Comparative Lesions Analysis Through a Targeted Sequencing Approach

Our protocol allows the capture of the genetic relatedness between primary and metastatic lesions through the identification of molecular alterations involved in disease progression. Our method provides the opportunity to explore genetic relatedness in clinical specimen with high sensitivity and specificity. This is due to the integration of molecular and histopathological analysis.

The assessment of intratumor heterogeneity in the cancer research field is of paramount importance in designing effective anti-tumor strategies and our method is broadly applicable to many solid tumor types. After preparing and quantifying the libraries of interest, dilute each library to a 100 picomolar concentration in nuclease-free water and combine 10 microliters of each diluted library for a single run in a single tube. Then mix the pooled libraries by pipetting.

To initiate a sequencing run, first open the Torrent browser on a computer connected to the sequencing system. Plan a new run using the generic template for the selected application and under the kits tab select Ion Proton system or Ion Gene Studio S5 system. Select the appropriate chip from the chip type dropdown menu and select Ion AmpliSeq 2.0 Library Kit from the library kit dropdown list.

Select the Ion Chef button for the template kit and select the appropriate Chef Kit from the template kit dropdown list. Select the appropriate sequencing kit from the sequencing kit dropdown menu and select Ion Express from the barcode set dropdown list. Under the plugin tab, select the coverage analysis plugin.

Under the plan tab, select the GRCH37HG19 genome from the reference library dropdown list. From the target regions dropdown list, select the appropriate panel to be sequenced. Then set the number of barcodes to be sequenced and set the barcode name and sample name for each library.

For a pooled libraries dilution, dilute the stock library with nuclease-free water to a 25 picomolar concentration for an ion proton chip and pipette 50 microliters of each diluted library to the bottom of the appropriate library sample tube. Load sample tubes containing the pooled diluted libraries onto the library preparation system and start the clonal amplification. For next generation sequencing, follow the on-screen instructions to initialize the instrument.

When the clonal amplification is finished, open the Ion Chef instrument door, then open the lid of the chip loading centrifuge and remove the two chips from the library preparation system. Place the chips into separate chip storage containers and load one of the chips into the chip compartment in the instrument. Then close the chip compartment lid and start the sequencing.

For mutation analysis, open the coverage analysis plugin and use the coverage analysis output to verify the depth of coverage and uniformity. Use the Torrent Variant Caller plugin to perform the variant calling selecting the germ line or somatic workflow as appropriate. Then download filtered variants variant call format files and use the variant effect predictor software in the NCBI RefSeq database to annotate the variants.

To estimate the copy-number variations, use the ion reporter uploader plugin to load the sequencing data into the ion reporter software and use the comprehensive cancer panel tumor normal pair workflow to analyze the data. Manually filter the mutations and copy-number variations based on the scores assigned by the software and verify the variations visually with the Integrative Genomics Viewer. Then visually inspect the alignment for unusual reads that may generate artifactual calls and inspect the normalized coverage for all of the amplicons across a given gene against the normalized coverage of the baseline to verify the copy-number variations.

For each case, index the mutation calls from the sequencing of normal tissue or blood or primary tumor or metastasis. Flag as germ line and discard calls that are also evident from the sequencing data of germ line DNA. Flag as clonal or founder the mutations that are shared among all lesions of a given patient.

Then flag as subclonal or progressor the mutations that are detected in some but not all of the lesions of a given patient. In this representative experiment, multi-lesion sequencing of five solid pseudopapillary neoplasm cases targeting the coding sequences of 409 cancer-related genes identified a total of 27 somatic mutations in eight genes. Mutations were defined as founder or clonal when shared among all of the lesions of a given patient and progressor or subclonal when detected in some but not all of the lesions of a given patient.

Consistently, immunohistochemical staining for beta-catenin and KMD6A was homogenous among the diverse lesions of cases with mutations of the corresponding genes. The moderate staining for KMD6A in mutated specimens suggest that genetic alteration is likely to alter the function rather than the expression of the protein. KMD6A loss of function in pancreatic tumors is associated with up regulation of the hypoxia marker GLUT-1.

And accordingly, GLUT-1 was over expressed in cases bearing KMD6A mutations. Immunohistochemistry for BAP1 and TP53 confirmed that mutations in those genes were subclonal. In this virtual karyotype of a representative case showing the location, proximity, and copy-number status of altered genes, the copy-number variation analysis using sequencing data revealed alterations in all of the specimens analyzed.

And in contrast to point mutations, the majority of copy-number variation alterations were subclonal. Validation and indexing of mutations and copy-number variation and using the tumor normal DNA comparison are important for avoiding the generation of artifactual calls that could invalidate the results. This procedure could also be applied to whole genome sequencing studies aimed at interrogating the entire genome for the identification of mutation between lesion of different solid tumor types.

In the cancer research field, these techniques are critical tools for understanding the biology of diseases with important clinical implications aimed at designing personalized and effective targeted therapies.