No evidence of NRAS mutation in squamous cell anal carcinoma (SCAC)

Epidermal growth factor receptor (EGFR) is usually expressed in squamous cell anal carcinoma (SCAC) and anti-EGFR agents could represent a valid treatment strategy, also considering that KRAS and BRAF mutations are rare events in this type of cancer. However, no data are available on NRAS status in SCAC. In this study we analyzed NRAS status (exons 2–4) by Pyrosequencing in a case series of 50 SCAC patients previously characterized in our laboratory for KRAS, BRAF, PIK3CA mutations and HPV and HIV infections. We found no mutation in NRAS gene. These results confirm that since the principal anti-EGFR resistance mechanisms are almost absent in SCAC, anti-EGFR agents should be considered for the treatment of this type of cancer.

The study protocol was reviewed and approved by the Medical Scientific Committee of IRST IRCCS, and written informed consent was obtained from patients or from their next of kin for the use of biological samples for research purposes. In addition, the experiments in this study were conducted in accordance with approved guidelines and regulations.

Statistical analyses.
Descriptive statistics were reported as frequencies and percentages for categorical variables and median and range for continuous variables. Progression-free survival (PFS) was calculated from the first day of treatment to the date of first observation of disease progression or death resulting from any cause, whichever occurred first, or the last follow-up for patients who were still alive and had not progressed. Overall survival (OS) was calculated from the first day of treatment to the date of death from any cause or the last follow-up. PFS, OS and their 95% confidence intervals (95% CI) were estimated using the Kaplan-Meier life-table method and survival curves were compared by the logrank test. Statistical significance was assumed for P < 0.05. Statistical analyses were carried out with SAS Statistical software (version 9.4, SAS Institute, Cary, NC, USA).

Discussion
The aim of our study was to analyze the status of NRAS gene in a case series of SCAC patients previously characterized in our laboratory for KRAS, BRAF and PIK3CA genes. To our knowledge, this is the first study to analyze NRAS status in SCAC. Given that mutations occurring on KRAS exon 2-4 in colorectal cancer -in addition to those occurring on NRAS exon 2-4 -may play a role in determining resistance to moAb-EGFRs 11 , these molecular characterizations could also be investigated in SCAC in view of the potential usefulness of these antibodies in this type of cancer.
Other studies have analyzed the frequency of KRAS, BRAF and PIK3CA mutations in SCAC, showing a very low rate of KRAS and BRAF mutations [15][16][17][18] , and a slightly higher frequency of PIK3CA mutation 15,18 . No correlation between PIK3CA mutation and patients prognosis was reported in this study with an updated follow up, confirming what previously stated 15 .
We demonstrated that no NRAS mutation is present in SCAC and confirmed the absence of potential resistance mechanisms to moAb-EGFR, such as cetuximab or panitumumab. This data, together with previous evidence on KRAS and BRAF, suggest that cetuximab could be a valid treatment strategy in SCAC.
We confirmed that PIK3CA mutations have no prognostic role in this type of cancer, whereas tumor grade and size, lymph node involvement and stage are significantly associated with PFS and OS. Moreover, infection of HPV 16 was significantly associated with a longer PFS but not with OS.
A recent phase I study has demonstrated that the adding of cetuximab to a chemo-radiotherapy with 5-fluorouracil (5-FU) and mitomycin-C in SCAC induced a complete remission rate of 73% with a relative high toxicity 10 . High toxicity was observed also in other clinical trials 20,21 , suggesting that the relation between toxicity, chemotherapy dosage and radiation techniques should be better clarified to optimize this promising combination treatment strategy for SCAC. In addition to this data, a recent case report has shown a dramatic response to cetuximab in combination with cisplatin and 5-FU in a patient with metastatic anal cancer 9 .
Moreover, there are no therapies available after progression to cisplatin and 5-FU that can improve survival. A case report showed a complete response to cetuximab in monochemotherapy in a patient with refractory metastatic anal carcinoma suggesting a possible use in this setting 22 . There are numerous similarities between SCAC and head and neck squamous cell carcinoma (HNSCC), both in terms of etiology and pathology. In particular, as observed in SCAC, high EGFR expression and a low percentage of RAS mutations have also been reported in HNSCC [23][24][25] , and this was the basic starting point for research  into mAb anti-EGFRs in this type of cancer. The approval of cetuximab for the treatment of advanced and metastatic HNSCC 26,27 further reinforces the potential effectiveness of anti-EGFR treatment in SCAC. A limitation of the study consists in the fact that, due to its retrospective nature, not all information on the chemoradiotherapy administered was available, in particular that pertaining to the different radiotherapy dosages delivered. Moreover, despite the known disadvantages of a composite endpoint, we decided to use such an endpoint because of the potential underreporting of disease recurrences caused by the non-homogeneous availability of follow-up data from the different centers involved in the study.
Our results demonstrated that NRAS gene is wt in SCAC patients. These data, together with those reported on KRAS status, seem to confirm the potential usefulness of anti-EGFR drugs in the treatment of SCAC. Results of ongoing clinical trials will clarify the tolerability and the efficacy of these agents in combination with conventional CRT.