Competitive allele-specific TaqMan PCR (Cast-PCR)

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F. 41. Cutaneus Melanoma leg positive. YES brain lung lymph node. 90%. V600E. V600E. 1,95 not available. 3. M. 43. Cutaneus Melanoma trunk negative yes.
Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients. Raffaela Barbano1, Barbara Pasculli1, Michelina Coco1, Andrea Fontana2, Massimiliano Copetti2, Michelina Rendina1,3, Vanna Maria Valori3, Paolo Graziano4, Evaristo Maiello3, Vito Michele Fazio1,5, Paola Parrella1*. 1

Laboratory of Oncology, 2Biostatistic Unit, 3Department of Oncology, 4Department of

Pathology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG) 71013; 5

CIR Laboratory for Molecular Medicine and Biotechnology, University Campus

Biomedico, Rome 00128, Italy.

Supplemental Table 1.

Supplemental Table 1. Clinicopathological characteristics and mutational analysis results for the 54 patients analysed by direct sequencing and Cast-PCR CASE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Gender F F M F F F M M M F F F M F F M F M M M F M M M M F M F M M M M F M F M M F F F M F F F M F M F M F M F F M

Age 59 41 43 57 61 75 53 41 33 53 81 48 72 42 62 62 61 51 44 35 74 80 52 46 77 84 81 66 71 62 63 21 35 68 60 82 53 44 74 74 69 38 79 34 28 58 54 60 55 85 72 81 74 54

2 6

Tumor Type

primary site

Regional Lymphnode

Progression

site progression

sample analyzed

% tumor cell

SANGER

Cast-PCR

Cast-PCR ∆Ct

anti-BRAF treatment

Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Occult Melanoma Cutaneus Melanoma Uveal Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Occult Melanoma Cutaneus Melanoma Spitz Nevous Cutaneus Melanoma Cutaneus Melanoma Mucosal Melanoma Occult Melanoma Mucosal Melanoma Uveal Melanoma Cutaneus Melanoma Mucosal Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Uveal Melanoma Mucosal Melanoma Cutaneus Melanoma Cutaneus Melanoma Uveal Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma Cutaneus Melanoma

leg leg trunk trunk trunk trunk trunk leg trunk cheek trunk trunk trunk trunk trunk trunk trunk unknown not available Neck LEG arm gallbladder trunk Iris scalp arm cheek trunk unknown ear leg trunk leg nose lymph nodes rectum choroid cheek nose arm trunk acral leg choroid vulva arm acral choroid not available leg nose cheek trunk

negative positive negative sentinel lymph node

YES YES yes YES

Skin brain lung Skin, Lymph node Lymph node

80% 90% A= 90% B= 70% 75%

positive

YES

Lymph node

positive positive positive positive positive negative positive negative positiveitive positive positive positive positive positive not available positive positive positive sentinel lymph node not available not available positive not available negative positive positive negative negative negative not available positive positive not available positive not available not available positive negative negative not available not available positive negative sentinel not available positive positive not available not available positive

YES YES YES YES yes YES YES YES YES YES YES YES YES YES not available YES YES not available YES YES NO YES not available yes YES YES NO NO YES YES YES YES YES YES YES YES YES YES NO YES not available YES YES YES YES YES not available NO NO

Skin, Lymph node Lung Skin Liver Lymph node primary site; lymphnode Breast Skin Lymph node Breast Lymph node Brain, Lung Lymph node Lymph node not available Skin Skin not available primary site; lymphnode Liver NO Skin not available Lymph node Retroperitoneal mass Liver NO NO Skin Bone Syncronous bone metastases not available Liver Brain Pleura Skin Peritoneum Skin NO Liver NO spleen skin LIVER lung Brain, Skin not available NO NO

primary site lymph node primary site primary site primary site primary site lymph node primary site primary site lymph node Primary (T1); lymphnode (T2) breast skin lymph node breast lymph node lymph node lymph node lymph node primary site skin skin gallbladder primary (T1); lymph node (T2) primary site primary site skin syncronous lung metastases lymph node Retroperitoneal mass primary site primary site primary site skin primary site lymph node primary site primary site primary site pleura primary site primary site skin primary site primary site primary site spleen primary site metastases lung primary site primary site primary site lymph node

Not Mutated V600E V600E V600E Not Mutated low height peak Not Mutated V600E V600E V600E Not Mutated V600E V600E V600E V600E V600E V600E V600E V600E V600E V600K low height peak V600K T1 V600K; T2 not mut Failed analysis V600K V600K V600K V600K Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated

V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600K V600K V600K T1 V600K; T2 not mutated V600K V600K V600K V600K V600K Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated Not Mutated

2.1 1,95 A= 1.85 B= 2.22 1.56 0.82 2.01 5.17 A= 1.74: B= 2.06 A=5.21; B=4.40; C=3.44; D=3.33 A= 1.58; B=0.10 T1=4.69; T2=2.72 A=1.72; B=2.04 1.71 2.01 0,21 2.26 2.14 3.33 1.88 1.73 3,17 6,13 2.4 T1= 0.77 2.04 2.06 2.52 1.12 3.77

NO not available YES not yet NO YES not available not available YES YES NO not available YES YES YES NO YES NO not available YES not available NO not available YES NO not yet not available YES YES

50%

25% 80% A= 60%; B= 50% A, B, C and D 80% A and B 90% T1=90%; T2=95% 80% 80% 50% 90% 90% 90% 60% 60% 90% 80% 70% 50% T1=90%; T2=90% 80% 80% 60% 80% 90% 75% A=50%; B=70% 50% 30% 60% 10% 80% 40% 90% 95% 60% A, B and C 90% 90% 80% 40% 90% 90% 75% 80% 80% 75% 80% 70% 70% 30%

Response to anti-BRAF treatment

TREATMENT NOTES

Partial Response

Progression after 7 months

Response

on going

Stable Disease Toxicity

at 3 months Suspended for toxicity after 1 month

Partial Response Response Stable Disease

Progression after 6 months at 3 months at 3 months

Toxicity

Suspended for toxicity after 3 months

Response

at 3 months

Toxicity, reduced dosage

on going Refused treatment

Stable Disease Stable Disease

at 3 months at 3 months