Relevance of Pharmacogenomics and Multidisciplinary Management in a Young-Elderly Patient With KRAS Mutant Colorectal Cancer Treated With First-Line Aflibercept-Containing Chemotherapy

Introduction: Intensive oncological treatment integrated with resection of metastases raised the clinical outcome of metastatic colorectal cancer (MCRC). In clinical practice, complex evaluation of clinical (age, performance status, comorbidities), and biological (tumoral genotype, pharmacogenomic) parameters addresses tailored, personalized multidisciplinary treatment strategies. Patients with MCRC unsuitable for first-line intensive medical treatments are prevalent and showed worse clinical outcome. After progression to oxaliplatin-based chemotherapy, aflibercept/FOLFIRI significantly improved clinical outcome, even if no survival benefit was reported in adjuvant fast relapsers by aflibercept addition. The case reported a young-elderly (yE) patient with KRAS mutant colorectal cancer rapidly progressing to adjuvant chemotherapy, unfit owing to comorbidities, with multiple pharmacogenomic alterations, who gained long-term survival in clinical practice by multidisciplinary treatment strategy consisting of first-line and re-introduction of aflibercept-containing chemotherapy and two-stage lung metastasectomies. Case presentation: A 71-years-old yE patient, unfit for intensive oncological treatments owing to Cumulative Illness Rating Scale (CIRS) stage secondary, affected by KRAS c.35 G>T mutant colorectal cancer, rapidly progressing with lung metastases after adjuvant XelOx chemotherapy, reached long-term survival 66 months with no evidence of disease after first-line and re-introduction of tailored, modulated aflibercept (4 mg/kg) d1,15-irinotecan (120 mg/m2) d1,15-5-fluorouracil (750 mg/m2/day) dd1–4, 15–18; and secondary radical bilateral two-stage lung metastasectomies. Safety profile was characterized by limiting toxicity syndrome at multiple sites (LTS-ms), requiring 5-fluorouracil discontinuation and aflibercept reduction (2 mg/kg), because of G2 hand-foot syndrome (HFS) for >2 weeks, and G3 hypertension. Pharmacogenomic analyses revealed multiple alterations of fluoropyrimidine and irinotecan metabolism: severe deficiency of fluorouracil degradation rate (FUDR), single nucleotide polymorphisms of UGT1A1*28 variable number of tandem repeats (VNTR) 7R/7R homozygote, ABCB1 c.C3435T, c.C1236T, MTHFR c.C667T homozygote, DPYD c.A166G, TSER 28bp VNTR 2R/3R heterozygote. Conclusions: In clinical practice, a complex management evaluating clinical parameters and RAS/BRAF genotype characterizing an individual patient with MCRC, particularly elderly and/or unfit owing to comorbidities, is required to properly address tailored, multidisciplinary medical and surgical treatment strategies, integrated with careful monitoring of superimposing toxicity syndromes, also related to pharmacogenomic alterations, to gain optimal activity, and long-term efficacy.

Introduction: Intensive oncological treatment integrated with resection of metastases raised the clinical outcome of metastatic colorectal cancer (MCRC). In clinical practice, complex evaluation of clinical (age, performance status, comorbidities), and biological (tumoral genotype, pharmacogenomic) parameters addresses tailored, personalized multidisciplinary treatment strategies. Patients with MCRC unsuitable for first-line intensive medical treatments are prevalent and showed worse clinical outcome. After progression to oxaliplatin-based chemotherapy, aflibercept/FOLFIRI significantly improved clinical outcome, even if no survival benefit was reported in adjuvant fast relapsers by aflibercept addition. The case reported a young-elderly (yE) patient with KRAS mutant colorectal cancer rapidly progressing to adjuvant chemotherapy, unfit owing to comorbidities, with multiple pharmacogenomic alterations, who gained long-term survival in clinical practice by multidisciplinary treatment strategy consisting of first-line and re-introduction of aflibercept-containing chemotherapy and two-stage lung metastasectomies.

INTRODUCTION
Activity of intensive medical treatment integrated with surgical resection of metastases raised the effectiveness of clinical outcome of patients with metastatic colorectal cancer (MCRC). We previously demonstrated that first-line intensive FIr-B/FOx triplet chemotherapy plus bevacizumab reached an objective response rate (ORR) of 82%, median progression-free survival (PFS) of 12 months, and overall survival (OS) of 28 months (1,2). High activity correlated with 26% secondary liver resections and 15% pathologic complete response (CR) (3). Integrated multidisciplinary treatments significantly improved clinical outcome of liver-limited patients (PFS 17 months, OS 44 months), compared with other/multiple metastatic sites (O/MM) (3), not significantly affected by KRAS/NRAS/BRAF genotype (4). In non-elderly RAS wild-type patients, FIr-C/FOx-C triplet chemotherapy plus cetuximab was highly active and tolerable at recommended doses, with PFS 12 months, confirming that intensive first-line regimens increase efficacy, also by increasing secondary resection of liver metastases (5). In patients progressing after oxaliplatin-based first-line treatment, aflibercept addition to FOLFIRI significantly improved OS to 13.5 months, PFS to 6.9 months, and ORR to 19.8% (6). Patients fast relapsing to adjuvant chemotherapy showed poorer efficacy and no survival benefit by aflibercept addition (OS 10.4 vs. 9.6 months) (7,8).
In clinical practice, a complex evaluation of clinical (age, performance status, comorbidity status) and biological (KRAS/NRAS/BRAF genotype) parameters addresses tailored, multidisciplinary treatment strategies (9). Patients unsuitable for first-line FIr-B/FOx regimen due to old-elderly status (≥75 years), performance status (PS) ≥2, and/or comorbidities were prevalent, mostly elderly, PS 1-2, CIRS stage intermediate/secondary (7), O/MM (9, 10); they were treated with tailored triplet or doublet first-line treatments, and showed worse clinical outcome (11). Thus, in unfit patients, it is challenging to select the proper, modulated treatment regimen, weighing expected efficacy with safety profile (9). To this aim, we recently added the evaluation of toxicity syndromes (TS), specifically limiting TS (LTS), evaluating the spectrum of limiting and non-limiting toxicities observed in the individual patient (12). Evaluation of LTS integrated with pharmacogenomic analysis of fluorouracil and irinotecan metabolism can be useful to personalize treatment schedule and doses (5).
We reported an experience in clinical practice of multidisciplinary management of a yE patient with KRAS mutant colorectal cancer rapidly progressing to adjuvant chemotherapy, unfit for intensive medical treatment, owing to yE and comorbidities, who reached long-term OS with no evidence of disease after first-line and re-introduction of aflibercept-containing chemotherapy integrated with secondary, bilateral, two-stage lung metastasectomies.

CLINICAL CASE PRESENTATION
A 71-years-old man, with secondary CIRS stage (10), resulting from hypertensive cardiopathy, dyslipidemia, diabetes on treatment, and positive cancer family history (mother with unspecified cancer at 75 years, brother with unspecified bone tumor, son with fibrosarcoma diagnosed at 8 months, relapsed at 17 years and surgically treated), underwent right colectomy for an ulcerative, stenotic lesion of 3 × 2.5 cm, microscopically defined as moderately differentiated adenocarcinoma with 5% mucinous component, infiltrating colic wall and pericolic fat, infiltrating pattern, poor lymphocytic infiltration, mesenteric tumoral nodes, negative resection margins, four out of 30 metastatic regional lymph nodes, stage pT3 pN2a, KRAS mutant c. 35 G>T genotype. Preoperative CT scan and postoperative PET did not show metastatic disease. Because of the elderly status and secondary CIRS stage, the patient underwent adjuvant chemotherapy according to the following schedule: oxaliplatin (120 mg/m 2 ) d1, capecitabine (825 mg/m 2 bid) d1-14, cycles repeated every 21 days, for six cycles. Safety profile was characterized by LTS-ms, specifically G2 HFS associated with G2 anemia (1,4,12,13). At disease-free survival (DFS) 10 months and disease-free interval (DFI) 4 months after completion of adjuvant chemotherapy, CT scan showed bilateral lung metastases at left antero-basal (8 mm), right inferior (7 mm), and posterior-superior lobe (3 mm), confirmed by PET. CEA, CA19.9 tumor markers were negative.
Because of long-term control of bilateral lung metastastes during aflibercept/irinotecan first-line and re-challenge treatments, low disease burden, even if bilateral lung metastases, multidisciplinary treatment strategy was shared with thoracic surgeons, and bilateral lung resections were planned. The patient underwent atypical resection of right dorsal segment of superior lobe and apical segment of inferior lobe; metastatic lesions of 1.4 cm and 0.8 cm of mucinous colon adenocarcinoma were diagnosed. Nine weeks after, the patient underwent second-stage atypical resection of inferior lobe. Histological examination confirmed a sub-pleural partially necrotic lesion of 2.4 cm, with mucinous features of colon adenocarcinoma. PET scan confirmed no evidence of disease, no further medical treatment was planned, and re-evaluation was performed 3 months after.
To date, clinical outcome shows PFS 50 months from aflibercept/irinotecan re-challenge, PFI 40 months after twostage lung metastasectomies, and OS of metastatic disease 66 months, with no evidence of disease (Figure 4).
The procedures followed were in accordance with the ethical standards. Written informed consent was provided by the patient for proposed medical and surgical treatments, and to represent his clinical case. Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article. Clinical management was shared with the patient, balancing oncological indication with patient's priorities, specifically regarding different available firstline treatment options, safety evaluation and implication on daily living, treatment modulation and interruptions caused by LTS, re-introduction, and integration with lung metastasectomies, followed by follow-up.

DISCUSSION
The case reported a yE patient with KRAS c.35 G>T (G12V) mutant MCRC rapidly progressing to adjuvant chemotherapy with bilateral lung metastases, unfit for intensive medical treatment owing to comorbidities, who was treated by firstline and re-introduction of aflibercept-containing chemotherapy followed by two-stage lung metastasectomies and gained in clinical practice by multidisciplinary treatment strategy longterm OS 66 months of metastatic disease with no evidence of disease at PFI 40 months.
Primary right-sided colonic adenocarcinoma with 5% mucinous component, Dukes C stage, harbored the second most prevalent (22.5%) KRAS c.35 G>T (G12V) mutation (15) and showed DFS 10 months, DFI 4 months after XelOx adjuvant chemotherapy. Prevalently occurring codon 12 KRAS mutations confer worse clinical behavior of CRC, and KRAS c.35 G>T mutation was an independent factor related with increased risk of recurrence and death (16), with significantly unfavorable DFS and OS in patients affected by Dukes C stage CRC (17); the poorer prognosis was not confirmed in other studies (18,19). KRAS codon 12 mutations, specifically c. 35 G>T, were related with worse OS compared with KRAS/BRAF wild-type cancers (20).
Effectiveness of intensive medical treatment integrated with radical resection of metastases raised clinical outcome of MCRC. In fit patients with MCRC, first-line FIr-B/FOx, developed from doublet and triplet schedules backbone (14,21), reached  ORR 82%, correlated with 26% secondary liver resections, PFS 12 months, OS 28 months (1); the prevalent codon 12 KRAS c.35 G>A mutant status was significantly associated with worse clinical outcomes of patients with MCRC treated with FIr-B/FOx compared with KRAS/BRAF wild-type and other KRAS mutant patients (22)(23)(24). FIr-B/FOx treatment integrated with metastasectomies significantly improved outcomes in liverlimited (PFS 17 months, OS 44 months) vs. O/MM patients (3,22). Clinical outcome was not significantly affected by KRAS exon 2 (22), nor KRAS/NRAS/BRAF genotype, even if trendly favorable in triple wild-type (5). KRAS exon 2 wild-type liverlimited patients gained significantly favorable outcome because of secondary surgery, with respect to mutant (22). Increased efficacy of intensive first-line treatment and improved liver resection rate was confirmed in non-elderly RAS/BRAF wildtype patients treated with FIr-C/FOx-C triplet chemotherapy plus cetuximab, highly active and tolerable, reaching PFS 12 months (5).
The reported yE patient with secondary CIRS stage required tailored medical treatment (9,11). Elderly status, PS ≥ 2, and/or  comorbidities represent major parameters justifying treatment modulation to avoid limiting toxicities, preserve adequate quality of life, and to maintain proper dose intensities for expected activity. Elderly patients with MCRC are prevalent, and proper selection between intensive vs. tailored treatments is challenging, weighing expected tolerability and clinical outcome. Consecutive patients unsuitable for first-line intensive regimens, as a result of elderly (≥65 years) and/or comorbidity status, were 56%: elderly 76%, old-elderly 54%, PS 1-2 59%, intermediate/secondary CIRS stage 89%, with O/MM extension 79% (9). They were prevalently treated with modulated triplet or doublet regimens (49 and 40%, respectively). Patients treated with doublet regimens showed worse clinical outcomes (9). Unfit patients who underwent secondary liver surgery did not experience increased morbidity/mortality, reported to be significantly higher in elderly (8%) (25). Moreover, KRAS wild-type compared with mutant patients showed significantly favorable PFS, but not OS (22). KRAS c.35 G >A mutant genotype correlated with significantly worse PFS and OS vs. wild-type and/or other mutant (23,24).
Then, because of long-term control of bilateral lung metastastes during first-line aflibercept/irinotecan and rechallenge, bilateral resections of lung metastases were performed. The diagnosis of mucinous lung metastases may justify such a long OS 66 months of metastatic disease without evidence of disease at PFS 50 months and PFI 40 months from second stage lung metastasectomies, thus realizing the effectiveness of the integrated lung metastasectomies. In a retrospective Spanish real-life analysis of 32 patients who underwent surgical resection after aflibercept/FOLFIRI (37), PR was 56.3%, CR 3.1%, and resection rates R0 75.0%, R1 15.6%, and R2 9.4%. Secondary resection of different metastatic sites was performed: liver, 46.9%; lung, 25.0%; cytoreductive surgery for carcinomatosis, 15.6%; supra-adrenalectomy, 3.1%; liver and peritoneal carcinomatosis, 9.4%. Median PFS from surgery was 8.0 months and OS 37.3 months; in 22% of patients, aflibercept was resumed after surgery.

CONCLUSION
In clinical practice, a complex management evaluating patientrelated clinical parameters and RAS/BRAF genotype of patients with MCRC, particularly the elderly and/or those who are unfit because of comorbidities, is required to properly tailor multidisciplinary medical and surgical treatment strategies to gain optimal activity and long-term efficacy, integrated with careful monitoring of toxicity syndromes, potentially related to pharmacogenomic alterations.

ETHICS STATEMENT
Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
GB and ER were the medical oncologists responsible for clinical and bioclinical management of patient. AD'A, SG, and EAR were abdominal and thoracic surgeons responsible for surgical management of patient. MS performed pharmacogenomic analyses. All authors read and approved the final manuscript.