Identifying Biomarkers and Disease Mechanisms with Protein Arrays

Frameshift muta­tions are com­monly the cause of severe patho­lo­gies, but they are in many cases neg­lected by research­ers, even though their dis­cov­ery often leads to a sig­ni­fic­ant break­through. This is mainly due to the great dif­fi­culties asso­ci­ated with frameshift ana­lys­is until now. Pro­tein arrays can be used to research these aber­rant pep­tides and provide insight into the patho­gen­ic pro­cesses they induce.

Frameshift research can improve patient care, new treat­ments or great­er under­stand­ing of a dis­ease mech­an­ism. These muta­tions play a key role in vari­ous dis­eases like cyst­ic fibrosis and Hunt­ing­ton’s Dis­ease — but in this art­icle, we focus on can­cer research and how engine pro­tein arrays can sup­port frameshift bio­mark­er dis­cov­ery.

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What are Frameshift Mutations?

Frameshift muta­tions are inser­tions or dele­tions of any num­ber of nuc­le­otides not divis­ible by three. This is due to the fact that nuc­le­otides are read and trans­lated to an amino acid in triplets. These ‘indels’ in the genet­ic code have been linked to a diverse array of dis­eases. Giv­en they dir­ectly cause, the pro­duc­tion of incor­rect pro­teins as well as trun­cated or abnor­mally long pro­teins, their key role in many patho­gen­ic mech­an­isms is unsur­pris­ing.

Frameshift Mutations in Cancer

Mutations in various Cancer Types

Examples of frameshift dis­cov­er­ies in can­cer lead­ing to improved patient care, new treat­ments or great­er under­stand­ing of a dis­ease mech­an­ism are numer­ous. For instance, stud­ies on lung can­cer, melan­oma and ren­al cell car­cinoma described an elev­ated num­ber of frameshift muta­tions to be a very sig­ni­fic­ant indic­at­or of immune check­point inhib­it­or treat­ment response (Chae et al., 2019; Hanna et al., 2018).

Immonogenic Frameshift Peptides

Fur­ther­more, mis­match-repair defi­cient tumors have been found to com­monly har­bor frameshift muta­tions. This can­cer type is marked by vari­ous muta­tions of DNA mis­match repair genes. The induced loss of DNA repair func­tion­al­ity leads to an increase of muta­tions with­in so-called micro-satel­lite regions (often con­sist­ing of repet­it­ive gene sequences). This, in turn, leads to fre­quent frame shift­ing. A recent study showed that tran­scrip­tion errors and exon mis-spli­cing could also lead to the pro­duc­tion of immun­o­gen­ic frameshift pep­tides (Shen et al., 2019). One can expect sev­er­al dis­cov­er­ies of these types of frameshift pep­tides in the future, as they are invest­ig­ated more extens­ively. Pre­sum­ably, some con­di­tions that were pre­vi­ously thought of as unre­lated to frameshift pep­tides may be uncovered as being asso­ci­ated with these. So, it is a great advant­age to include such pep­tides in screen­ing arrays, just as in our engine pro­tein array hEXse­lect.

Neopeptides as reactive Antigens

In a study by Schwit­alle et al. it was found that mis­match-repair defi­cient can­cer patients (MRI‑H can­cers) fre­quently dis­play a high dens­ity of tumor infilt­rat­ing lymph­o­cytes (Sæter­dal et al., 2001). This sup­ports the the­ory that frameshift-neopeptides act as highly react­ive neoanti­gens and evoke a tumor-spe­cif­ic immune response (Schwit­alle et al., 2008). The immune response mark­ers could also be detec­ted in peri­pher­al blood. This facil­it­ates the invest­ig­a­tion of this pro­cess using pro­tein arrays screen­ings. This inher­ent immun­o­gen­i­city of tumor-asso­ci­ated frameshift neopeptides can be exploited to enhance an anti-tumor immune response in patients.

Frameshift mutations as therapeutic input

The great poten­tial of such an approach is high­lighted by the advent of sev­er­al highly effect­ive per­son­al­ized can­cer vac­cines.  In a recent study by Zhang, Shen and John­ston (2019), a pep­tide array plat­form was used for identi­fy­ing tumor frameshift neoanti­gens for the pur­pose of devel­op­ing such per­son­al­ized can­cer vac­cines. They could detect both com­mon react­ive frameshift pro­teins and can­cer-spe­cif­ic immune responses. Using such arrays greatly facil­it­ates the pro­cess of screen­ing for frameshift pep­tides and determ­in­ing what role they play in dif­fer­ent patient groups.

The Power of Protein Arrays in Frameshift Research

Usu­ally, dis­cov­er­ing frameshift muta­tions is a very time-con­sum­ing, labor­i­ous task. An extens­ive DNA sequence ana­lys­is of tumor samples is required in tan­dem with RNA expres­sion veri­fic­a­tion. After­wards, one has to extract and puri­fy can­did­ate pro­teins from anim­al mod­els or patients for fur­ther test­ing. And even if a frameshift muta­tion was dis­covered, one must still uncov­er wheth­er it is dis­ease-rel­ev­ant or not.
If the goal is to identi­fy a frameshift neoep­itope, one also has to employ com­pu­ta­tion­al meth­ods to fil­ter all can­did­ate neoep­itopes accord­ing to pre­dicted immun­o­gen­i­city. This pro­cess takes up to three months, is very work-intens­ive and requires the expert­ise of spe­cial­ists (Lee, K. L., Schlom, J., & Hamilton, D. H., 2020; Zhang, J., Shen, L., & John­ston, S. A., 2018).

The use of pro­tein arrays for dis­cov­er­ing frameshift pep­tides facil­it­ates this pro­cess immensely. Screen­ing can now be con­duc­ted effi­ciently, and research­ers have a great­er chance of dis­cov­er­ing dis­ease-rel­ev­ant frameshift pep­tides.

engine hEXselect Protein Array for your Frameshift Research

Our hEXse­lect pro­tein arrays con­tain mul­tiple frameshift pro­teins and are thus a power­ful tool in address­ing related research ques­tions.
engine pro­tein arrays can be util­ized for e.g.

  • com­pil­ing anti­body pro­files of dif­fer­ent MRI‑H can­cer sub­types (e.g. HNPCC vs. sporad­ic CRC),
  • inter­ac­tion site determ­in­a­tion for new frameshift tar­get­ing drugs and anti­body-based drugs
  • immune response pro­fil­ing of patients express­ing dif­fer­ent frameshift products
  • dis­cov­ery of inter­ac­tion net­work and degrad­a­tion path­ways of frameshift pro­teins

engine Service for your Frameshift Marker

Take advant­age of our ser­vice pack­age, and we will find your out of frame mark­er for you! Upon send­ing in your samples, you will receive your res­ults with­in only two weeks (e.g. for 6 samples) – no work on your behalf is required. You will bene­fit from many advant­ages, such as:

  • a team of experts at your dis­pos­al
  • all sample matrices can be used
  • design of your sub-arrays to boost effi­ciency
  • detailed report of in-house ana­lys­is and raw data
  • eas­ily expan­ded to accom­mod­ate for lar­ger sample sizes
  • reli­able, repro­du­cible and highly effi­cient

Find out more details about our Bio­mark­er Test­ing Ser­vice.


  • Chae, Y. K., Viveir­os, P., Lopes, G., Sukhadia, B., Sheikh, M. M., Sara­via, D., Flor­ou, V., Sokol, E. S., Framp­ton, G. M., Chalmers, Z. R., Ali, S. M., Ross, J. S., Chang, S., Wang, S., Chiec, L., Rah­bari, A., Mohindra, N., Vil­la­flor, V., Shin, S. H., … Park, W. (2019). Clin­ic­al and Immun­o­lo­gic­al Implic­a­tions of Frameshift Muta­tions in Lung Can­cer. Journ­al of Thoracic Onco­logy, 14(10), 1807–1817.
  • Shen, L., Zhang, J., Lee, H. et al. RNA Tran­scrip­tion and Spli­cing Errors as a Source of Can­cer Frameshift Neoanti­gens for Vac­cines. Sci Rep 9, 14184 (2019).–50738‑4
  • Sæter­dal, I., Bjørheim, J., Lislerud, K., Gjertsen, M. K., Buk­holm, I. K., Olsen, O. C., Nes­land, J. M., Eriksen, J. A., Møller, M., Lind­blom, A., & Gaud­er­nack, G. (2001). Frameshift-muta­tion-derived pep­tides as tumor-spe­cif­ic anti­gens in inher­ited and spon­tan­eous colorectal can­cer. Pro­ceed­ings of the Nation­al Academy of Sci­ences of the United States of Amer­ica, 98(23), 13255–13260.
  • Schwit­alle Y, Kloor M, Eier­mann S, Lin­nebach­er M, Kienle P, Knaebel HP, Tariver­d­i­an M, Ben­ner A, von Knebel Doe­ber­itz M. Immune response against frameshift-induced neopeptides in HNPCC patients and healthy HNPCC muta­tion car­ri­ers. Gast­roen­ter­o­logy. 2008 Apr;134(4):988–97.
  • Zhang, J., Shen, L. & John­ston, S.A. Using Frameshift Pep­tide Arrays for Can­cer Neo-Anti­gens Screen­ing. Sci Rep8, 17366 (2018).–35673‑0
  • Lee KL, Schlom J, Hamilton DH. Com­bin­a­tion ther­apies util­iz­ing neoep­itope-tar­geted vac­cines. Can­cer Immun­ol Immun­oth­er. 2020 Oct 8.–02729‑y
  • Hanna, G. J., Lizotte, P., Cavanaugh, M., Kuo, F. C., Shiv­dasani, P., Frieden, A., Chau, N. G., Schoen­feld, J. D., Lorch, J. H., Uppaluri, R., Mac­Co­n­aill, L. E., & Had­dad, R. I. (2018). Frameshift events pre­dict anti-PD‑1/L1 response in head and neck can­cer. JCI Insight, 3(4).

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