- Email: [email protected]
Toward the molecular dissection of peritoneal pseudomyxoma
original articles
Annals of Oncology
Annals of Oncology 27: 2097–2103, 2016 doi:10.1093/annonc/mdw314 Published online 8 August 2016
Toward the molecular dissection of peritoneal pseudomyxoma F. Pietrantonio1*, F. Perrone2, A. Mennitto1, E. M. Gleeson3, M. Milione2, E. Tamborini2, A. Busico2, G. Settanni2, R. Berenato1, M. Caporale1, F. Morano1, I. Bossi1, A. Pellegrinelli2, M. Di Bartolomeo1, F. de Braud1,4, D. Baratti5, W. B. Bowne3, S. Kusamura5 & M. Deraco5 1 Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy; 2Pathology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano; 3Department of Surgery, Drexel University College of Medicine, Philadelphia, USA; 4Oncology Department, University of Milan; 5Surgery Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Background: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Patients and methods: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene’s hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). Results: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not—being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Conclusions: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations. Key words: pseudomyxoma peritonei, KRAS, GNAS, prognosis
introduction Pseudomyxoma peritonei (PMP) is an extremely rare condition characterized by progressive accumulation of mucinous ascites and tumor implants throughout the peritoneum with an incidence of 1–2 per million per year [1]. It has been recently demonstrated that most PMP originate from mucinous appendiceal neoplasms. Survival improvement has been reported with a strategy combining cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) to treat macroscopic and microscopic disease, respectively. Outcome following curative surgery is predominantly determined by the
*Correspondence to: Dr Filippo Pietrantonio, Medical Oncology Unit 1, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian, 1, 20133 Milan, Italy. Tel: +39-0223903807; Fax: +39-02-23902149; E-mail: fi[email protected]
completeness of cytoreduction (CC), peritoneal carcinomatosis index (PCI) and pathological grade—although the latter has been categorized using different and often inconsistent classifications [2–4]. Despite the low-malignant potential, PMP comprises a heterogeneous group of neoplasms with highly variable biologic behavior and up to 30% of patients will die due to progressive disease. It is conceivable that prognosis of PMP may be related to the individual molecular profile. Therefore, prognostic and predictive biomarkers are urgently needed for improving the management of patients with this orphan disease. Appendiceal mucinous neoplasms show one of the highest prevalence of KRAS mutations among human cancers and may drive the progression of PMP [4–8]. GNAS mutations are also frequently observed in low-grade appendiceal mucinous tumors and may play a crucial role in mucin production in patients with PMP [9, 10]. Therefore, even if the knowledge of the molecular
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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Received 11 February 2016; revised 27 April 2016 and 13 July 2016; accepted 28 July 2016
original articles landscape of PMP is evolving, the clinical impact of specific gene mutations is still poorly understood. This is due to the rarity of the disease, heterogeneity of patients’ series and lack of standardization of the techniques used for mutational analysis. In this study, we aimed at investigating the prognostic role of KRAS and GNAS mutations, as detected by next-generation sequencing (NGS), in PMP patients undergoing CRS and HIPEC.
materials and methods patients
next-generation sequencing The 50-gene Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies) with the Ion-Torrent™ Personal Genome Machine platform (Life Technologies) was used in all experiments, as described in the supplementary materials, available at Annals of Oncology online.
statistical analysis The differences of significance across categorized groups were compared using the χ 2 test or Fisher’s exact test when appropriate. Progression-free survival (PFS) was defined as the time period between the date or CRS and the date of death or the date of first progression, whichever occurred first. Overall survival (OS) was defined as the time between the date or CRS and the date of death from any cause or the date of the last follow-up. Analysis of the survival curves was carried out using the Kaplan–Meier survival analysis and differences in survival distributions according to molecular parameters were tested using log-rank statistics. A P value of 0.05 by the univariate analysis was adopted as enter criteria for the multivariate analysis using the Cox proportional hazard model. The final model was obtained by the selection of independent predictors of PFS by means of backward elimination method. P values <0.05 were considered significant. We did not perform prognostic analysis in terms of OS due to a limited number of events (7 patients died). The statistical software used was SPSS 20.0 (SPSS, Inc., Chicago, IL).
results patient population Data for this study were collected from a prospective institutional database that has gathered information from 230 patients from 2004. Figure 1 shows detailed analysis of the selection process of the study population. After exclusion of patients non-fulfilling the inclusion criteria and the inadequate tumor samples, the final study population (training cohort) included a total of 40 assessable patients. Patients and disease characteristics are listed in Table 1. Regarding prognostic variables, cytoreduction was complete (CC-0
Initial study population n = 230 patients Treated with CRS and HIPEC from 2004 to 2014 N = 121 patients excluded due to follow up < 5 years N = 109 patients with adequate follow up time N = 18 patients excluded due to incomplete follow up data N = 5 patients did not provide consent N = 3 patients excluded due to early death N = 83 samples suitable for molecular analysis N = 10 samples without sufficient cellularity N = 5 samples DNA not amplified N = 28 samples DNA not evaluable for insufficient quality Final study population N = 40 patients’ samples Figure 1. Flow-chart of patients’ progress into the study steps.
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All 40 patients included in the mono-institutional training cohort were treated with CRS and HIPEC at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan from 2004 up to December 2014. CRS included peritonectomy procedures and multivisceral resections aiming at removing the macroscopic disease, according to the technique described by Sugarbaker [11]. Closed abdomen HIPEC was carried out using cisplatin and mitomycin C as previously reported [2]. Institutional follow-up guidelines were adopted in all patients, with thorax–abdomen CT scan and tumor markers (CEA, CA 19–9 and CA 125) carried out every 6 months until the fifth year and annually thereafter. On pathological review done by two blinded pathologists, only mucinous neoplasms of appendiceal origin according to WHO classification were considered eligible for the present study. Other eligibility criteria were as follows: (i) complete follow-up data at our institution; (ii) follow-up of at least 5 years from the surgery in progression-free patients; (iii) complete clinical data; (iv) availability of paraffin-embedded blocks with sufficient cellularity for molecular analysis; and (v) signed informed consent for alive patients. The following clinical parameters were chosen for prognostic analysis according to data literature: gender, age, previous systemic chemotherapy, pathological grade, PCI and CC [12]. This study received IRB approval. The validation cohort comprised 25 patients: 18 treated at our institution and 7 at Drexel University College of Medicine, Philadelphia. Eligibility criteria were the same used for the training set, except for the reduction in the follow-up cut-off from 5 to 3 years.
Annals of Oncology
original articles
Annals of Oncology
Table 1. Patients and disease characteristics in the training and validation cohorts Main characteristics
Training cohort Number = 40 (%)
54 (30–70) 8 (32%) 17 (68%) 25 (100%) 0 24 (4–39) 13 (52%) 10 (40%) 2 (8%) 2 (8%) 23 (92%)
and CC-1) in 35 (88%) cases. The median PCI was 27 (range: 6– 39). Thirty-five (88%) tumors were classified as low grade and five (12%) as high grade PMP. Seven (17%) patients received previous systemic chemotherapy. Finally, the main variables distribution in the validation cohort was similar, as shown in Table 1.
results of NGS
discussion
Exon 2 KRAS mutations were detected in 29 (72%) samples, while exon 8 GNAS mutations were detected in 21 (52%). The relative proportion of different hotspot mutations is summarized in supplementary Table S1, available at Annals of Oncology online ( panel A, study population; panel B, validation cohort). In the study population, KRAS mutation was not significantly associated with the other variables, including age, gender, CC, PCI or pathological grade. On the other hand, the presence of GNAS mutation was associated with incomplete cytoreduction (P = 0.05). Most importantly, GNAS mutations were associated with KRAS mutations (P = 0.002). Neither KRAS nor GNAS mutational status was associated with pathological grade (P = 0.338 and 0.427, respectively). Table 2 shows the type of mutations found in each patients with their relative allele frequencies. The median KRAS mutant allelic fraction was 9% (range 1%–57%), and was <10% cut-off in 16 (55%) cases. Similarly, the median GNAS mutant allelic fraction was 11% (range, 4%–57%) and was <10% cut-off in 9 (43%). Interestingly, additional mutations beyond KRAS and GNAS were found. Among others, we observed mutations in TP53 in five cases, as well as mutations in PI3K/AKT/mTOR pathways in five (including three PI3KCA, one AKT and one LKB1 mutation) and mutations in angiogenic tyrosine kinase receptors, such as FGFR3 and PDGFRA.
In this study, we showed that KRAS mutations, but not GNAS mutations, are associated with poorer outcome following CRS and HIPEC in PMP patients. Despite its indolent behavior, some adverse prognostic factors have been validated, including advanced age, PCI≥20, incomplete cytoreduction, high pathological grade and use of preoperative chemotherapy [12]. Since prognosis is extremely heterogeneous even after adjusting for these clinical and pathological variables, it is conceivable that disease behavior may be also linked to molecular features. The pathological classification of mucinous appendiceal neoplasms and the associated PMP syndrome has been a controversial issue. The recent international consensus conducted by Peritoneal Surface Oncology Group International managed to build an agreement on PMP classification [13]. However, the problem of variability of inter- and intra-observer still persists, even in the case of well-trained pathologists. Mutational analyses can overcome the limitations of morphological assessment, given the higher level of reproducibility and objectiveness. Moreover, several studies including ours showed that KRAS and GNAS mutations are independent from pathological grade [9, 10, 14–16]. It is well known that KRAS and GNAS mutations play a predominant role in the development of mucinous tumors of several districts, including the appendix [5–7, 9, 10, 14, 15]. We showed for the first time that KRAS mutations were independently associated with poorer PFS of PMP patients following CRS and HIPEC. Most importantly, we were able to validate our results by analyzing an independent cohort of patients. Conversely, the
survival outcomes After a median follow-up of 68.1 months, 25 patients had a documented disease progression and only 7 patients died of
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Age Median (range), years 52 (32–71) Gender Male 19 (48%) Female 21 (52%) Previous systemic chemotherapy No 33 (83%) Yes 7 (17%) Peritoneal carcinomatosis index (PCI) Median (range) 27 (6–39) Completeness of cytoreduction 0 18 (45%) 1 17 (43%) 2 5 (12%) Pathological grade High 5 (12%) Low 35 (88%)
Validation cohort Number = 25 (%)
disease. Five-year survival and median PFS were 41.2% and 37.4% months, respectively. Five-year OS was 89% and the median OS was not reached (Figure 2A and B). At univariate analysis, the variables correlated with PFS were CC, PCI, KRAS status and GNAS status (Table 3). Figure 3 shows the Kaplan–Meier curves for PFS according to KRAS and GNAS mutational status (Figure 3A and B, respectively). The Cox hazard model identified only PCI>20 [hazard ratio (HR): 4.32, 95% confidence interval (CI) 1.28–14.55, P-value: 0.018] and KRAS mutation (HR: 14.96, 95% CI 1.95–114.77) as independent predictors of PFS. Neither CC nor GNAS mutation resulted to be independently correlated with PFS (Table 3). Interestingly, our initial results were validated in an independent cohort of 25 patients (supplementary Figure S1 and Table S2, available at Annals of Oncology online). Briefly, KRAS mutation was again an independent prognostic variable maintaining a significant impact on PFS in multivariate analysis (P = 0.029). By grouping the KRAS-mutated cases in the study and validation populations, we explored whether KRAS mutational rate may have a prognostic impact. The median KRAS mutant allelic fraction was 10% and was adopted as a cut-off. However, patients with KRAS mutant allelic fraction ≥10% did not show a poorer median PFS when compared with those with rate <10% (20.5 versus 23 months; HR 0.78, 95% CI 0.39–1.57; P = 0.486). A subset analysis in the KRAS-mutated population failed to show any survival impact of coexisting events in GNAS, PI3KCA and TP53.
original articles
Annals of Oncology
Table 2. Gene mutations detected by means of next-generation sequencing KRAS mutation
Mutant alleles fraction
GNAS mutation
Mutant alleles fraction
Other mutations (mutant alleles)
1 2 3 4 5 6
G12V G12D G12D G12D G12D G12D
2% 10% 4% 17% 7% 6%
R201C WT WT R201H R201H WT
6% — — 22% 5% —
7 8 9
WT G12D G12V
— 14% 6%
WT R201H R201H
— 17% 7%
10 11 12 13 14 15 16 17 18 19 20 21
WT WT WT WT WT G12D WT G13D WT G12C G12D G12D
— — — — — 25% — 19% — 12% 26% 57%
WT WT WT WT WT R201C WT R201C WT R201H R201H R201H
— — — — — 18% — 9% — 16% 24% 57%
22 23
G12D G13D
12% 7%
R201H WT
11% —
24
G12D
13%
WT
—
25
G12D
13%
R201C
14%
26
G12D
6%
R201H
6%
27
G12D
9%
WT
—
28 29
G12D G12V
4% 7%
WT WT
— —
30 31
G12D WT
2% —
R201H R201H
3% 25%
32 33
G12V G12D
8% 1%
R201H R201H
3% 6%
— — — — — PIK3CA H1047R (5%) — — TP53 P151S (7%) — — — — — — — — — — — CTNNB1 D32N (41%) — TP53 R248W (30%) TP53 Q192Stop (8%); PDGFRA R558C (6%); AKT T172I (7%) HNF1A R278W (5%);TP53 R273H (25%) PIK3CA E545K (5%) FGFR3 A257V (9%); LKB1 P319S (8%) — SMO E208K (51%) — PIK3CA N345Y (17%); CDH1 P373L (67%); SMAD4 R135STOP (23%) — — Continued
| Pietrantonio et al.
ID
KRAS mutation
Mutant alleles fraction
GNAS mutation
Mutant alleles fraction
Other mutations (mutant alleles)
34 35 36 37
G12V G12D WT G12C
5% 26% — 29%
R201H R201H WT R201C
10% 22% — 26%
38 39 40
G12D G12D G12V
4% 10% 6%
WT WT Q227STOP
— — 4%
— — — TP53 Y220C (26%) — — —
poor prognostic effect of GNAS mutation observed at univariate analysis was not retained at the multivariate model. The conduction of translational studies in PMP is not straightforward and hampered by several factors such as the low incidence of the disease, wide range of prognostic features of the tumor, variety of pathological classification, heterogeneity of the treatments and differences in the techniques used for gene sequencing. The few studies published thus far have not shown a prognostic role of KRAS mutations. The first used a highly sensitive technique, i.e. the mutant-enriched polymerase chain reaction (PCR). The patients included were 64, but no data on disease stage and type of surgery were available [7]. The second study involved patients undergoing standard CRS (with or without HIPEC) [4], but KRAS mutations were assessed in a subgroup of 111 patients of the overall population. However, the method of assessment was direct Sanger sequencing, which has a sensitivity cut-off of 10%. In a more recent series from a highly specialized center, both KRAS and GNAS mutations were not shown to be prognostic in patients with disseminated appendiceal mucinous neoplasms. The limitation of the study was the inclusion of all-grades histologies, including grade 3 adenocarcinomas and signet-ring histology, and the use of Sanger sequencing [16]. Our data are in contrast with these studies, excepting for GNAS, that was not independently correlated with PFS. The lack of prognostic value of GNAS mutations could be attributed to its frequent association with KRAS mutations. Although these genes are related to different aspects of molecular mechanisms underlying tumor development, they present a sort of redundant and collinear effect on PFS from the statistical standpoint. One possible criticism to our study is that of a possible selection bias due to technical issues. It is well known that the cellularity of PMP is extremely low, hampering the results of translational or preclinical studies. Therefore, it could be reasonable to speculate that we included cases with higher cellularity and other biological features that may have favored the success of the mutational analysis. However, the hypothesis of a skewed sample of patients was excluded as the 5-year OS and PFS of 89% and 41%, respectively, were perfectly in line with literature data (Figure 2). Furthermore, in contrast to the traditional Sanger sequencing that has a limited reliability in cases with fraction of mutated alleles of <10%, the NGS approach is,
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ID
Table 2. Continued
original articles
Annals of Oncology
B 1.0
1.0
Survival function
0.8
Censored
0.6 0.4
0.4 0.2
0.0
0.0 180
0
60 120 Time, months
180
Figure 2. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) in the training cohort.
Table 3. Results of univariate and multivariate analysis in the training cohort, according to progression-free survival Variable
Age <52 ≥52 Gender Male Female Previous systemic chemotherapy No Yes Peritoneal carcinomatosis index (PCI) <20 ≥20 Completeness of cytoreduction 0–1 2 Pathological grade Low High GNAS Wild-type Mutated KRAS Wild-type Mutated
Univariate analysis Hazard ratio (95% CI)
P value
Multivariate analysis Hazard ratio (95% CI)
—
0.458 —
1 1.35 (0.61–2.95)
—
1.000 —
1 1.00 (0.59–1.71)
—
0.95 —
1 1.04 (0.36–3.02) 0.006 1 5.41 (1.61–18.16)
0.027 1 3.97 (1.17–13.52) 0.114
0.012 1 3.66 (1.33–10.05)
1 2.38 (0.81–6.94 —
0.384 —
1 0.59 (0.18–2.00)
0.846
0.011 1 3.06 (1.29–7.27)
1 0.91 (0.36–2.34) 0.006
1 17.67 (2.33–134.40)
P value
0.012 1 15.09 (1.80–126.27)
Bold values highlighted the statistically significant results
beyond any doubt, much more sensitive with a capability to detect mutations present in as few as 1% of tumor cells [17]. Our data have shown that KRAS and GNAS mutant allelic fractions were <10% cut-off in about half of our samples, likely due to the scarce quantity of epithelial cells within abundant mucinous deposits. Differently from colorectal cancer, KRAS mutant allelic fraction did not seem to have a prognostic effect [18]. However, the most important issue is that NGS or mutantenriched PCR are clearly the most appropriate techniques to
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obtain accurate data and to avoid false-negative results. KRAS mutations were found in all samples included in a previous NGS-based study [10], even if the 100% mutation positivity may partly be the result of a selection bias. Finally, our study allowed us to obtain valuable data regarding the presence of new and potentially targetable mutations in this orphan disease. In particular, this may be of particular interest for future development of newer medical treatments in unresectable patients [10, 19]. Although the occurrence of PI3KCA and
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60 120 Time, months
Censored
0.6
0.2
0
Survival function
0.8 Cum survival
Cum survival
A
original articles
Annals of Oncology
A
B 1.0
1.0 KRAS Non mutated Mutated 0-Censored 1-Censored
0.6 0.4
0.8 Cum survival
Cum survival
0.8
0.6 0.4
0.2
0.2
0.0
0.0 0
24
48 72 Time, months
96
GNAS Non mutated Mutated 0-Censored 1-Censored 0
24
48 72 Time, months
96
AKT mutations was already known [10], we showed for the first time the presence of an LKB1 mutation possibly linked to activation of mTOR pathway. Also, the involvement of SMAD4 was already described [10, 19]. Interestingly, we discovered new mutations in angiogenic tyrosine kinase receptors such as FGFR3 or PDGFRA, which may be targeted by specific inhibitors. Further limitations of our study are constituted by the retrospective nature and the small sample size. The effect on OS could not be investigated due to low number of events—which is consistent with the indolent course of the disease. In conclusion, KRAS mutations were independently prognostic for PFS in PMP patients undergoing curative peritonectomy and HIPEC. The conduction of further studies on tumor profiling should be strongly encouraged in referral centers to increase the available knowledge on disease development and progression, to develop a molecular classification and to validate new prognostic and/or predictive biomarkers. Even if validated, our results should be still viewed with caution, considering the limitations to generalize the prognostic role of KRAS mutations to common clinical scenarios.
acknowledgements The authors would like to thank all the patients involved in this study.
funding No funding was received.
disclosure The authors have declared no conflicts of interest.
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Figure 3. Kaplan–Meier curves for progression-free survival according to KRAS (A) and GNAS (B) mutational status in the training cohort.
original articles
Annals of Oncology 16. Singhi AD, Davison JM, Choudry HA et al. GNAS is frequently mutated in both lowgrade and high-grade disseminated appendiceal mucinous neoplasms but does not affect survival. Hum Pathol 2014; 45: 1737–1743. 17. Perrone F, Lampis A, Orsenigo M et al. PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients. Ann Oncol 2009; 20: 84–90.
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Annals of Oncology 27: 2103–2110, 2016 doi:10.1093/annonc/mdw322 Published online 6 September 2016
J. C.-H. Yang1, L. V. Sequist2, C. Zhou3, M. Schuler4, S. L. Geater5, T. Mok6, C.-P. Hu7, N. Yamamoto8, J. Feng9, K. O’Byrne10, S. Lu11, V. Hirsh12, Y. Huang13, M. Sebastian14, I. Okamoto15, N. Dickgreber16, R. Shah17, A. Märten18, D. Massey19, S. Wind20 & Y.-L. Wu21* 1 National Taiwan University Hospital and National Taiwan University Cancer Center, Taipei, Taiwan; 2Massachusetts General Hospital and Harvard Medical School, Boston, USA; 3Shanghai Pulmonary Hospital, Tongji University, Shanghai, China; 4West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 5Prince of Songkla University, Songkhla, Thailand; 6State Key Laboratory of South China, Hong Kong Cancer Institute, The Chinese University of Hong Kong, Hong Kong; 7Xiangya Hospital, Central South University, Changsha, China; 8Wakayama Medical University, Wakayama, Japan; 9Jiangsu Province Cancer Hospital, Nanjing, Jiangsu, China; 10Princess Alexandra Hospital and Queensland University of Technology, Brisbane, Australia; 11Shanghai Lung Tumor Clinical Medical Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China; 12McGill University, Montreal, Canada; 13Yunnan Tumor Hospital (The Third Affiliated Hospital of Kunming Medical University), Kunming, Yunnan Province, China; 14Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany; 15Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 16Thoracic Oncology and Respiratory Care Medicine, Mathias Spital Rheine, Rheine, Germany; 17Kent Oncology Centre, Maidstone Hospital, Kent, UK; 18Boehringer Ingelheim GmbH & Co. KG, Ingelheim am Rhein, Germany; 19Boehringer Ingelheim Ltd UK, Bracknell, Berkshire, UK; 20Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; 21Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
Received 7 April 2016; revised 30 June 2016; accepted 29 July 2016
Background: Afatinib 40 mg/day is approved for first-line treatment of EGFR mutation-positive non-small-cell lung cancer (NSCLC). In the case of drug-related grade ≥3 or selected prolonged grade 2 adverse events (AEs), the dose can be reduced by 10 mg decrements to a minimum of 20 mg. Here, we evaluate the influence of afatinib dose reduction on AEs, pharmacokinetics and progression-free survival (PFS) in the phase III LUX-Lung 3 and 6 (LL3/6) trials. Patients and methods: Treatment-naïve patients with advanced EGFR mutation-positive NSCLC in LL3 (global) and LL6 (China, Thailand, South Korea) were randomized to afatinib or chemotherapy. All afatinib-treated patients (LL3, n = 229; LL6, n = 239) were included in the post hoc analyses. Incidence and severity of common AEs before and after afatinib dose reduction were assessed. Afatinib plasma concentrations were compared in patients who reduced to 30 mg versus those remaining at 40 mg. PFS was compared between patients who dose reduced within the first 6 months of treatment and those who did not. Results: Dose reductions occurred in 53.3% (122/229) and 28.0% (67/239) of patients in LL3 and LL6, respectively; most (86.1% and 82.1%) within the first 6 months of treatment. Dose reduction led to decreases in the incidence of drug-related AEs, and was more likely in patients with higher afatinib plasma concentrations. On day 43, patients who dose reduced to 30 mg (n = 59) had geometric mean afatinib plasma concentrations of 23.3 ng/ml, versus 22.8 ng/ml in patients who remained on 40 mg (n = 284). The median PFS was similar in patients who dose reduced during the first 6 months versus those who did not {LL3: 11.3 versus 11.0 months [hazard ratio (HR) 1.25]; LL6: 12.3 versus 11.0 months (HR 1.00)}. *Correspondence to: Dr Yi-Long Wu, Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, 106 Zhongshan Er Rd, Guangzhou 510080, China. Tel: +86-20-83877855; Fax: +86-20-83827712; E-mail: [email protected]
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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Effect of dose adjustment on the safety and efficacy of afatinib for EGFR mutation-positive lung adenocarcinoma: post hoc analyses of the randomized LUX-Lung 3 and 6 trials
Annals of Oncology
Annals of Oncology 27: 2097–2103, 2016 doi:10.1093/annonc/mdw314 Published online 8 August 2016
Toward the molecular dissection of peritoneal pseudomyxoma F. Pietrantonio1*, F. Perrone2, A. Mennitto1, E. M. Gleeson3, M. Milione2, E. Tamborini2, A. Busico2, G. Settanni2, R. Berenato1, M. Caporale1, F. Morano1, I. Bossi1, A. Pellegrinelli2, M. Di Bartolomeo1, F. de Braud1,4, D. Baratti5, W. B. Bowne3, S. Kusamura5 & M. Deraco5 1 Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy; 2Pathology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano; 3Department of Surgery, Drexel University College of Medicine, Philadelphia, USA; 4Oncology Department, University of Milan; 5Surgery Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Background: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Patients and methods: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene’s hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). Results: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not—being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Conclusions: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations. Key words: pseudomyxoma peritonei, KRAS, GNAS, prognosis
introduction Pseudomyxoma peritonei (PMP) is an extremely rare condition characterized by progressive accumulation of mucinous ascites and tumor implants throughout the peritoneum with an incidence of 1–2 per million per year [1]. It has been recently demonstrated that most PMP originate from mucinous appendiceal neoplasms. Survival improvement has been reported with a strategy combining cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) to treat macroscopic and microscopic disease, respectively. Outcome following curative surgery is predominantly determined by the
*Correspondence to: Dr Filippo Pietrantonio, Medical Oncology Unit 1, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian, 1, 20133 Milan, Italy. Tel: +39-0223903807; Fax: +39-02-23902149; E-mail: fi[email protected]
completeness of cytoreduction (CC), peritoneal carcinomatosis index (PCI) and pathological grade—although the latter has been categorized using different and often inconsistent classifications [2–4]. Despite the low-malignant potential, PMP comprises a heterogeneous group of neoplasms with highly variable biologic behavior and up to 30% of patients will die due to progressive disease. It is conceivable that prognosis of PMP may be related to the individual molecular profile. Therefore, prognostic and predictive biomarkers are urgently needed for improving the management of patients with this orphan disease. Appendiceal mucinous neoplasms show one of the highest prevalence of KRAS mutations among human cancers and may drive the progression of PMP [4–8]. GNAS mutations are also frequently observed in low-grade appendiceal mucinous tumors and may play a crucial role in mucin production in patients with PMP [9, 10]. Therefore, even if the knowledge of the molecular
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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Received 11 February 2016; revised 27 April 2016 and 13 July 2016; accepted 28 July 2016
original articles landscape of PMP is evolving, the clinical impact of specific gene mutations is still poorly understood. This is due to the rarity of the disease, heterogeneity of patients’ series and lack of standardization of the techniques used for mutational analysis. In this study, we aimed at investigating the prognostic role of KRAS and GNAS mutations, as detected by next-generation sequencing (NGS), in PMP patients undergoing CRS and HIPEC.
materials and methods patients
next-generation sequencing The 50-gene Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies) with the Ion-Torrent™ Personal Genome Machine platform (Life Technologies) was used in all experiments, as described in the supplementary materials, available at Annals of Oncology online.
statistical analysis The differences of significance across categorized groups were compared using the χ 2 test or Fisher’s exact test when appropriate. Progression-free survival (PFS) was defined as the time period between the date or CRS and the date of death or the date of first progression, whichever occurred first. Overall survival (OS) was defined as the time between the date or CRS and the date of death from any cause or the date of the last follow-up. Analysis of the survival curves was carried out using the Kaplan–Meier survival analysis and differences in survival distributions according to molecular parameters were tested using log-rank statistics. A P value of 0.05 by the univariate analysis was adopted as enter criteria for the multivariate analysis using the Cox proportional hazard model. The final model was obtained by the selection of independent predictors of PFS by means of backward elimination method. P values <0.05 were considered significant. We did not perform prognostic analysis in terms of OS due to a limited number of events (7 patients died). The statistical software used was SPSS 20.0 (SPSS, Inc., Chicago, IL).
results patient population Data for this study were collected from a prospective institutional database that has gathered information from 230 patients from 2004. Figure 1 shows detailed analysis of the selection process of the study population. After exclusion of patients non-fulfilling the inclusion criteria and the inadequate tumor samples, the final study population (training cohort) included a total of 40 assessable patients. Patients and disease characteristics are listed in Table 1. Regarding prognostic variables, cytoreduction was complete (CC-0
Initial study population n = 230 patients Treated with CRS and HIPEC from 2004 to 2014 N = 121 patients excluded due to follow up < 5 years N = 109 patients with adequate follow up time N = 18 patients excluded due to incomplete follow up data N = 5 patients did not provide consent N = 3 patients excluded due to early death N = 83 samples suitable for molecular analysis N = 10 samples without sufficient cellularity N = 5 samples DNA not amplified N = 28 samples DNA not evaluable for insufficient quality Final study population N = 40 patients’ samples Figure 1. Flow-chart of patients’ progress into the study steps.
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All 40 patients included in the mono-institutional training cohort were treated with CRS and HIPEC at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan from 2004 up to December 2014. CRS included peritonectomy procedures and multivisceral resections aiming at removing the macroscopic disease, according to the technique described by Sugarbaker [11]. Closed abdomen HIPEC was carried out using cisplatin and mitomycin C as previously reported [2]. Institutional follow-up guidelines were adopted in all patients, with thorax–abdomen CT scan and tumor markers (CEA, CA 19–9 and CA 125) carried out every 6 months until the fifth year and annually thereafter. On pathological review done by two blinded pathologists, only mucinous neoplasms of appendiceal origin according to WHO classification were considered eligible for the present study. Other eligibility criteria were as follows: (i) complete follow-up data at our institution; (ii) follow-up of at least 5 years from the surgery in progression-free patients; (iii) complete clinical data; (iv) availability of paraffin-embedded blocks with sufficient cellularity for molecular analysis; and (v) signed informed consent for alive patients. The following clinical parameters were chosen for prognostic analysis according to data literature: gender, age, previous systemic chemotherapy, pathological grade, PCI and CC [12]. This study received IRB approval. The validation cohort comprised 25 patients: 18 treated at our institution and 7 at Drexel University College of Medicine, Philadelphia. Eligibility criteria were the same used for the training set, except for the reduction in the follow-up cut-off from 5 to 3 years.
Annals of Oncology
original articles
Annals of Oncology
Table 1. Patients and disease characteristics in the training and validation cohorts Main characteristics
Training cohort Number = 40 (%)
54 (30–70) 8 (32%) 17 (68%) 25 (100%) 0 24 (4–39) 13 (52%) 10 (40%) 2 (8%) 2 (8%) 23 (92%)
and CC-1) in 35 (88%) cases. The median PCI was 27 (range: 6– 39). Thirty-five (88%) tumors were classified as low grade and five (12%) as high grade PMP. Seven (17%) patients received previous systemic chemotherapy. Finally, the main variables distribution in the validation cohort was similar, as shown in Table 1.
results of NGS
discussion
Exon 2 KRAS mutations were detected in 29 (72%) samples, while exon 8 GNAS mutations were detected in 21 (52%). The relative proportion of different hotspot mutations is summarized in supplementary Table S1, available at Annals of Oncology online ( panel A, study population; panel B, validation cohort). In the study population, KRAS mutation was not significantly associated with the other variables, including age, gender, CC, PCI or pathological grade. On the other hand, the presence of GNAS mutation was associated with incomplete cytoreduction (P = 0.05). Most importantly, GNAS mutations were associated with KRAS mutations (P = 0.002). Neither KRAS nor GNAS mutational status was associated with pathological grade (P = 0.338 and 0.427, respectively). Table 2 shows the type of mutations found in each patients with their relative allele frequencies. The median KRAS mutant allelic fraction was 9% (range 1%–57%), and was <10% cut-off in 16 (55%) cases. Similarly, the median GNAS mutant allelic fraction was 11% (range, 4%–57%) and was <10% cut-off in 9 (43%). Interestingly, additional mutations beyond KRAS and GNAS were found. Among others, we observed mutations in TP53 in five cases, as well as mutations in PI3K/AKT/mTOR pathways in five (including three PI3KCA, one AKT and one LKB1 mutation) and mutations in angiogenic tyrosine kinase receptors, such as FGFR3 and PDGFRA.
In this study, we showed that KRAS mutations, but not GNAS mutations, are associated with poorer outcome following CRS and HIPEC in PMP patients. Despite its indolent behavior, some adverse prognostic factors have been validated, including advanced age, PCI≥20, incomplete cytoreduction, high pathological grade and use of preoperative chemotherapy [12]. Since prognosis is extremely heterogeneous even after adjusting for these clinical and pathological variables, it is conceivable that disease behavior may be also linked to molecular features. The pathological classification of mucinous appendiceal neoplasms and the associated PMP syndrome has been a controversial issue. The recent international consensus conducted by Peritoneal Surface Oncology Group International managed to build an agreement on PMP classification [13]. However, the problem of variability of inter- and intra-observer still persists, even in the case of well-trained pathologists. Mutational analyses can overcome the limitations of morphological assessment, given the higher level of reproducibility and objectiveness. Moreover, several studies including ours showed that KRAS and GNAS mutations are independent from pathological grade [9, 10, 14–16]. It is well known that KRAS and GNAS mutations play a predominant role in the development of mucinous tumors of several districts, including the appendix [5–7, 9, 10, 14, 15]. We showed for the first time that KRAS mutations were independently associated with poorer PFS of PMP patients following CRS and HIPEC. Most importantly, we were able to validate our results by analyzing an independent cohort of patients. Conversely, the
survival outcomes After a median follow-up of 68.1 months, 25 patients had a documented disease progression and only 7 patients died of
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Age Median (range), years 52 (32–71) Gender Male 19 (48%) Female 21 (52%) Previous systemic chemotherapy No 33 (83%) Yes 7 (17%) Peritoneal carcinomatosis index (PCI) Median (range) 27 (6–39) Completeness of cytoreduction 0 18 (45%) 1 17 (43%) 2 5 (12%) Pathological grade High 5 (12%) Low 35 (88%)
Validation cohort Number = 25 (%)
disease. Five-year survival and median PFS were 41.2% and 37.4% months, respectively. Five-year OS was 89% and the median OS was not reached (Figure 2A and B). At univariate analysis, the variables correlated with PFS were CC, PCI, KRAS status and GNAS status (Table 3). Figure 3 shows the Kaplan–Meier curves for PFS according to KRAS and GNAS mutational status (Figure 3A and B, respectively). The Cox hazard model identified only PCI>20 [hazard ratio (HR): 4.32, 95% confidence interval (CI) 1.28–14.55, P-value: 0.018] and KRAS mutation (HR: 14.96, 95% CI 1.95–114.77) as independent predictors of PFS. Neither CC nor GNAS mutation resulted to be independently correlated with PFS (Table 3). Interestingly, our initial results were validated in an independent cohort of 25 patients (supplementary Figure S1 and Table S2, available at Annals of Oncology online). Briefly, KRAS mutation was again an independent prognostic variable maintaining a significant impact on PFS in multivariate analysis (P = 0.029). By grouping the KRAS-mutated cases in the study and validation populations, we explored whether KRAS mutational rate may have a prognostic impact. The median KRAS mutant allelic fraction was 10% and was adopted as a cut-off. However, patients with KRAS mutant allelic fraction ≥10% did not show a poorer median PFS when compared with those with rate <10% (20.5 versus 23 months; HR 0.78, 95% CI 0.39–1.57; P = 0.486). A subset analysis in the KRAS-mutated population failed to show any survival impact of coexisting events in GNAS, PI3KCA and TP53.
original articles
Annals of Oncology
Table 2. Gene mutations detected by means of next-generation sequencing KRAS mutation
Mutant alleles fraction
GNAS mutation
Mutant alleles fraction
Other mutations (mutant alleles)
1 2 3 4 5 6
G12V G12D G12D G12D G12D G12D
2% 10% 4% 17% 7% 6%
R201C WT WT R201H R201H WT
6% — — 22% 5% —
7 8 9
WT G12D G12V
— 14% 6%
WT R201H R201H
— 17% 7%
10 11 12 13 14 15 16 17 18 19 20 21
WT WT WT WT WT G12D WT G13D WT G12C G12D G12D
— — — — — 25% — 19% — 12% 26% 57%
WT WT WT WT WT R201C WT R201C WT R201H R201H R201H
— — — — — 18% — 9% — 16% 24% 57%
22 23
G12D G13D
12% 7%
R201H WT
11% —
24
G12D
13%
WT
—
25
G12D
13%
R201C
14%
26
G12D
6%
R201H
6%
27
G12D
9%
WT
—
28 29
G12D G12V
4% 7%
WT WT
— —
30 31
G12D WT
2% —
R201H R201H
3% 25%
32 33
G12V G12D
8% 1%
R201H R201H
3% 6%
— — — — — PIK3CA H1047R (5%) — — TP53 P151S (7%) — — — — — — — — — — — CTNNB1 D32N (41%) — TP53 R248W (30%) TP53 Q192Stop (8%); PDGFRA R558C (6%); AKT T172I (7%) HNF1A R278W (5%);TP53 R273H (25%) PIK3CA E545K (5%) FGFR3 A257V (9%); LKB1 P319S (8%) — SMO E208K (51%) — PIK3CA N345Y (17%); CDH1 P373L (67%); SMAD4 R135STOP (23%) — — Continued
| Pietrantonio et al.
ID
KRAS mutation
Mutant alleles fraction
GNAS mutation
Mutant alleles fraction
Other mutations (mutant alleles)
34 35 36 37
G12V G12D WT G12C
5% 26% — 29%
R201H R201H WT R201C
10% 22% — 26%
38 39 40
G12D G12D G12V
4% 10% 6%
WT WT Q227STOP
— — 4%
— — — TP53 Y220C (26%) — — —
poor prognostic effect of GNAS mutation observed at univariate analysis was not retained at the multivariate model. The conduction of translational studies in PMP is not straightforward and hampered by several factors such as the low incidence of the disease, wide range of prognostic features of the tumor, variety of pathological classification, heterogeneity of the treatments and differences in the techniques used for gene sequencing. The few studies published thus far have not shown a prognostic role of KRAS mutations. The first used a highly sensitive technique, i.e. the mutant-enriched polymerase chain reaction (PCR). The patients included were 64, but no data on disease stage and type of surgery were available [7]. The second study involved patients undergoing standard CRS (with or without HIPEC) [4], but KRAS mutations were assessed in a subgroup of 111 patients of the overall population. However, the method of assessment was direct Sanger sequencing, which has a sensitivity cut-off of 10%. In a more recent series from a highly specialized center, both KRAS and GNAS mutations were not shown to be prognostic in patients with disseminated appendiceal mucinous neoplasms. The limitation of the study was the inclusion of all-grades histologies, including grade 3 adenocarcinomas and signet-ring histology, and the use of Sanger sequencing [16]. Our data are in contrast with these studies, excepting for GNAS, that was not independently correlated with PFS. The lack of prognostic value of GNAS mutations could be attributed to its frequent association with KRAS mutations. Although these genes are related to different aspects of molecular mechanisms underlying tumor development, they present a sort of redundant and collinear effect on PFS from the statistical standpoint. One possible criticism to our study is that of a possible selection bias due to technical issues. It is well known that the cellularity of PMP is extremely low, hampering the results of translational or preclinical studies. Therefore, it could be reasonable to speculate that we included cases with higher cellularity and other biological features that may have favored the success of the mutational analysis. However, the hypothesis of a skewed sample of patients was excluded as the 5-year OS and PFS of 89% and 41%, respectively, were perfectly in line with literature data (Figure 2). Furthermore, in contrast to the traditional Sanger sequencing that has a limited reliability in cases with fraction of mutated alleles of <10%, the NGS approach is,
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ID
Table 2. Continued
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Annals of Oncology
B 1.0
1.0
Survival function
0.8
Censored
0.6 0.4
0.4 0.2
0.0
0.0 180
0
60 120 Time, months
180
Figure 2. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) in the training cohort.
Table 3. Results of univariate and multivariate analysis in the training cohort, according to progression-free survival Variable
Age <52 ≥52 Gender Male Female Previous systemic chemotherapy No Yes Peritoneal carcinomatosis index (PCI) <20 ≥20 Completeness of cytoreduction 0–1 2 Pathological grade Low High GNAS Wild-type Mutated KRAS Wild-type Mutated
Univariate analysis Hazard ratio (95% CI)
P value
Multivariate analysis Hazard ratio (95% CI)
—
0.458 —
1 1.35 (0.61–2.95)
—
1.000 —
1 1.00 (0.59–1.71)
—
0.95 —
1 1.04 (0.36–3.02) 0.006 1 5.41 (1.61–18.16)
0.027 1 3.97 (1.17–13.52) 0.114
0.012 1 3.66 (1.33–10.05)
1 2.38 (0.81–6.94 —
0.384 —
1 0.59 (0.18–2.00)
0.846
0.011 1 3.06 (1.29–7.27)
1 0.91 (0.36–2.34) 0.006
1 17.67 (2.33–134.40)
P value
0.012 1 15.09 (1.80–126.27)
Bold values highlighted the statistically significant results
beyond any doubt, much more sensitive with a capability to detect mutations present in as few as 1% of tumor cells [17]. Our data have shown that KRAS and GNAS mutant allelic fractions were <10% cut-off in about half of our samples, likely due to the scarce quantity of epithelial cells within abundant mucinous deposits. Differently from colorectal cancer, KRAS mutant allelic fraction did not seem to have a prognostic effect [18]. However, the most important issue is that NGS or mutantenriched PCR are clearly the most appropriate techniques to
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obtain accurate data and to avoid false-negative results. KRAS mutations were found in all samples included in a previous NGS-based study [10], even if the 100% mutation positivity may partly be the result of a selection bias. Finally, our study allowed us to obtain valuable data regarding the presence of new and potentially targetable mutations in this orphan disease. In particular, this may be of particular interest for future development of newer medical treatments in unresectable patients [10, 19]. Although the occurrence of PI3KCA and
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60 120 Time, months
Censored
0.6
0.2
0
Survival function
0.8 Cum survival
Cum survival
A
original articles
Annals of Oncology
A
B 1.0
1.0 KRAS Non mutated Mutated 0-Censored 1-Censored
0.6 0.4
0.8 Cum survival
Cum survival
0.8
0.6 0.4
0.2
0.2
0.0
0.0 0
24
48 72 Time, months
96
GNAS Non mutated Mutated 0-Censored 1-Censored 0
24
48 72 Time, months
96
AKT mutations was already known [10], we showed for the first time the presence of an LKB1 mutation possibly linked to activation of mTOR pathway. Also, the involvement of SMAD4 was already described [10, 19]. Interestingly, we discovered new mutations in angiogenic tyrosine kinase receptors such as FGFR3 or PDGFRA, which may be targeted by specific inhibitors. Further limitations of our study are constituted by the retrospective nature and the small sample size. The effect on OS could not be investigated due to low number of events—which is consistent with the indolent course of the disease. In conclusion, KRAS mutations were independently prognostic for PFS in PMP patients undergoing curative peritonectomy and HIPEC. The conduction of further studies on tumor profiling should be strongly encouraged in referral centers to increase the available knowledge on disease development and progression, to develop a molecular classification and to validate new prognostic and/or predictive biomarkers. Even if validated, our results should be still viewed with caution, considering the limitations to generalize the prognostic role of KRAS mutations to common clinical scenarios.
acknowledgements The authors would like to thank all the patients involved in this study.
funding No funding was received.
disclosure The authors have declared no conflicts of interest.
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2. Baratti D, Kusamura S, Nonaka D et al. Pseudomyxoma peritonei: biological features are the dominant prognostic determinants after complete cytoreduction and hypertermic intraperitoneal chemotherapy. Ann Surg 2009; 249: 243–249. 3. Bradley RF, Stewart JH, 4th, Russell GB et al. Pseudomyxoma peritonei of appendiceal origin: a clinicopathologic analysis of 101 patients uniformly treated at a single institution, with literature review. Am J Surg Pathol 2006; 30: 551–559. 4. Austin F, Manavur A, Magesh S et al. Aggressive management of peritoneal Carcinomatosis from mucinous appendiceal neoplasm. Ann Surg Oncol 2012; 19: 1386–1393. 5. Kabbani W, Houlihan PS, Luthra R et al. Mucinous and nonmucinous appendiceal adenocarcinomas: different clinicopathological features but similar genetic alterations. Mod Pathol 2002; 15: 599–605. 6. Lilleberg SL, Foster JM, Sharma P et al. Use of mutations analysis of pseudomixoma peritonei and appendiceal adenocarcinomas to identify somatic variants within MAPK, AKT and WNT signaling pathways: potential markers for focused treatment strategies. In 2009 ASCO Gastrointestinal Cancers Symposium. Abstract 334. 7. Shetty S, Thomas P, Ramanan B et al. KRAS mutations and p53 overexpression in pseudomyxoma peritonei: association with phenotype and prognosis. J Surg Res 2013; 180: 97–103. 8. Pietrantonio F, Maggi C, Fanetti G et al. FOLFOX-4 chemotherapy for patients with unresectable or relapsed peritoneal pseudomyxoma. Oncologist 2014; 19: 845–850. 9. Nishikawa G, Sekine S, Ogawa R et al. Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms. Br J Cancer 2013; 108: 951–958. 10. Nummela P, Saarinen L, Thiel A et al. Genomic profile of pseudomyxoma peritonei analyzed using next-generation sequencing and immunohistochemistry. Int J Cancer 2015; 136: E282–E289. 11. Sugarbaker PH. Peritonectomy procedures. Cancer Treat Res 1996; 82: 235–253. 12. Chua TC, Moran BJ, Sugarbaker PH et al. Early- and long-term outcome data of patients with pseudomyxoma peritonei from appendiceal origin treated by a strategy of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. J Clin Oncol 2012; 30: 2449–2456. 13. Baratti D, Kusamura S, Maggi C et al. In reply. Oncologist 2015; 20: e5. 14. Zauber P, Marotta S, Sabbath-Solitare M. GNAS mutations are associated with mucin production in low-grade appendiceal mucinous neoplasms, villous adenomas, and carcinomas. Hum Pathol 2015; 46: 339. 15. Bazan V, Migliavacca M, Zanna I et al. Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 Kras mutations are associated with mucinous histotype. Ann Oncol 2002; 13: 1438–1446.
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Figure 3. Kaplan–Meier curves for progression-free survival according to KRAS (A) and GNAS (B) mutational status in the training cohort.
original articles
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Annals of Oncology 27: 2103–2110, 2016 doi:10.1093/annonc/mdw322 Published online 6 September 2016
J. C.-H. Yang1, L. V. Sequist2, C. Zhou3, M. Schuler4, S. L. Geater5, T. Mok6, C.-P. Hu7, N. Yamamoto8, J. Feng9, K. O’Byrne10, S. Lu11, V. Hirsh12, Y. Huang13, M. Sebastian14, I. Okamoto15, N. Dickgreber16, R. Shah17, A. Märten18, D. Massey19, S. Wind20 & Y.-L. Wu21* 1 National Taiwan University Hospital and National Taiwan University Cancer Center, Taipei, Taiwan; 2Massachusetts General Hospital and Harvard Medical School, Boston, USA; 3Shanghai Pulmonary Hospital, Tongji University, Shanghai, China; 4West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 5Prince of Songkla University, Songkhla, Thailand; 6State Key Laboratory of South China, Hong Kong Cancer Institute, The Chinese University of Hong Kong, Hong Kong; 7Xiangya Hospital, Central South University, Changsha, China; 8Wakayama Medical University, Wakayama, Japan; 9Jiangsu Province Cancer Hospital, Nanjing, Jiangsu, China; 10Princess Alexandra Hospital and Queensland University of Technology, Brisbane, Australia; 11Shanghai Lung Tumor Clinical Medical Center, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China; 12McGill University, Montreal, Canada; 13Yunnan Tumor Hospital (The Third Affiliated Hospital of Kunming Medical University), Kunming, Yunnan Province, China; 14Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany; 15Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 16Thoracic Oncology and Respiratory Care Medicine, Mathias Spital Rheine, Rheine, Germany; 17Kent Oncology Centre, Maidstone Hospital, Kent, UK; 18Boehringer Ingelheim GmbH & Co. KG, Ingelheim am Rhein, Germany; 19Boehringer Ingelheim Ltd UK, Bracknell, Berkshire, UK; 20Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; 21Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China
Received 7 April 2016; revised 30 June 2016; accepted 29 July 2016
Background: Afatinib 40 mg/day is approved for first-line treatment of EGFR mutation-positive non-small-cell lung cancer (NSCLC). In the case of drug-related grade ≥3 or selected prolonged grade 2 adverse events (AEs), the dose can be reduced by 10 mg decrements to a minimum of 20 mg. Here, we evaluate the influence of afatinib dose reduction on AEs, pharmacokinetics and progression-free survival (PFS) in the phase III LUX-Lung 3 and 6 (LL3/6) trials. Patients and methods: Treatment-naïve patients with advanced EGFR mutation-positive NSCLC in LL3 (global) and LL6 (China, Thailand, South Korea) were randomized to afatinib or chemotherapy. All afatinib-treated patients (LL3, n = 229; LL6, n = 239) were included in the post hoc analyses. Incidence and severity of common AEs before and after afatinib dose reduction were assessed. Afatinib plasma concentrations were compared in patients who reduced to 30 mg versus those remaining at 40 mg. PFS was compared between patients who dose reduced within the first 6 months of treatment and those who did not. Results: Dose reductions occurred in 53.3% (122/229) and 28.0% (67/239) of patients in LL3 and LL6, respectively; most (86.1% and 82.1%) within the first 6 months of treatment. Dose reduction led to decreases in the incidence of drug-related AEs, and was more likely in patients with higher afatinib plasma concentrations. On day 43, patients who dose reduced to 30 mg (n = 59) had geometric mean afatinib plasma concentrations of 23.3 ng/ml, versus 22.8 ng/ml in patients who remained on 40 mg (n = 284). The median PFS was similar in patients who dose reduced during the first 6 months versus those who did not {LL3: 11.3 versus 11.0 months [hazard ratio (HR) 1.25]; LL6: 12.3 versus 11.0 months (HR 1.00)}. *Correspondence to: Dr Yi-Long Wu, Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, 106 Zhongshan Er Rd, Guangzhou 510080, China. Tel: +86-20-83877855; Fax: +86-20-83827712; E-mail: [email protected]
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Effect of dose adjustment on the safety and efficacy of afatinib for EGFR mutation-positive lung adenocarcinoma: post hoc analyses of the randomized LUX-Lung 3 and 6 trials