Sandra Vukusic
email: sandra.vukusic@chu-lyon.fr

David Brassat

Jerome De Seze

Guillermo Izquierdo

Andreas Lysandropoulos

Ludo Vanopdenbosch Wibe Moll

Ludo Vanopdenbosch g Maria Jesus Arque

Mehdi Kertous

Pascal Rufi

Celia Oreja-Guevara

* Pr

Single-arm study to assess comprehensive infusion guidance for the prevention and management of the infusion associated reactions (IARs) in relapsing-remitting multiple sclerosis (RRMS) patients treated with alemtuzumab (EMERALD)

Keywords:

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

mild to moderate infusion-associated reactions (IARs) were the most frequently reported adverse events (AEs) associated with alemtuzumab. EMERALD (NCT02205489) was a phase 4, multicenter, multinational, single-arm study designed to assess an algorithm for the prevention and management of IARs in RRMS patients treated with alemtuzumab.

Methods: Patients were treated with a study regimen of enhanced IAR prophylaxis relative to phase 2 and 3 studies. H1 and/or H2 antagonists or equivalent gastroprotection (proton pump inhibitors) were given 1 day before alemtuzumab infusion, 1 hour prior to the infusion, and post-infusion. Methylprednisolone was given orally 1 day before infusion, 1 hour prior to the infusion, and as needed post-infusion.

Antipyretics were given 1 hour before infusion and as needed post-infusion. Antiemetics and normal saline were given as needed during and post-infusion.

Results: Of the 61 patients screened, 58 (95.1%) were enrolled into the study. Of the 58 patients who received the first infusion of Period 1, 57 (98.3%) completed the 5 days of Course 1. A total of 54 patients received the first infusion of Period 2 and 53 completed the 3-day course. All patients (n=58) completed the Month 6 visit and 54 the Month 12 visit. 93.1% of patients had at least one IAR (91.4% in Period 1 and 81.5% in Period 2), the majority of which were grade 1 (69.1%) or grade 2 (28.0%). The three most common IARs of headache, pyrexia, and rash occurred in 48.8%, 40.7%, and 24.1% of patients during the first course and 14.8%, 17.2%, and 5.6% of patients during the second course, respectively. The majority of IARs occurred within 6 hours after the start of alemtuzumab infusion, with a peak during the first 2 hours. The types and overall incidence of IARs were consistent with phase 2 and 3 trials. Frequency and distribution of rash were reduced in the EMERALD study compared with previous clinical trials.

Serious IARs occurred in 15.5%, a higher rate than reported in clinical trials of alemtuzumab.

Introduction

Alemtuzumab is a humanized monoclonal antibody that selectively binds to CD52 to deplete circulating T and B lymphocytes [START_REF] Hu | Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model[END_REF] , 2016). This rebalancing of the immune system may contribute to the sustained efficacy of alemtuzumab without re-treatment [START_REF] Bittner | Neuroimmunotherapies Targeting T Cells: From Pathophysiology to Therapeutic Applications[END_REF][START_REF] Ruck | Alemtuzumab in Multiple Sclerosis: Mechanism of Action and Beyond[END_REF][START_REF] Turner | Reduction of inflammation and preservation of neurological function by anti-CD52 therapy in murine experimental autoimmune encephalomyelitis[END_REF].

Alemtuzumab is a highly effective therapy for relapsing remitting multiple sclerosis (RRMS). Alemtuzumab is indicated for treating adult patients with RRMS with active disease defined by clinical or imaging features based on phase 3 studies, in which treatment-naive patients (CARE-MS I; NCT00530348) or those with inadequate response to prior treatment (CARE-MS II; NCT00548405) showed significantly greater improvements in clinical and magnetic resonance imaging (MRI) outcomes, including brain volume loss (BVL), compared with those receiving subcutaneous interferon β-1a. [START_REF] Cohen | Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial[END_REF][START_REF] Coles | Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial[END_REF]. Patients who participated in the core CARE-MS I and II trials could enroll in an extension study (NCT00930553), which demonstrated that alemtuzumab-treated patients experienced durable efficacy through 5 years (Coles et al., 2017b;[START_REF] Havrdova | Alemtuzumab CARE-MS I 5-year follow-up: Durable efficacy in the absence of continuous MS therapy[END_REF]. Patients who completed the extension study could enroll in the 5-year TOPAZ study (NCT02255656) for further long-term evaluation (Coles et al., 2017a;[START_REF] Singer | Durable Improvements in Clinical Outcomes With Alemtuzumab in Patients With Active RRMS in the Absence of Continuous Treatment: 7-Year Follow-up of CARE-MS II Patients (TOPAZ Study)[END_REF]. In TOPAZ, 86% (CARE-MS I) and 81%

(CARE-MS II) remained on study through Year 7, with 59% (CARE-MS I) and 47%

(CARE-MS II) receiving only the initial two courses of alemtuzumab and no other treatments (Coles et al., 2017a;[START_REF] Singer | Durable Improvements in Clinical Outcomes With Alemtuzumab in Patients With Active RRMS in the Absence of Continuous Treatment: 7-Year Follow-up of CARE-MS II Patients (TOPAZ Study)[END_REF]. By Year 7, 74% and 69% of patients from CARE-MS I and II, respectively, were free of 6-month confirmed disability worsening (CDW), whereas 37% and 44% of patients from CARE-MS I and II, respectively, achieved 6-month confirmed disability improvement (CDI). Additionally, 78% and 73% of patients from CARE-MS I and II, respectively, demonstrated improved or stable disability, as measured by the expanded disability status scale (EDSS).

Median yearly BVL remained low at Year 6 (CARE-MS I: -0.17% and CARE-MS II: -0.10%) [START_REF] Ziemssen | Alemtuzumab in the long-term treatment of relapsingremitting multiple sclerosis: an update on the clinical trial evidence and data from the real world[END_REF]. Most patients were free of MRI lesion activity (CARE-MS I: 68% and CARE MS II: 67%) and achieved no evidence of disease activity in Year 7 (CARE-MS I: 61% and CARE-MS II: 60%) (Coles et al., 2017a;[START_REF] Singer | Durable Improvements in Clinical Outcomes With Alemtuzumab in Patients With Active RRMS in the Absence of Continuous Treatment: 7-Year Follow-up of CARE-MS II Patients (TOPAZ Study)[END_REF].

Infusion-associated reactions (IARs) are the most frequently reported AEs in alemtuzumab-treated patients, affecting ~90% of patients in clinical trials [START_REF] Cohen | Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial[END_REF]Coles et al., 2017b;[START_REF] Coles | Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial[END_REF][START_REF] Havrdova | Alemtuzumab CARE-MS I 5-year follow-up: Durable efficacy in the absence of continuous MS therapy[END_REF]. Most IARs were mild-to-moderate in severity and generally decreased in number with each subsequent alemtuzumab treatment course. Incidence of treatment discontinuation due to IARs was low; IARs led to 1 (0.03%) treatment discontinuation in CARE-MS I due to allergic dermatitis and 5 (1.1%) discontinuations in CARE-MS II due to noncardiac chest pain and dyspnea in 1 patient, and pharyngeal edema, rash, purpura, and urticaria each in 1 patient. The most common IARs included headache, rash, nausea, and pyrexia. These reactions may reflect cytokine release associated with alemtuzumab-induced lymphocyte lysis [START_REF] Cohen | Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial[END_REF]Coles et al., 2017b;[START_REF] Coles | Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial[END_REF][START_REF] Havrdova | Alemtuzumab CARE-MS I 5-year follow-up: Durable efficacy in the absence of continuous MS therapy[END_REF].

The primary objective of EMERALD was to assess the impact of a different medication algorithm on development and/or management of IARs in RRMS patients receiving two courses of alemtuzumab.

Materials and Methods

Patients

Inclusion criteria were adult RRMS patients who initiated treatment with alemtuzumab according to the approved label in the country of treatment and who signed written informed consent. Patients were excluded if they previously received alemtuzumab, had any known contraindications to alemtuzumab according to labeling in the country of treatment, were participating in another investigational interventional study, had any known contraindications to symptomatic therapy used in infusionmanagement guidance based on their local approved label, were women of childbearing potential not using birth control and/or were unwilling or unable to be tested for pregnancy, pregnant (as defined as positive β-HCG blood test) or breastfeeding, had known infection with latent or active tuberculosis, or had known infection with hepatitis B or C virus.

Study Design

EMERALD was a multicenter, multinational, single-arm, phase 4, open-label study conducted at 19 sites in Belgium, France, the Netherlands, and Spain. EMERALD was a two-period study with mandatory administration of a comprehensive medication regimen aimed to prevent and/or manage alemtuzumab-associated IARs during the standard two-treatment course (Figure 1). Period 1 was defined from the first alemtuzumab dose to Day 0 of Period 2. During Period 1, patients began pre-infusion medications on Day 0 (the day before the first infusion). Period 2 was defined from the first alemtuzumab dose in Period 2 to end of study (Day 30 of Period 2). Infusion period was defined as the start of alemtuzumab dose through 24 hours after each infusion during Periods 1 and 2. The On-study Period was defined as the first alemtuzumab dose of Period 1 to end of study. After a maximum of a 2-week screening, patients were enrolled. Patients began the pre-infusion regimen the day before starting the first alemtuzumab course, infusions of 12 mg/day for 5 consecutive days (total dose of 60 mg), accompanied by the infusion-management medications. The second course was administered 12 months later, wherein patients received alemtuzumab infusions of 12 mg/day for 3 consecutive days (total dose of 36 mg) with the same pre-infusion regimen and infusionmanagement medications. Guidance for administering the infusion-management medication algorithm is shown in Table 1. Patients underwent monthly safety monitoring throughout the study. The end-of-study visit was performed on Day 30 of Period 2. After study completion, patients were followed by their physician in a regular healthcare setting until the 48-month safety monitoring period was completed according to the Summary of Product Characteristics (SmPC) and Risk Management Plan (RMP).

Primary endpoints were proportion of patients with at least one IAR during the study period, overall and by severity grade, and distribution of IARs by severity grade.

Serious adverse events (SAEs) were evaluated. Monitoring of potential autoimmune AEs according to the RMP for alemtuzumab included urinalysis with microscopy, complete blood count with differential, and serum creatinine before treatment initiation and monthly during the study; thyroid-stimulating hormone levels were tested before treatment initiation and every 3 months.

Treatments

Alemtuzumab was administered by intravenous infusion over approximately 4 hours. If IARs were evident during the infusion, the individual investigators could prolong the infusion duration based on their clinical judgment. The infusionmanagement medication regimen was expanded upon recommendations informed by experts from the SmPC for treatment of active RRMS with alemtuzumab (European Medicines Agency) (Tables 1 and2). (Table 1). Patients in CARE-MS I and II received pretreatment with systemic steroids during each treatment course (1 g/day intravenous methylprednisolone on the first 3 days), and patients with IARs were treated symptomatically with concomitant antipyretics, antihistamines, and antiemetics at treating neurologist's discretion (Table 2). In contrast, the EMERALD protocol included a more comprehensive pretreatment regimen with mandatory antipyretics and analgesics, and H1 and H2 receptor antagonists. As mentioned previously, the EMERALD protocol required additional oral methylprednisolone administration 1 day before the first infusion, and intravenous methylprednisolone after each treatment course as needed (Table 1). Acyclovir was administered both before and after alemtuzumab infusion for prophylaxis against herpes infection as recommended by the SmPC.

Safety Analysis

The following safety populations were defined and used for endpoint reporting.

The full safety set (SS) was defined as enrolled patients who received at least part of an alemtuzumab dose during Periods 1 or 2. The first SS (Course 1) was defined as patients who received at least part of an alemtuzumab dose during Period 1. The second SS (Course 2) was defined as patients from Course 1 who received at least part of an alemtuzumab dose during Period 2.

IAR data are discussed for EMERALD alone, and in comparison with patients who received alemtuzumab 12 mg in the CARE-MS I and II studies.

AE Definitions

AE and IAR definitions were based on standard designations. An IAR was defined as any AE occurring during or within 24 hours of alemtuzumab infusion. An AE was defined as any untoward medical occurrence in a patient that may or may not have a causal relationship with alemtuzumab. Recording of AEs began from signed informed consent through end of study. An AE was considered serious (SAE) if it either resulted in death, was life-threatening, required hospitalization, resulted in disability/incapacity, was a congenital anomaly/birth defect, or was medically important. Adverse events of special interest (AESIs) were AEs (serious or nonserious) of scientific or medical concern specific to this treatment, which required ongoing monitoring, such as autoimmune cytopenias and other autoimmune disorders, infections, malignancy, hypersensitivity, or anaphylaxis.

Treatment-emergent adverse events (TEAEs) were any AEs occurring between first dose (Period 1) and end of study. Infusion-associated reactions were any TEAEs occurring during or within 24 hours after each infusion for both infusion periods, and were described for each 2-hour interval. IAR definition and severity grade ratings were consistent with previous clinical trials and described accordingly.

Ethics

The protocol was approved by the appropriate Independent Ethics France, Netherlands, and Spain was performed by the Sponsor's staff according to the Sponsor's procedures. EMERALD is registered at ClinicalTrials.gov (NCT02205489).

Results

Patient Characteristics

Demographics, disease characteristics, and disease activity at baseline are shown in Table 3. Most patients (91.4%) had received treatment for MS prior to EMERALD, the most common of which were natalizumab (32 patients; 55.2%) and fingolimod (31 patients; 53.4%) (Table 3). Among the 31 patients who received fingolimod, 18 (58.1%) discontinued this treatment <4 months prior to alemtuzumab initiation. Median duration between discontinuation of fingolimod and initiation of alemtuzumab was 2.4 months. Of note, total white blood cell and lymphocyte counts were consistent between patients who had received different types of prior diseasemodifying therapies (DMTs). During Periods 1 and 2, all patients received medication for concomitant conditions; the most common drug classes were nervous system drugs (98.3% in Period 1 and 98.1% in Period 2, mainly analgesics and psycholeptics) and respiratory system drugs (72.4% in Period 1 and 72.2% in Period 2, mainly antihistamines).

Infusion-Associated Reactions

A summary of IARs by severity grade for the On-study Period is presented in Most IARs occurred within 6 hours of initiating alemtuzumab infusion, with a peak during the first 2 hours (Table 6). Nearly all IARs decreased in rate from Course 1 to Course 2; incidence was 48.3% and 40.7% for headache, 24.1% and 14.8% for pyrexia, 17.2% and 5.6% for rash, and 15.5% and 5.6% for bradycardia, respectively. Hot flush was the only IAR that did not decrease in incidence from Course 1 to Course 2 (5.2% and 5.6%, respectively). Serious IARs occurred in 15.5% (n=9) of patients who received alemtuzumab 2 (most occurred during the first course) (Table 7). This is a higher rate than reported in 3 clinical trials of alemtuzumab. Serious IARs occurred in 3% (n=12) of patients in each 4 CARE-MS I and II. In EMERALD, the most common serious IARs were cardiac 5 disorders, with bradycardia occurring in 3 patients and tachycardia in 1 patient. All other 6 serious IARs occurred in ≤1 patient. Bradycardia led to permanent withdrawal of 7 alemtuzumab for 1 patient. Bradycardia and tachycardia were each experienced as 8 serious IARs by 1 patient in CARE-MS I. Bradycardia and tachycardia were not reported 9 as serious IARs in any patient who received alemtuzumab 12 mg in CARE-MS II or in 10 the phase 2 trial (CAMMS223) [START_REF] Coles | Alemtuzumab vs. interferon beta-1a in early multiple sclerosis[END_REF][START_REF] Coles | Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial[END_REF]. No IAR led to 11 death in EMERALD, CAMMS223, or CARE-MS I or II. 12 13

Discussion

As with most monoclonal antibodies, IARs are frequently reported AEs associated with alemtuzumab treatment [START_REF] Caon | Prevention and Management of Infusion-Associated Reactions in the Comparison of Alemtuzumab and Rebif((R)) Efficacy in Multiple Sclerosis (CARE-MS) Program[END_REF]. The observation that most IARs in EMERALD were mild or moderate and decreased in rate during subsequent infusions is consistent with previous studies and with patient response to other monoclonal antibodies [START_REF] Lenz | Management and preparedness for infusion and hypersensitivity reactions[END_REF]. Consistent with previous controlled clinical trials, no deaths occurred in this study.

EMERALD evaluated use of a comprehensive medication regimen that was developed to prevent and manage IARs during alemtuzumab infusion in a real-world population. However, small sample size, lack of a comparator arm, and differences in population between EMERALD and CARE-MS I and II limit data interpretation. For example, more than half of patients in EMERALD were treated previously with natalizumab, as is common in the real-world population [START_REF] Berger | Alemtuzumab Use in Clinical Practice: Recommendations from European Multiple Sclerosis Experts[END_REF]. In comparison with patients in CARE-MS I and II, patients in EMERALD presented with more comorbidities, were older, had longer disease duration, and had been treated with a greater number of prior MS treatments including immunomodulating therapy. These differences in patient populations may have altered risk of IARs and reduced the feasibility of cross-trial comparison. Furthermore, potential differences in alemtuzumab infusion rates further confounded study assessment, as clinicians may slow infusions to manage IARs [START_REF] Caon | Prevention and Management of Infusion-Associated Reactions in the Comparison of Alemtuzumab and Rebif((R)) Efficacy in Multiple Sclerosis (CARE-MS) Program[END_REF]. al., 2015) did not increase during EMERALD, suggesting that methylprednisolone's effect was most apparent in the reduced incidence of rash. It is possible that insomnia associated with prophylaxis may be more easily tolerated by patients than rash.

Prophylaxis shown to reduce rash in alemtuzumab-treated patients may be beneficial in these cases.

A higher rate of bradycardia and serious IARs was observed in EMERALD compared with previous trials. Differences in baseline characteristics, such as increased comorbidities, may have contributed to increases in cardiac events among EMERALD

patients. An additional comparison of IAR frequency between EMERALD patients with prior DMT use and CARE-MS II patients, all of whom also had prior DMT use, showed increased rates of bradycardia and palpitations for patients in EMERALD. This suggests that factors other than prior DMT use (eg, comorbidities) may increase the risk of cardiac-related IARs after alemtuzumab. In EMERALD, 3 of the 9 patients with brachycardia had history of endocrine, cardiac, and metabolic disorders. Furthermore, 2 of these 3 patients had history of bradycardia prior to the study.

Additionally, most patients in EMERALD were hospitalized during the entire duration of infusion so they could be closely monitored, which was not required by Pharmacologic intervention for IAR management in previous clinical trials included a variety of doses and schedules, which were largely dictated by individual patient need and physician choice [START_REF] Caon | Prevention and Management of Infusion-Associated Reactions in the Comparison of Alemtuzumab and Rebif((R)) Efficacy in Multiple Sclerosis (CARE-MS) Program[END_REF]. Thus, the effectiveness of IAR prophylaxis had been unclear. The EMERALD treatment regimen is generally aligned with recommendations from clinical trial experience and labeling; the key difference is the consistent application of a specific medication algorithm developed from consensus group recommendations that included increased corticosteroid dosing, and mandatory administration of antihistamines, antipyretics, and H1/H2 receptor antagonists. [START_REF] Berger | Alemtuzumab Use in Clinical Practice: Recommendations from European Multiple Sclerosis Experts[END_REF][START_REF] Caon | Prevention and Management of Infusion-Associated Reactions in the Comparison of Alemtuzumab and Rebif((R)) Efficacy in Multiple Sclerosis (CARE-MS) Program[END_REF][START_REF] Mcewan | Best Practices in Alemtuzumab Administration: Practical Recommendations for Infusion in Patients With Multiple Sclerosis[END_REF] However, based on the overall findings in this study, there is no compelling advantage to adopt the IAR management approach used in EMERALD. Using medication to manage IARs in the real-world setting should follow the recommendations in labeling and, as is the case in clinical practice, should be left to physician discretion on an individual patient basis.

Conclusion

In this phase 4 study, we assessed distribution of IARs by severity grade when alemtuzumab is administered to RRMS patients in the real world with a specified medication algorithm aimed at preventing and managing IARs. Inclusion of real-world patients in EMERALD provides an understanding of the AEs physicians will likely encounter with alemtuzumab treatment. It is reassuring that no previously unknown or unexpected SAEs or TEAEs of special interest were reported during this study. We found that types and overall incidence of IARs were generally consistent with previous controlled clinical trials. However, incidence of rash was reduced in this study compared with the previous trials, which may have been associated with this study's prophylaxis treatment regimen. The increased incidence of serious IARs (eg, cardiac events) potentially reflected differences in baseline characteristics in this population.

  . After lymphocyte depletion, a distinctive pattern of T-and B-cell repopulation begins within weeks. The immunomodulatory effects of alemtuzumab are potentially achieved through alterations in the number, proportions, and properties of some lymphocyte subsets, with increased representation of regulatory T-cell (Treg) subsets, memory T-and B-lymphocytes, and transient effects on components of innate immunity (ie, neutrophils, macrophages, natural killer cells) (LEMTRADA Summary of Product Characteristics

Figure 1 .

 1 Figure 1.-Study Design. a After the end of Period 2, patients were followed by their physician in a regular healthcare setting until the 48-month safety monitoring period is completed. Abbreviations: EOS, end of study; FU, follow-up; IAR, infusion-associated reaction; IV, intravenous; RRMS, relapsing remitting multiple sclerosis.

  For example, the SmPC states that patients should be pretreated with corticosteroids immediately before alemtuzumab administration for the first 3 days of each treatment course. During EMERALD, patients received intravenous methylprednisolone on Day 0 and then every day before each infusion for each treatment course of 5 and 3 days, respectively, in Period 1 and Period 2, and in the evenings post-infusion as needed. Pretreatment with antihistamines and/or antipyretics, suggested in the SmPC, was mandatory during EMERALD (cetirizine [10 mg orally], diphenhydramine [25 mg intravenously], gastroprotection [proton pump inhibitors] according to the local label, and paracetamol [500 mg orally]). Oral prophylaxis for herpes infection is also recommended by the SmPC for all patients starting the first day of each treatment course and continuing for a minimum of 1 month following alemtuzumab treatment. Patients in EMERALD received acyclovir (200 mg orally) twice daily beginning the first day of each alemtuzumab course and continuing for 30 days following completion of each course. Additional agents not mentioned in the SmPC, but given as needed during EMERALD, included antiemetics and normal saline

  Committees/Institutional Review Boards and complied with International Conference onHarmonisation guidelines for Good Clinical Practice (GCP). All patients provided written informed consent prior to screening. The Sponsor conducted training sessions for clinical research associates and held individual site initiation meetings to develop a common understanding of the clinical study protocol, case report form, and study procedures, in compliance with GCP. Monitoring of all investigative sites in Belgium,

  EMERALD's protocol and may have increased the rate of reported AEs. Twenty-fourhour hospitalization during infusion periods was not part of the protocol in CARE-MS I and II. The in-hospital monitoring of patients in EMERALD, including heart rate, may have facilitated identification of transient heart rate alterations contributing to the higher AE rates observed for these events. The increase in reported incidence of pyrexia, although not a two-fold increase, in EMERALD versus CARE-MS I and II may be associated with the increased frequency of patient temperature measurements during hospitalization in EMERALD, wherein temperature measurements were recorded approximately at 1 hour pre-infusion, hourly during infusion, and 1 hour and 2 hour postinfusion.

Table 1 . Administration Algorithm of Mandatory and "As Needed" Associated Medication During EMERALD

 1

		Day 0	Days of alemtuzumab infusions
	Medication	1 day before infusion a	Pre-infusion b	During infusion c	Post-infusion d
	H1 antagonist according to the local label (eg, cetirizine 10 mg or equivalent) in the afternoon	Mandatory	-	-	Mandatory
	H1 antagonist 25 mg IV diphenhydramine or equivalent	-	Mandatory	In case of pruritus/rash	-
	H2 antagonist in the morning and afternoon or equivalent gastro-
	protector medications prescribed according to the local label (ie,	Mandatory	Mandatory	-	Mandatory
	proton pump inhibitors)
	Methylprednisolone 1,000 mg orally (Medrol ® ) (10 tablets of 100 mg each in the morning)	Mandatory	-	-	-
			Mandatory
			following immediately IV	-	As needed
			diphenhydramine
	NSAID/antipyretic: 500 mg oral paracetamol or equivalent	-	Mandatory	In case of pain/headache e	As needed e
	Antiemetic but no phenothiazine (eg, ondansetron 8 mg or equivalent)	-	-	In case of nausea	As needed
	Acyclovir 200 mg orally or equivalent	-	Mandatory f	-	Mandatory f
	0.9% sodium chloride (normal saline) bolus IV g	-	-	In case of dehydration	-

Methylprednisolone IV 1,000 mg (50% dose reductions from Day 3 [500 mg] to Day 5 [125 mg] in Period 1 and from Day 1 [750 mg] to Day 3 [250 mg] in Period 2) a 1 day before infusion patients took medications at home which were recorded in a patient diary.

b 1 hour prior to infusion. c If IARs occurred during infusion, the patient may receive as needed.

d The evening of alemtuzumab infusion.

e Not to exceed maximum daily recommended dose according to the local label.

f Patients must continue to receive acyclovir 200 mg PO BID or equivalent until Day 30 to minimize herpes virus infection.

g Fluid intake was encouraged and monitored. Abbreviations: BID, twice daily; H1, histamine receptor H1; H2, histamine receptor H2; IAR, infusion-associated reaction; IV, intravenous; NSAID, nonsteroidal antiinflammatory drug; PO, orally.

Table 2 . Mandatory and "As Needed" Associated Medications for CARE-MS I and II and Guidelines from the SmPC for the Treatment of Active RRMS with Alemtuzumab Treatment CARE-MS I and II SmPC Guidelines a

 2 If a patient assigned to the IFN β-1a arm had received corticosteroids for symptomatic treatment of a relapse within 1 month (30 days) of a scheduled steroid infusion, that infusion was omitted. Abbreviations: H2, histamine receptor H2; IAR, infusion-associated reaction; RRMS, relapsing-remitting multiple sclerosis; SmPC, Summary of Product Characteristics.

	Antipyretics	Permitted at the Investigator's	Pretreatment with antihistamines
	Antihistamines	discretion to minimize IARs known to	and/or antipyretics may
		be associated with alemtuzumab	be considered
		administration (eg, rash)
	H2 receptor blockers	Permitted at the Investigator's
	Antiemetics	discretion to minimize IARs known to be associated with alemtuzumab	-
		administration (eg, rash)
	Methylprednisolone	As prophylaxis of IARs administered	Patients should be pretreated with
		to all patients. All patients in both	corticosteroids immediately prior to
		treatment groups received 1 g/day of	alemtuzumab administration on
		IV methylprednisolone for 3	each of the first 3 days of any
		consecutive days at Month 0 and	treatment course
		Month 12
		The dose of methylprednisolone could
		be reduced at the investigator's
		discretion for patients who did not
		tolerate the standard regimen b

a As published by the European Medicines Agency. b

Table 3 . Demographics, Disease Characteristics, and Clinical and MRI Data at Baseline

 3 Patients without prior antineoplastic and/or immunomodulating therapy received prior nervous system drugs, systemic corticosteroids, or an investigational drug. d Not comprehensive; prior antineoplastic and immunomodulating agents ≤ 13.8% not listed. Data are mean ± SD unless stated otherwise. Data are comparable between Course 1 and Course 2. Abbreviations: EDSS, Expanded Disability Status Scale; MRI, magnetic resonance imaging; MS, multiple sclerosis; SD, standard deviation.

	Total (n = 58)

c Of the 61 patients screened, 58 (95.1%) were enrolled into the study. Of the 58 patients who received the first infusion of Period 1, 57 (98.3%) completed the 5 days of Course 1. Fifty-four patients received the first infusion of Period 2 and 53 completed the 3-day course. All patients (n=58) completed the Month 6 visit and 54 the Month 12 visit.

Table 4 .

 4 IARs

	Headache was the

occurred in 91.4% (n=53) and 81.5% (n=44) of patients in Course 1 and Course 2, respectively. Types and overall incidence of IARs were similar to those reported previously for alemtuzumab in controlled clinical trials (Table

5

). The three most common IARs were headache, pyrexia, and rash (Table

5

).

and II (Table

5

).

Table 4 . Summary of IARs for On-Study Periods 1 and 2

 4

		SS (n = 58)	Course 1 (n = 58)	Course 2 (n = 54)
	IAR within 24 hours of any infusion	54 (93.1%)	53 (91.4%)	44 (81.5%)
	Grade 1: Mild	51 (87.9%)	47 (81.0%)	34 (63.0%)
	Grade 2: Moderate	36 (62.1%)	32 (55.2%)	19 (35.2%)
	Grade 3: Severe	6 (10.3%)	6 (10.3%)	0 (0.0%)
	Serious IAR within
	24 hours of any	9 (15.5%)	7 (12.1%)	2 (3.7%)
	infusion
	Grade 1: Mild	4 (6.9%)	4 (6.9%)	0 (0.0%)
	Grade 2: Moderate	4 (6.9%)	2 (3.4%)	2 (3.7%)
	Grade 3: Severe	2 (3.4%)	2 (3.4%)	0 (0.0%)

Patients may have experienced IARs on multiple occasions whereby each IAR was recorded. Abbreviations: SS, safety set; IAR, infusion-associated reaction.

Table 5 . Infusion-Associated Reactions (IARs) Occurring in ≥5% of Patients for Periods 1 and 2 of the EMERALD Study, and for Course 1 and 2 of the CARE-MS I and CARE-MS II Studies Preferred term n (%)

 5 Dash represents IARs that appeared in <5% of patients. A patient can be counted in several categories of preferred term by system organ class.

				CARE MS-I	CARE MS-I
		EMERALD	EMERALD	and II	and II
		Course 1	Course 2	Course 1	Course 2
		12 mg	12 mg	12 mg	12 mg
		(n = 58)	(n = 54)	(n = 811)	(n = 791)
	Any class	53 (91.4)	44 (81.5)	687 (84.7)	543 (68.6)
	Headache	28 (48.3)	22 (40.7)	289 (35.6)	221 (27.9)
	Pyrexia	14 (24.1)	8 (14.8)	124 (15.3)	104 (13.1)
	Rash	10 (17.2)	3 (5.6)	294 (36.3)	112 (14.2)
	Bradycardia	9 (15.5)	3 (5.6)	24 (3.0)	6 (0.8)
	Insomnia	9 (15.5)	-	56 (6.9)	-
	Erythema	8 (13.8)	-	28 (3.5)	-

Note:

Table 6 . Number of Patients With Infusion-Associated Reactions (IARs) by 2-Hour Interval and by Grade According to Time From Start of Infusion

 6

	Period 1 Total n = 58	Grade 1	Grade 2	Grade 3
	0-2 h	30	14	5
	2-4 h	9	10	1
	4-6 h	18	8	0
	6-8 h	7	3	0
	8-10 h	7	1	0
	10-12 h	9	4	1
	12-14 h	7	6	1

Table 7 . Summary of Serious Infusion-Associated Reactions (IARs) by Frequency for On- 1 Study Period 2 MedDRA System Organ Class Name and Preferred Term Name

 7

	Total n = 58
	n (%)

Table 8 . Adverse Events (AEs) of Special Interest for On-Study Period MedDRA System Organ Class Name and Preferred Term Name

 8 Other infections not reported as AEs of special interest are considered TEAEs. Medical Dictionary for Regulatory Activities (MedDRA) version 19.0. Note: A patient can be counted in several categories of preferred term by system organ class.

	Total n = 58
	n (%)

a

ACKNOWLEDGMENTS

Financial support for medical editorial assistance was provided by Sanofi. We thank Sarah Jablonski Schandle, PhD, ProEd Communications, Inc., for her medical editorial assistance with this manuscript. This manuscript was reviewed by Darren P. Baker, PhD, Erika Bueno, PhD, and Colin Mitchell, PhD, of Sanofi.


Ivan Brihuega

Pierre Mallet

Cristina Bena

Sangita Bose

Christian Michaelis

Lucia Vitali

François Varchon

Laurence Magaud

Klaus Kern

Jean-Yves Y Veuillen

Quasiparticle chirality in Epitaxial Graphene Probed at the Nanometer Scale

Keywords: numbers: 73.20.-r, 68.37.Ef, 72.10.Fk

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Graphene, one single layer of carbon atoms packed in a honeycomb structure, has two identical carbon atoms in each unit cell and thus two equivalent atom sublattices. This gives rise to an extra degree of freedom absent in conventional two-dimensional (2D) systems, and leads to exceptional electronic properties. The major consequence of the honeycomb structure is that the low energy quasiparticles of graphene are described by a Diraclike Hamiltonian. The energy spectrum is thus linear, and consists of two Dirac cones centered at the opposite corners K and K of the Brillouin zone. This is, however, insufficient to understand properly the specific electronic properties of graphene. Due to the presence of the two C atoms per unit cell, the quasiparticles have to be described by two-component wave functions, each component specifying the (complex) amplitude on each atomic sublattice. This results in an additional degree of freedom known as pseudospin. The projection of the pseudospin on the direction of the momentum defines the chirality of the quasiparticules; in graphene the direction of the quasiparticle pseudospin is parallel (positive chirality) or antiparallel (negative chirality) to their momentum [1,2]. Within one single Dirac cone, electrons (or equivalently holes) of opposite direction have opposite pseudospin, but the same chirality. In graphene bilayer with Bernal stacking, chiral Dirac fermions are also found, but they are massive and their chirality differs from the one found in graphene monolayer [1,2].

In order to fully exploit graphene in future electronic devices it is of primary importance to assess the chirality of the quasiparticles, since this feature is central to the transport properties. It is responsible for the new "chiral" quantum Hall effects (QHE) measured in exfoliated monolayer and bilayer graphene [3][4][5]. In epitaxial graphene multilayers on the SiC(0001) face, Shubnikov-de-Haas oscillations and weak antilocalization effects consistent with the electronic chirality of "uncou-pled" graphene layers [6,7] have been reported, but surprisingly the QHE has not been yet observed [8]. Therefore, a direct measure of the electronic chirality in a single epitaxial graphene layer is essential. Here we show that the quasiparticles pseudospin -and the associated chirality -can be probed at the nanometer scale with a low temperature scanning tunneling microscope (STM). Our results on graphene monolayer on SiC(0001) are consistent with the electronic chirality of ideal graphene.

Epitaxial graphene was grown on a 6H-SiC(0001) substrate by thermal desorption of silicon at high temperatures, in ultra high vacuum (UHV) [9,10]. Parameters were chosen to obtain a mixed surface with a similar coverage of monolayer and bilayer (with Bernal stacking) terraces, ∼ 50 nm wide. A carbon layer is present at the interface between graphene and the SiC. This layer is strongly bound to the substrate, and acts as a buffer layer with negligible interactions with the graphene layers [11]. Although not metallic, the carbon buffer layer has electronic states that give a significant contribution to the STM images [10,12]. Our experiments were done using a home-made microscope operating at 4K in ultra-high vacuum. High bias STM images, as shown in Fig. 1(a), allow discriminating monolayer from bilayer graphene terraces due to the different interface states contribution [10].

Two groups have reported angle-resolved photoemission spectroscopy (ARPES) measurements on epitaxial graphene layers on SiC(0001). They demonstrate that the graphene layers are electron doped. The measured difference between the Fermi energy (E F ) and the Dirac energy (E D ) is ∼ 0.45 eV for a monolayer and ∼ 0.30 eV for a bilayer [13][14][15]. For graphene monolayer, a low energy linear dispersion has been found, a fingerprint of Dirac-like fermions [13,14]. However, different results are obtained by the two groups concerning the crossing of the bands: An energy gap of 0.25 eV is found at the Dirac point by only one of the groups [14]. It has been proposed by the authors that the gap arises from a substrate-induced breaking of the sublattice symmetry of graphene. ARPES, also shows that the Fermi surfaces (FSs) of monolayer and bilayer graphene on SiC(0001) are essentially identical [15,16].They consist of two tiny circular pockets, of radius q F , surrounding the K and K points [Fig. 1(b), six pockets are sketched to account for the symmetry of the Brillouin zone].

At low sample bias (a few mVs), the STM probes essentially the electronic states of the FS, and a constant current image reflects a map of the surface local density of states (LDOS) at E F [17]. In a 2D system such as graphene, low bias images probe the quantum interferences (QIs) arising from elastic scattering of surface quasiparticles at static defects [18]. Such QIs are related to the Friedel oscillations in 2D metals. In a simple picture, any impurity scattering between two states -→ k and -→ k of the FS should give rise to LDOS spatial modula-tions with wavevector -→ k --→ k , with a scattering probability depending on the topology of the FS. For a circular FS of radius k F , scattering between states -→ k F and states --→ k F (termed backscattering in the following) are the most efficient processes due to enhanced phase space, resulting in LDOS modulations with wavector 2k F [18,19].

For the FS sketched in Fig. 1(b), and depending on the impurity potentials present in the system, we may expect elastic electron scattering processes between the two non-equivalent Dirac cones at K and K (i.e. intervalley scattering) as depicted in Fig. 1(c), together with elastic scattering processes between states located on the same Dirac cone (i.e. intravalley scattering). Figure 1(d) is a constant current STM image taken at +2mV that includes two adjacent graphene monolayer and bilayer terraces. A striking difference is measured in the LDOS patterns between the left and right terraces. A clear long-range modulation, of wavelength 5.2±0.3 nm, is found on the graphene bilayer terrace. This modulation has already been reported by Rutter et al. [12], and is attributed to quantum interferences of wave vector 2q F due to intravalley backscattering between states -→ q F and states --→ q F within one single pocket [Fig. 1(f)]. Surprisingly, we do not find such LDOS modulation on the monolayer, which is puzzling if we keep in mind that the two systems have almost the same FS.

The lack of LDOS modulation of wave vector 2q F for the monolayer implies that QIs associated with intravalley backscattering are virtually absent. It has been reported that wave function symmetries may lead to selection rules that can prevent QIs, for instance in systems with strong spin-orbit coupling where the electronic spin governs the elastic scattering processes [20,21]. In the present case, the absence of QIs in graphene monolayer is associated to the pseudospin instead of real spin. Intuitively, this is because the pseudospin of the backscattered wave is opposite to that of the incident wave, so that their overlap should be zero [see Fig. 1(e)]. For a scattering potential with range larger than the C-C distance (which leads to scattering without pseudospinflip), it has been shown that intravalley backscattering processes and the associated QIs are strictly forbidden in monolayer graphene [22,23]. On the contrary, the electronic chirality does not prevent intravalley backscattering in bilayer graphene [Fig. 1

(f)] [24].
For both monolayer and bilayer epitaxial graphene on SiC(0001), previous STM experiments have reported that atomic-size impurities give rise to short-wavelength modulations of the LDOS, associated with intervalley scattering [10,12]. According to Ando et al. [22], such impurities, acting as short-range potentials, should also allow intravalley backscattering, which raises the question of possible QIs of wave vector 2q F . Recent theoretical studies show that for a general impurity potential, LDOS modulations with wave vector 2q F should exist for graphene monolayer, but they should be strongly attenuated [25][26][27], with a 1/r 2 decay law instead of the 1/r law found in conventional 2D systems. On the other hand, a 1/r decay law is obtained for the QIs due to intravalley backscattering in a graphene bilayer, and also for the QIs due to intervalley scattering for both monolayer and bilayer graphene. In reference [25] one of us showed that the 1/r 2 decay law should have a clear impact on the 2D Fourier transform (FT) of the LDOS modulations: while on bilayer graphene the efficiency of intravalley backscattering should give rise to a central ring of radius 2q F in the FT map similar to the one observed for conventional 2D systems, the strong attenuation of intravalley backscattering on monolayer graphene should generate a FT image with no central ring [25].

In the following, we present high resolution fast Fourier transform (FFT) of STM images recorded at low sample bias on 50 nm wide graphene terraces. We first show the real-space images, for graphene monolayer [Fig. 2(a)] and graphene bilayer [Fig. 2(b)]. Note that both im-ages exhibit a triangular pattern of periodicity ∼ 1.9 nm which is related to the interface reconstruction [10,12], and which appears as a sextuplet of bright spots in the corresponding FFT images [Figs. 2(c) and2(d)].

The central region of the FFT in Figs. 2(c) and 2(d) is related to intravalley scattering: a clear ring-like feature of average radius 1.2 nm -1 is found for the bilayer [Figs. 2(d) and 2(g)]. This radius value is in agreement with the value given in ref. [12], and with the 2q F value derived from ARPES [13,14]. On the monolayer terrace, no central ring is found [Figs. 2(c) and 2(e)], despite the unprecedented momentum resolution obtained here. This has been checked on many different monolayer terraces (see also the EPAPS document [START_REF]for more details. For more information on EPAPS[END_REF]), and we conclude that the result is robust and systematic. As discussed above, pseudospin and electronic chirality are the key parameters for the absence (presence) of the central ring in the FFT image of monolayer (bilayer) graphene.

In the high frequency regions of the FFTs of Figs. 2(c) and 2(d), we find six outer pockets with ring-like shapes centered at K(K ) points. They result from intervalley scattering, associated to real space LDOS modulations with a ( √ 3 × √ 3)R30 • periodicity with respect to graphene [10,12]. As shown in Figs. 2(f) and 2(h), the intensity of the high frequency rings in the FFT is not isotropic. The anisotropy is much more pronounced for graphene monolayer, for which a splitting of each ring into two arcs is found [Fig. 2

(f))

]. This splitting is discussed in more detail in the EPAPS document [START_REF]for more details. For more information on EPAPS[END_REF].

The experimental results are compared with T-matrix calculation made along the lines of Ref. [25] (see also the EPAPS document [START_REF]for more details. For more information on EPAPS[END_REF]). Figure 3(a) is the FT of a calculated LDOS map at E F for monolayer graphene with E D = E F -0.45 eV, in the presence of a single localized impurity. It is clear that no central 2q F ring is generated in this case. This is the signature of LDOS modulations resulting from inefficient intravalley backscattering, and it is the most direct fingerprint of graphene's pseudospin and chirality [25]. This is in very good agreement with the experimental absence of the central ring [Fig. 2(e)]. Also, the high frequency split rings, resulting from intervalley scattering, are nicely reproduced by the theoretical calculation. The splitting of the rings is another consequence of the quasiparticle chirality [START_REF]for more details. For more information on EPAPS[END_REF].

Figure 3(b) is an equivalent map as Fig. 3(a) but for a graphene bilayer, with E D = E F -0.3 eV. From ARPES it is known that the two layers have a different electron density, giving rise to a gap at E D of about 0.1 eV [15]. The asymmetry between the two layers has been included in the present calculation. Note the appearance of the inner ring of radius 2q F , as well as the presence of the outer rings which have not obvious splitting (a faint intensity remains perpendicularly to the ΓK and ΓK directions). The agreement with the experimental FFT is excellent.

Finally, we propose the use of STM to check any possible breaking of graphene's sublattice symmetry. To this end, we have calculated the FT of the LDOS map for a monolayer with the same parameters as in Fig. 3(a), but including an arbitrary sublattice asymmetry of 0.25 eV, which is the gap value measured by Zhou et al. [14].

The calculation shows a clear inner ring in the FT, corresponding to efficient QIs due to intravalley backscattering [START_REF]for more details. For more information on EPAPS[END_REF]. This dramatically illustrates the effect of the asymmetry on the FT STM data since this ring is absent in the symmetrical case [Fig. 3(a)]. The data of Fig. 3(c) are for a defect on one sublattice, but we found that the shape of the ring depends on the location of the scattering potential [START_REF]for more details. For more information on EPAPS[END_REF]. Due to the large size of the images it is likely that a distribution of defects is present in the experimental data. Calculations indicate that a large sublattice asymmetry (leading to a gap of 0.6 eV) should be readily detected in FT-STM images, but that for a moderate asymmetry (as for Fig. 3(c)) the intensity of the ring could depend on the defect configuration. Since we have taken images in many different surface areas without detecting any ring, we believe that our data are rather consistent with a symmetrical monolayer, although a small asymmetry can not be totally ruled out. In summary, by analyzing the Fourier transform of low bias STM images, we have demonstrated that the pseudospin and the chirality of epitaxial graphene quasiparticles can be probed directly at the nanometer scale. We show that graphene monolayer on SiC(0001) exhibits the chirality of an ideal graphene sheet. Together with the previous evidence by ARPES of the low energy linear spectrum, a complete assessment of the Dirac nature of the quasiparticles in this system is obtained.

Supporting Material of "Quasiparticle Chirality in Epitaxial Graphene

Experimental method

Synthesis of epitaxial graphene has been performed at Institut Néel, Grenoble, France.

The details of the growth process and characterization of our atomically thin graphene samples have been discussed elsewhere [1]. Low temperature STM measurements have been done at Max Planck Institute, Stuttgart, Germany. After air exposure during the travelling time, the clean surface has been recovered in the UHV-STM chamber using a smooth annealing at 300-400°C.

LDOS maps at the Fermi energy were obtained at 4 K (with a base pressure lower than 5.0 × 10 -11 mbar), by recording standard constant current images at low sample bias (in the mV range) [2]. All measurements were performed in the tunnelling regime (i.e. under tunnelling conditions for which an exponential dependence of the tunnelling current with distance and reasonable work function values -between 3.5 and 5 eV-are obtained) at low enough currents (<1nA). Under these tunnelling conditions our results were completely independent of the tunnelling current used.

As indicated in the main text, a convenient way to differentiate monolayer areas from bilayer areas in large scale images is to take advantage of the larger interface contribution at high sample bias. We have cross-checked these observations by investigating the atomic contrast in the STM images [1,[3][4][5]. As shown on Fig. S1, high resolution images taken at atomic scale show the graphene lattice periodicity of 2.45 Å for monolayer and bilayer terraces. However, the contrast is markedly different, with a triangular pattern on graphene Bernal-stacked bilayer (upper-right image), and with a perfect honeycomb-like pattern on graphene monolayer (lower-right image). This allows us to unambiguously discriminate monolayer terraces from bilayer terraces. Large images (typically 50 nm wide) with a large number of points (up to 2048 points per line) were acquired to get sufficient resolution -and range -in the fast Fourier transform (FFT) images [6]. It takes only a few minutes (typically 20 minutes) to get such an image. This relatively short acquisition time allows for the investigation of many different spots on the sample in order to verify that the FFT results were not dependent on a specific (local) form of disorder. The data were processed using the WsXM software [7].

Intervalley scattering in the monolayer Figure S2(a) shows a 30x50 nm² image of a monolayer area taken at low bias. Figure S2(c) is a zoom-in on an area of size 6.3 x 6.3 nm². The honeycomb contrast (where all C atoms of the graphene layer are visible), which is typical for a graphene monolayer, is clearly seen at this scale. Also visible in the lower part of Fig. S2(b) is the √3x√3R(30°) superstructure commonly found around point defects in monolayer and bilayer graphene [1,3]. It results from elastic electron scattering between the non equivalent K and K' valleys of the band structure of graphene (also called intervalley scattering). The Fermi contours at K and K' points are to first approximation circles with radius q F (see Fig. 1). In a simple picture, the intervalley scattering events should therefore give rise to circles with radius 2q F centered at the K and K' points of the surface Brillouin zone (SBZ) in the FFT of low-bias STM images [1,3]. Indeed, structures that appear to be circular (indicated by arrows) are detected close to the K (K') points of the SBZ in the FFT of the STM image shown in Fig. S2(c). One notices however that these circles are not complete (see an expanded view in figure S2(d)): for any circle around K (K'), there is a line with minimum intensity, perpendicular to the ΓK (or ΓK') direction and going through the K (K') point, which divides the circle into an inner and an outer part. The same feature is observed in Fig. 2(f), and it is characteristic of the monolayer (it is not found for the bilayer, see Fig. 2(h)).

This peculiar split structure is also obtained in the computed FT-STM image of the monolayer shown in Fig. S2(d). It is again a consequence of the particular quasiparticle chirality in single-layer graphene. This conclusion stems from the fact that the split structure is absent in FT-STM images of bilayer graphene, either in the measurements (Fig. 2(h)) or in the calculations ( [8] and Fig. 3(b)), despite the similarity between the Fermi surface of the two phases. In the calculated FT image of monolayer graphene, the signal intensity of the outer pockets in the direction perpendicular to ΓK is lower than 4% of the value along the ΓK direction. For bilayer graphene, a 40% intensity ratio between directions perpendicular and parallel to ΓK is still observed. Hence the chirality of the single layer graphene is reflected in various features of the FT-STM images: the absence of a circle around the Γ point and the split structures close to the K (K') points.

T-matrix calculations of the FT-STM images

We compare our experimental results to theoretical calculations performed using a Tmatrix approximation. For the details of the procedure see references [8][9][10]. It is sufficient to note here that the FT-STM images are obtained by computing the trace of a convolution of two free Green's functions of graphene and the T-matrix, which incorporates the effect of the scattering potential. In general we focus on a delta-function impurity potential situated on one atom, which makes the T-matrix diagonal in the sublattice basis, and independent of momentum.

The parameters we use in our evaluations of the LDOS for monolayer (ML) graphene correspond to a Dirac point at -0.45eV below the Fermi surface [11], with the FT-STM images evaluated at the Fermi surface. A quasiparticle inverse lifetime of 30meV is introduced to avoid divergences in the calculation; this translates into 30meV broadening of the peaks observed in the FT-STM images of Fig. 3.

For the graphene monolayer with an asymmetry between the two carbon sublattices, the computed FT-STM images actually depend on the site where the impurity resides. The FT-STM image depicted in figure 3(c) corresponds to a defect on the sublattice with the highest on-site energy (sublattice A), and in this case the asymmetry gives rise to a positive ring-like contribution at 2q F , superimposed over the central disk associated to the attenuated Friedel oscillations. This is the configuration we have chosen to present in the main text because it illustrates most clearly the effect of the symmetry breaking. It turns out however that for a defect on a site of the other sublattice (sublattice B), the asymmetry results in a negative ring-like contribution to the FT-STM signal, superimposed over the central disk. Nevertheless, the nonequivalency between the two sublattices leads in both cases to standing wave patterns around the defect that will be decaying as 1/r. This is at variance with the case of a symmetrical monolayer where long wavelength LDOS oscillations decaying as1/r are absent due to a cancelation of the two sublattices contributions [8,12,13].

To model the situation in real samples, we have considered that: (i) Atomic resolution images show a honeycomb contrast, which indicates that the LDOS's on A and B sublattices are similar close to the Fermi level. Adsorption of impurities should thus occur equally on both sublattices. (ii) Images of ML samples of different origins [1,5] suggest that there are different kinds of (native) localized defects (of unknown structure). Therefore, the conclusions stated in the main text derive from numerical simulations of the central spot of the FT-STM images for different structures of point defects where a given localized potential is placed on a: A site, on a B site or on two closely spaced A and B sites.

For bilayer graphene we consider that, in agreement with ARPES experiments [14], the lower layer (upper layer) has a chemical potential of -350 meV (-240 meV), with the Dirac point at -295meV and a gap of 110 meV. The impurity is chosen to lay on a site in the upper layer which is not coupled with a site in the bottom layer. The FT-STM images are again evaluated at the Fermi level in the top layer. The interlayer coupling is taken to be 600meV, while the intralayer hopping parameter is taken to be 3eV for both monolayer and bilayer graphene.

FIG. 1 :

 1 FIG. 1: (color online) (a) Constant current 90×43 nm 2 STM image of epitaxial graphene on SiC(0001), with two adjacent monolayer (ML) and bilayer (BL) graphene terraces. The spatial derivative of the image is shown, to enhance the corrugation due to the interface states which is higher on ML terraces [10]. Sample bias: +980 mV, tunneling current: 0.15 nA. (b) Schematic Fermi surface for electron-doped ML and BL graphene. (c) Illustration of an intervalley scattering process. (d) 3D rendered 50×50 nm 2 constant current image of two adjacent ML and BL terraces, taken at low sample bias (+ 1mV). A long wavelength scattering pattern is found on the BL terrace (left), and not on the ML terrace (right). Tunneling current: 0.2 nA. (e) Schematic of forbidden intravalley backscattering for ML graphene. Red arrows sketch the pseudospin direction. (f) Schematic of intravalley backscattering for BL graphene.

FIG. 2 :

 2 FIG. 2: (color online) (a),(b) Low-bias STM images of 50 nm wide monolayer (a) and bilayer (b) terraces. Sample bias and tunneling current are respectively +2 mV and 0.4 nA for (a), +4 mV and 0.13 nA for (b). (c),(d) Two-dimensional fast Fourier transform (FFT) maps of the STM images (a) and (b). (e) Central region of (c), showing no intravalleybackscattering related ring (the green arrow points out the position where such a ring should appear). (f) One of the outer pockets of (c). (g) Central region of (d), showing a clear ring-like feature of radius 2qF related to intravalleybackscattering. (h) One of the outer pockets of (d). Outer pockets shown in (f) and (h) are centered at the K (or K') point and result from intervalley scattering.

FIG. 3 :

 3 FIG. 3: (color online) (a-c) Theoretical calculation of the Fourier Transform (FT) of the LDOS at E F for different graphene layers with a single delta-function impurity. (a) 3D rendered FT map for graphene monolayer with a Dirac energy ED = EF -0.45 eV. The calculation predicts no central ring of radius 2qF , showing the unefficiency of intravalley backscattering. (b) Same representation as (a) for a bilayer, showing a clear central 2q F ring. E D = E F -0.3 eV. An asymmetry of ∼ 0.1eV between the two layers has been included, to account for the different electron density reported by ARPES (12,13). (c) Same representation as (a) for an asymmetric monolayer, with E D = E F -0.45eV. A site energy difference of 0.25 eV is introduced between the carbon atoms of each unit cell, and the impurity is placed on a carbon atom with high site energy. The symmetry breaking between the two carbon sublattices restores the efficiency of intravalley backscattering as shown by the reappearance of a central ring in the FT map.

  Probed at the Nanometer Scale", I. Brihuega et al.

Fig. S1 .

 S1 Fig. S1. A large scale constant-current STM image of our graphitized SiC(0001) sample, exhibiting monolayer (ML) and bilayer (BL) graphene terraces. Image size: 100 x 70 nm 2 , sample bias +100mV, tunneling current 0.1 nA. The two images with atomic resolution sketched on the right correspond to images of the boxed areas on the left image. Sample bias and tunnelling current are respectively -50 mV and + 1.0 nA for the upper-right image, and -125 mV and + 0.1 nA for the lower-right image.

Fig. S2 .

 S2 Fig. S2. STM analysis of the quantum interferences related to intervalley scattering in graphene monolayer. (a) 30 × 50 nm² image of a monolayer terrace at 4.5 K, sample bias: -2 mV, tunnelling current: 0.4 nA. (b) Fast

We thank D. Mayou for helpful discussions, F. Hiebel for participating to sample preparation, R. Cox for careful reading of the manuscript and the French ANR (programme Blanc) for partial financial support of this work. C. B. and I. B. were supported by a Marie Curie action under the Sixth Framework Programme.