using the Qubit™ dsDNA HS Assay on a Qubit™ 4 Fluorometer . At the time of diagnosis, the mutational status of reported ,99. AfteRNA was purified from diagnostic biopsies and tumor rebiopsies utilizing the Maxwell RSC instrument  with a Maxwell RSC RNA FFPE kit (Promega) and quantified with the Qubit™ RNA HS Assay. NGS analysis of RNA from these specimens was performed to identify gene fusions causing primary or acquired EGFR-TKI resistance using the Archer FusionPlex Lung kit, which analyses 14 gene fusions according to the manufacturer’s instructions , as reported .Additionally, liquid biopsies of cfDNA from 3.5 mL of plasma were analyzed at baseline when EGFR-TKI treatment was started, after two months of treatment, and at progression for relevant DNA mutations. Plasma was isolated and cfDNA was purified with the Cobas cfDNA Sample Preparation Kit  and quantified with the Qubit™ dsDNA HS Assay, as described above. NGS analysis of cfDNA was performed using the Oncomine Lung cfDNA NGS-assay, which analyzes hotspot mutations in 11 genes, according to the assay’s instructions , as previously described ,99.https://igv.org) [https://www.oncokb.org), COSMIC (https://cancer.sanger.ac.uk/cosmic), and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar) databases [All NGS data were analyzed using the Torrent Browser (v.5.14.0) and the Ion Reporter (v.5.14) software . Variants were visualized by the Integrative Genomics Viewer (igv.org) , classifigv.org) , and furatabases ,103,104.EGFRm+ NSCLC progressing on EGFR-TKIs due to acquired MET amplification is feasible and currently a reasonable option while we are awaiting the results of clinical trials with new, more potent MET inhibitors. Based on real-world data, the combination of EGFR-TKI and Crizotinib in patients with EGFRm+ NSCLC patients with high-level MET amplification received the highest benefit from Crizotinib compared to less MET amplified cases. Furthermore, the response to combined EGFR-TKI and Crizotinib is longer in cases without multiple co-existing genomic or phenotypical alterations. MET amplification, rebiopsies should be examined with all three methods  for thoroughly defining the level of amplification and co-existing alterations. Given the heterogeneity of EGFRm+ NSCLC with de novo MET amplification, an uncommon but clinically relevant molecular configuration, also deserves special diagnostic awareness and therapeutic consideration, as combined EGFR-TKI/MET-TKI treatment will be needed up-front in this setting.


Despite causing high morbidity, IgG4-related disease (IgG4-RD) and its gastroenterological manifestations lack better and greater theoretical contributions. Therefore, the objective of this work was to evaluate the clinical–epidemiological, diagnostic and treatment aspects of the gastrointestinal manifestations of this disease. A systematic review was carried out using the PubMed, Scopus and Embase databases between January 2012 and March 2023 with the following descriptors: “Immunoglobulin G4-Related Disease” (MeSH) AND #2 “Gastrointestinal Tract” (MeSH). Our data collection grouped a total of 3607 patients from mostly epidemiological cohort studies and cross-sectional follow-ups. In the subgroup analysis, IgG4-RD was associated with male gender, with an estimated prevalence between 54% and 80%. In our findings, the prevalence by topography was presented in the following ranges: lacrimal glands (17–57%); salivary glands (28–72%); pancreas (19–60%); biliary tract (5–40%); retroperitoneal cavity (9–43%). Longitudinal studies are needed to better map the natural history of the gastrointestinal manifestations of IgG4-RD and enable the formulation of individualized treatments. Immunoglobulin G subclass 4-related disease (IgG4-RD) is a chronic, multisystemic, idiopathic disease characterized by a lymphoplasmacytic inflammatory process that leads to fibrosis of organs and systems. Historically, IgG4-RD was described as a heterogeneous set of diseases with an emphasis on gastrointestinal involvement, especially pancreatitis. Its characterization came from histopathological and immunological studies and mapping of the response to immunosuppressive agents ,2,3.Due to the heterogeneous clinical spectrum of gastroenterological manifestations of IgG4-RD, the diagnosis is challenging, requiring a correlation between clinical, laboratory and imaging aspects ,6. This Despite the high morbidity  and the A qualitative systematic review of the literature was conducted following the PRISMA protocol. Electronic databases including PubMed, Scopus, ScienceDirect (Elsevier) and the Virtual Health Library (VHL) were searched using the following strategy: #1 “Immunoglobulin G4-Related Disease” (MeSH) AND #2 “Gastrointestinal Tract” (MeSH) between January 2012 and June 2023. The year 2012 was chosen as the starting point because it marked the publication of the disease description.The study was carried out with the acronym PICOS, with “P” being patients with IgG4-RD, “I” being the clinical–epidemiological characterization of the gastrointestinal manifestations of IgG4-RD, “C” being the healthy control group and “O” being the outcomes of the gastrointestinal manifestations of IgG4-RD.Data collection took place in June 2023. The articles were pre-analyzed from their titles and abstracts. Two researchers collected data individually, with a third senior researcher responsible for evaluating discrepancies and doubts. After this selection, each article was read in full. The most relevant data from each article are given in https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools, accessed on 15 June 2023) created by the National Heart, Lung and Blood Institute (NHLB) was used. Cohort studies were evaluated using the Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies and case–control studies were evaluated using the Quality Assessment of Case–Control Studies. These tools classify studies as “good”, “fair” or “poor” based on the presence or absence of relevant methodological elements for each type of study.To analyze the quality of each study, for the convenience of the authors, the Study Quality Assessment tool (n > 100); and prevalence of the topography of involvement due to IgG4-RD. Review articles, brief comments, editorials, communications and letters to the editor were excluded.Articles in English, Portuguese and Spanish that were original, complete and related to the subject of the study were selected. Inclusion criteria considered suitability for the purpose of this review; availability and transparency of data; methodological rigor applied with an emphasis on clinical, comparative and observational studies; number of participants greater than one hundred  dismissed the requirement for approval from a Human Research Ethics Committee.Initially, 1211 articles were identified in the databases searched . After exclusion by title and abstract, 227 articles were selected for full-text analysis . In the Most (91.2%) of the sample comprised cohort studies, while the rest were case–control studies. As for the quality analysis, 41.66% were classified as good, 41.66% as fair and 16.68% as poor.For heuristic reasons, the data were divided as follows: “epidemiology of the gastrointestinal manifestations of IgG4-RD”, “clinical and diagnostic aspects of the gastrointestinal manifestations of IgG4-RD” and “therapeutic approach to the gastrointestinal manifestations of IgG4-RD”.According to our search strategy, 3607 patients had gastrointestinal manifestations of IgG4-RD, and the majority were Asian (mainly Japanese), with a follow-up period between 4 and 5 years of the disease. Survival rates ranged from 95.3% to 89.0% for 5 to 10 years of disease, respectively ,19,20. AThis fact is intriguing as it is not commonly observed in other autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, autoimmune myopathies and sarcoidosis, among others. This could be interesting to consider in the clinical reasoning for the differential diagnoses between the gastrointestinal involvement of IgG4-RD and connective tissue diseases.Individuals affected by IgG4-RD often have other comorbidities, particularly type 2 diabetes mellitus and allergic diseases, such as rhinosinusitis, bronchial asthma and drug allergies . FurtherIgG4-RD is progressive, insidious leading to organ dysfunction or mass effects. It commonly affects glands , the biliary tract, retroperitoneum, lungs, aorta, kidneys, meninges and orbits, and the concomitant involvement of these structures is a strong indication of IgG4-RD . It is sThe main laboratory changes in IgG4-RD are 1) normocytic–normochromic anemia with eosinophilia; 2) increase in immunoglobulin levels such as IgG and IgG4, with 3) polyclonal peak in serum protein electrophoresis; (4) increase in C-reactive protein (CRP) and erythrocyte sedimentation volume; 5) when certain organs are affected, an increase in alkaline phosphatase, gamma-glutamyl transferase (in autoimmune pancreatitis and primary sclerosing cholangitis), salivary amylase (in parotitis) and complement consumption (C3 and C4) may be seen in renal dysfunction [ normocyt increase polyclon when cerHelicobacter pylori, Mycobacterium tuberculosis and Gram-negative bacteria) and immunological factors  that trigger an immune response at the expense of Th-2 lymphocytes, with an increase in interleukins (IL) such as IL-4, IL-5 and IL-13. This immune response is accompanied by the proliferation and maturation of eosinophils, the recruitment of macrophages and differentiation of B lymphocytes, the consumption of complement (C3 and C4) and the increased production of IgG4. The increase in IgG4 is also considered an epiphenomenon. Subsequently, tissue infiltration with lymphoplasmacytic infiltrate occurs, leading to tumors and/or tissue destruction [Regarding the pathophysiology of IgG4-RD, the most widely accepted theory is that there is a relationship between environmental factors . The major pancreatic manifestation of IgG4-RD is type 1 autoimmune pancreatitis (AIP). In type 1 AIP, the clinical manifestations can be acute (obstructive jaundice and/or pancreatic mass) or chronic . The differentiation between them is a challenge for three reasons: (i) imaging tests  may show similar characteristics, including parenchymal enlargement, sometimes resembling a sausage shape, peripancreatic edematous borders and narrowing of the main pancreatic duct without upstream dilation ; (ii) reThree aspects can be used to differentiate type 1 and 2 AIPs: (i) serological abnormalities; (ii) serum IgG4 levels; (iii) histopathological analysis. Histopathological analysis demonstrates a pattern of lymphoplasmacytic infiltration affecting the pancreatic tissue in a diffuse or irregular manner, storiform fibrosis and phlebitis obliterans. Finally, it is possible to observe an increase in serum levels of IgG4 in cases of 1 AIP ,25.Also known as immunoglobulin G4-related hepatobiliary disease, this condition manifests as glucocorticoid-responsive cholangitis of the extrahepatic and perihilar bile ducts, often associated with type 1 AIP. The symptomatology presents as jaundice, pruritus, weight loss and abdominal pain . HoweverThere is an elevation in serum markers of cholestasis, including alkaline phosphatase, gamma-glutamyl transferase and conjugated bilirubin. Histopathological criteria include lymphoplasmacellular infiltrates with more than 10 IgG4-positive plasma cells in the area visible under the microscope, storiform fibrosis and phlebitis obliterans. Minor criteria include eosinophilia and partial obliterative phlebitis .Cholangiographic characteristics are classified into five subtypes based on the involvement of the biliary tree: (i) inferior stenosis of the distal bile duct, (ii) segmental or (iii) diffuse intrahepatic stenosis, (iv) minor stenosis of the distal bile duct or (v) a combination of hilar and inferior stenoses of the distal bile duct or isolated hilar stenoses. The use of therapeutic response to corticosteroids as a diagnostic criterion is controversial . The CT Retroperitoneal fibrosis (RPF) is the process of fibro-inflammation in the peri-aortic and peri-iliac retroperitoneum . It is aRegarding renal involvement, the disease is manifested by typical clinical characteristics such as uremic syndrome or elevation of nitrogenous slags. In histopathological terms, there are lesions similar to tumors, dense infiltration with IgG4-positive plasmocytes and extensive fibrosis. In laboratory terms, it is important to highlight that it is the only manifestation of IgG4-RD that involves complement consumption (C3 and C4). This type of condition is usually related to diagnostic delays that allow the accumulation of involvement in additional organs and increase the risk of permanent damage to these organs ,18.Treatment for IgG4-RD depends on the degree of disease activity, the affected organs and the patient’s symptomatology. In the case of gastrointestinal symptoms, the therapy of choice is glucocorticoid, at a dose of 0.6 to 0.8 mg/kg per day orally for 1 month, aiming to induce remission. Then, the dose should be gradually reduced by 5 mg every 2 weeks, evaluating the disease activity clinically and through laboratory tests during follow-up .Glucocorticoid therapy proved to be the most effective for initial control of the disease, achieving response rates of 97–100%. However, there are not enough studies demonstrating its effectiveness as a maintenance treatment. Even so, about 33% of patients will relapse during weaning from corticosteroid therapy, requiring the reintroduction of the loading dose. There are no known effective means of preventing IgG4-RD relapse. There are recommendations for low-dose maintenance corticosteroid therapy that can last for several years, capable of reducing this chance from 58% to 23%. Furthermore, immunosuppressants can also be used, especially to induce remission in refractory cases, but the combined use with glucocorticoids is initially not a unanimous recommendation [2 of body surface area, weekly, for 4 weeks, followed by infusions every 2–3 months. Studies with immunosuppressants such as azathioprine (2–3 mg/Kg/day), mycophenolate mofetil (2–3 g/day), methotrexate (15–25 mg/week) and calcineurin inhibitors are insufficient. However, it is known that relapses are common when using these medications in medical practice [Currently, in patients with recurrent IgG4-RD, high-dose glucocorticoids can be used, followed or not by rituximab. This biologic is used at a dose of 375 mg/mexate 15– mg/week practice .In the case of involvement of the lacrimal and salivary glands in the head and neck region, treatment usually involves the use of glucocorticoids, such as prednisone. This medication is administered in gradually reduced doses over time, with the aim of reducing the inflammatory process and relieving symptoms. In some more severe cases or refractory to glucocorticoids, immunosuppressants such as azathioprine and rituximab may be used .In specific cases with obstruction of the lacrimal and salivary glands, additional procedures may be required to relieve the obstruction and restore glandular function. This may include performing endoscopic procedures, such as dilation or placement of prostheses, to open obstructed lacrimal or salivary pathways .For the treatment of involvement of the oral cavity, esophagus, stomach and intestines, glucocorticoids are also the first line of treatment. In more severe cases or when symptoms do not respond adequately to glucocorticoids, azathioprine and methotrexate may be considered. When there is an obstruction or stenosis of the digestive tract, it may be necessary to perform dilation or endoscopic clearance ,12.Type 1 AIP has a good response to initial glucocorticoid therapy, yet has high relapse rates. In addition to immunosuppressive therapy, the assessment of nutritional deficiencies is part of the approach, such as (i) liposoluble vitamins  and (ii) trace elements such as zinc, calcium and magnesium. Surgery is generally not indicated for patients with AIP, but may be considered when suspected pancreatic cancer cannot be excluded after diagnostic evaluation. It is important to remember that endocrine insufficiency or pancreatic duct stones may be present, reinforcing the need for longitudinal follow-up in these cases ,26.Involvement of the liver and biliary tract in IgG4-RD can lead to serious complications such as secondary sclerosing cholangitis (SSC). SSC treatment aims to control inflammation and prevent disease progression. The most commonly used medications are glucocorticoids, such as prednisone. In cases of recurrence or relapse, azathioprine, methotrexate or rituximab can be used. In severe cases of SSC with significant obstruction of the bile ducts, surgical intervention or endoscopic procedures to clear bile flow may be performed ,16,25.As in the other organs, in the retroperitoneum, glucocorticoids are first-line drugs followed by immunosuppressive therapy with azathioprine or rituximab in refractory or recurrent cases. A surgical approach or endoscopic procedures may be necessary in specific situations, such as when there is compression of adjacent structures or obstruction of vital organs such as post-renal acute kidney injury. These interventions are aimed at relieving the obstruction and restoring normal function ,17,25.In the analysis of subgroups, IgG4-RD was associated with the male gender with an estimated prevalence ranging from 54 to 80%, while only 20–42% of the involvements were related to females. These results strengthen the association, consistency and analogy to the Bradford–Hill criteria. The literature indicates male predominance in these manifestations, typically occurring between the ages of 50 and 80, except for head and neck involvement. The disease rarely begins before the age of 40, with less than 10% of cases occurring in this age group ,20,27.It is important to understand that most studies are retrospective and were conducted in Asian populations . TherefoRegarding the gastrointestinal manifestations of IgG4-RD, this review found that the most common involvement was observed in the lacrimal glands 17–57%) and salivary glands 28–72%), followed by pancreatic lesions (19–60%), the biliary tract (5–40%) and the retroperitoneum (9–43%). Most of these manifestations were diagnosed postoperatively as a presumed tumor ,26 Figu. Accordi8–72%, fo7% and saThe diagnosis is clinical , and tren > 100, papers with large samples are difficult because IgG4-RD is a rare rheumatic disease. Consequently, data analysis is limited in terms of natural history and treatment approach. In addition, the difficulty of conducting longitudinal and prospective studies should be emphasized.It must be taken into account that the samples in the studies are small . AlthougMore than half of IgG4-RD gastrointestinal manifestations occur in men around the fifth decade of life. The commonly affected organs are the head and neck glands, the pancreas and the hepatobiliary tract. The diagnosis is one of exclusion, with biopsy being the gold standard.There is still a scarcity in the literature to define a better immunosuppressive approach. As limitations of the present study, we can mention (i) the small number of selected articles and, consequently, of samples; (ii) the mostly Asian population; and (iii) data heterogeneity. However, it should be noted that these are elements inherent to the available literature. Thus, longitudinal studies are needed to better map the natural history of gastrointestinal manifestations of IgG4-RD and to allow the development of individualized treatments.