Lymphly: Interpretable Genetic Classifier for DLBCL

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Background

DLBCL is a genetically and clinically heterogeneous disease. Lymphly is a transparent, evidence-based classifier developed to enhance biological resolution and improve patient stratification for clinical trials and treatment selection. Built on recent genomic insights, Lymphly complements existing approaches such as LymphGen and DLBclass, offering a flexible framework that integrates core mutations, CNAs, and translocations.

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Key Features

• Nine-step hierarchical algorithm based on core and extended genomic features
• Classification into six subtypes: EZB, MCD, BN2, N1, JS3, JS6
• Additional flags for high-risk cases: TP53+, A+, MYC+ statuses
• Integrated biological knowledge: derived from over 300 publications and 850 genomic events
• Handles genetically composite cases
• Compatible with whole-exome sequencing (WES) input, whole-genome sequencing (WGS), targeted panels, and circulating tumor DNA (ctDNA)

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Subtype Descriptions

Subtype	Key Features	Biological Interpretation
EZB	BCL2, EZH2 mutations	GCB-like, epigenetic dysregulation
MCD	MYD88, CD79B mutations	ABC-like, BCR/TLR activation
BN2	BCL6, NOTCH2 mutations	NF-κB activation, NOTCH signaling
N1	NOTCH1 mutations	Poor prognosis
JS3	STAT3, IL6/IL10 loops	ABC-like, PI3K activation, BCR-resistance
JS6	STAT6, PDL1 amplification	GCB-like/PMBL-like, immune evasion

Additionally, TP53+, A+, and MYC+ samples are flagged due to their prognostic and therapeutic significance.

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Repository Contents

• images/: Visual assets and figures used in documentation and the README, including algorithm diagram and subtype classification schema
• test_data/: Example input/output files
• License.md: Legal terms for using, modifying, and distributing Lymphly
• Lymphly Notice file.docx: Legal notice listing third-party Python libraries used in Lymphly, including license and attribution information.
• Lymphly_feature_table.tsv: The table defines the complete list of features used by Lymphly to assign molecular subtypes to DLBCL samples. Each row corresponds to a biologically relevant event (mutation, CNA, or translocation).
• Lymphly_Classifier.ipynb: Python notebook for subtype classification based on mutation, CNA, and translocation data
• lymphly.py: Core module with all functions used for DLBCL subtype classification, including feature processing, subtype assignment, and utilities
• make_venv.sh: Script for setting up a Python virtual environment and Jupyter kernel for Lymphly
• requirements.txt: contains a list of all Python packages required to run the Lymphly classifier and reproduce the analysis

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Installation

Before installing, make sure you have the following installed on your system:

• Python ≥ 3.10 (Python 3.10.11 recommended)

• pip — Python package manager

• venv — module for creating virtual environments (included in standard Python)

• Jupyter — expected to be available for running the Lymphly notebook

To install Lymphly locally:

git clone
cd Lymphly
chmod +x make_venv.sh
./make_venv.sh

This will create a virtual environment named Lymphly in the current directory and install all required dependencies.

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Environment

• Python 3.10.11 recommended

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Input File Specifications

mutation.maf — Somatic Mutation Annotation

Tab-delimited text file in MAF (Mutation Annotation Format) style, containing somatic mutation data for all samples. The classifier is designed to work primarily with mutation data aligned to the GRCh38 (hg38) human genome assembly.

• However, it can also process data aligned to the older GRCh37 (hg19) assembly without issues.
• The classifier handles coordinates and annotations transparently, ensuring compatibility with both genome builds.

Required columns:

• Tumor_Sample_Barcode or Sample:
  Sample ID. One of these columns must be present. Will be renamed to Sample.

• Chromosome:
  Chromosome number or identifier. Will be normalized to chrN format.

• Start_Position:
  Genomic start coordinate of the mutation. Required to compute End_Position.

• Reference_Allele:
  Reference allele sequence. Used in variant localization and classification.

• Tumor_Seq_Allele2 or Tumor_Seq_Allele1:
  Observed alternative allele. Will be standardized to Tumor_Seq_Allele2.

• Hugo_Symbol:
  HGNC gene symbol.

• Variant_Classification:
  Mutation effect (e.g., Missense_Mutation, Nonsense_Mutation).

  The Variant_Classification column in mutation.maf must contain one of the supported values listed below. These are grouped internally into functional categories:

  • MISSENSE: includes Missense_Mutation, Intron, 3'UTR, and 5'UTR.
    These represent coding and non-coding mutations that are often retained for exploratory analysis or because they are subtype-relevant.

  • NONSENSE: includes only Nonsense_Mutation.
    This indicates mutations that introduce a premature stop codon, likely truncating the protein.

  • FRAME_SHIFT: includes Frame_Shift_Ins, Frame_Shift_Del, and Splice_Site.
    These are disruptive mutations, including frameshifts and splicing errors, that can significantly alter protein function.

  Any Variant_Classification not listed above will be ignored during subtype classification.

Optional columns:

• End_Position:
  If not provided, it will be computed as:
  End_Position = Start_Position + len(Reference_Allele) - 1

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cna-gene.tsv — Copy Number Alteration Gene Matrix

Tab-separated file where rows are genes and columns are samples.

Each cell contains an integer copy number call, representing the relative copy number status of that gene compared to the sample’s baseline ploidy.

Expected values (example schema):

• 0 — diploid (normal copy number, relative to the sample’s ploidy)

• 1 or 2 — gain or amplification (increased copy number relative to ploidy)

• -1 or -2 — loss or deletion (reduced copy number relative to ploidy)

This matrix is used to identify subtype-defining copy number alterations in specific genes.

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Note: Negative values (-1, -2) represent copy number loss or deletion relative to the estimated ploidy of the sample. For example, if a sample has a baseline ploidy of 3, a value of -1 may indicate a reduction to 2 copies in that region.

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CNA locus deduplication

Some CNA features correspond to multiple genes located within the same recurrent genomic locus and therefore reflect a single underlying copy number event rather than independent alterations. To avoid double-counting, the following gene groups are collapsed and counted as one CNA hit:

Locus	Genes
10q23	PTEN, FAS
18q21	BCL2, MALT1
9p24.1	JAK2, CD274 (PD-L1), PDCD1LG2 (PD-L2)
20q11	ASXL1, DNMT3B

If any gene in a group shows a CNA, the entire locus contributes a single CNA event to the subtype scoring.
This rule is applied only at the feature-scoring stage; the underlying cna-arm.tsv matrix is not modified.

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cna-arm.tsv — Copy Number Alteration Arm Matrix

Tab-separated file where rows represent chromosome arms and columns represent samples.

Each cell contains an integer copy number call, indicating the relative copy number status of a chromosome arm compared to the sample’s ploidy.

The ploidy is not fixed (e.g., typically 2) but is calculated specifically for each sample. It is typically determined using the weighted average of copy numbers across the genome:

• For every segment in the sample, multiply its Total Copy Number by the segment's length.

• Sum these values and divide by the total length of the genome.

• The result is rounded to the nearest integer to define the sample's ploidy used in the scoring function.

The cna-arm.tsv file is required to compute the arm-level aneuploidy status A+/–. If this file is not provided, the A+/– status cannot be calculated, and any downstream analyses depending on this metric will be unavailable.

For each sample, any non-zero deviation from the baseline ploidy contributes +1 to the aneuploidy score. Thus, the total A+/– score equals the number of chromosome arms whose copy number differs from the sample’s ploidy

A sample is considered A+ if its score is > 14

A sample is considered A− if its score is ≤ 14

This functionality is available for any cohort that includes arm-level CNA calls

Expected values (example schema):

• 0 — Neutral: The arm's copy number matches the sample's ploidy.

• -1 — Loss (Hemizygous Deletion): The arm is missing one copy relative to the ploidy. In a diploid sample (Ploidy ≈ 2), a Total Copy Number of 1 is a Loss (-1). In a tetraploid sample (Ploidy ≈ 4), a Total Copy Number of 3 is a Loss (-1).

• -2 — Deletion (Homozygous/Deep Deletion): The arm is missing two copies relative to the sample's ploidy.

• 1 — Gain: The arm has one extra copy compared to the ploidy.

• 2 — Amplification: The arm has two or more extra copies (high-level amplification).

This matrix is used to compute broad aneuploidy metrics, identify recurrent arm-level CNAs, and support subtype classification driven by chromosomal gains and losses.

Allowed chromosome arm names

Chromosome arms must follow the format:

{1–24}{p/q}

where:

• 1–22 represent autosomes

• 23 represents chromosome X

• 24 represents chromosome Y

Examples of valid names: 1p, 1q, 7p, 19q, 22p, 23q, 24p.

Invalid chromosome arm names

The following are not allowed:

• Literal X or Y arm names (Xp, Xq, Yp, Yq)

• Chromosome numbers without an arm (1, 7, 23, 24)

• Cytobands (1p36, 8q24)

• Any non-numeric or non-arm identifiers (e.g., contigs, scaffolds, or mitochondrial labels)

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Note: Negative values (-1, -2) represent copy number loss or deletion relative to the estimated ploidy of the sample. For example, if a sample has a baseline ploidy of 3, a value of -1 may indicate a reduction to 2 copies in that region.

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annotation.tsv — Annotation file

The sample annotation file is a tab-separated text file containing exactly four columns:

Required Column

• Sample
• BCL2
• BCL6
• MYC

This file serves two purposes:

Defines the list of samples to be included in the classification. Only samples listed in this file will be processed.

Provides translocation status for the genes BCL2, BCL6, and MYC.

Values indicating presence of the translocation should be:

• True, TRUE, true, or the string "True"
  Any other values are ignored (treated as no translocation).

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Note: Each row corresponds to a single sample. The Sample column must exactly match sample IDs used elsewhere in the analysis (e.g. in mutation.maf or cna-gene.tsv).

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Usage

Run the main notebook using Jupyter:

source Lymphly/bin/activate
jupyter notebook Lymphly_Classifier.ipynb

Important: Make sure the Lymphly environment is selected in the Jupyter Notebook interface

Provide input files with mutation, CNA, and translocation information for your DLBCL samples. The notebook will classify each sample and annotate subtype and risk features (TP53+, A+, MYC+).

Input file paths and parameters can be specified in the notebook via the cohorts_settings dictionary, for example:

cohorts_settings = {
    'path_to_maf': 'test_data/mutations.maf',
    'path_to_cna_gene': 'test_data/cna-gene.tsv',
    'path_to_cna_arm': 'test_data/cna-arm.tsv',
    'path_to_annotation': 'test_data/annotation.tsv',
    'name_to_save': 'test_data/Lymphly_test.tsv',
    'ref': 'HG38'
}

The fields path_to_maf, path_to_cna_gene, 'path_to_cna_arm', and path_to_annotation should contain paths to input files formatted as described in the Input File Specifications section.
name_to_save is the output path for the resulting classifier file.
ref specifies the reference genome used in the input MAF file (either HG38 or HG19).

Set additional parameters to control classification behavior:

• USE_TRANSLOCATIONS
• USE_CNA
• USE_STATUSES

Modify these parameters as needed to enable or disable usage of translocations, copy number alterations, and status flags (TP53+, A+, and MYC+) during classification.

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Performance

96% classification rate across internal and public datasets (n=840)

Subtypes correlate with survival outcomes and known biology

Detects cases missed by LymphGen and DLBclass

Highlights novel high-risk and immune-evasion subtypes (JS3, JS6)

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Contact

For questions or collaboration inquiries, please contact: Pavel Zemskiy – pavel.zemskiy@bostongene.com

Licencse

BY UTILIZING THE CODE, YOU ARE CONSENTING TO BE AND AGREE TO BE BOUND BY ALL OF THE TERMS OF THIS LIMITED LICENSE, SEE "License.md" FILE