Exploring Biochemical Characteristics of Pediatric Hyperdiploid Acute Lymphoblastic Leukemia by Raman Spectroscopy

Abstract

Hyperdiploid (HD) B-cell acute lymphoblastic leukemia (ALL) is widely recognized as the most common molecular subtype of leukemia, characterized by the presence of supernumerary chromosomes in the leukemic karyotype. While HD B-ALL is often associated with a favorable prognosis, an important subset of patients still experience relapse, reflecting the biological heterogeneity of this subtype. Current genomic and epigenetic research has shed light on the molecular complexity of HD B-ALL, yet rapid methods for capturing both the metabolic state and the chromosomal content of individual cells remain limited. Here, we introduce a novel Raman spectroscopy (RS)-based approach for the single-cell analysis of HD B-ALL. By detecting characteristic spectroscopic signatures of nucleic acids, proteins, and lipids, RS not only distinguishes malignant cells from normal B cells, but also discriminates between HD B-ALL and other molecular subtypes, including TCF3-PBX1, KMT2A-r, BCR-ABL1, and TEL-AML1. Notably, we developed a partial least-squares regression (PLS-R) model capable of accurately predicting chromosome number from each cell's Raman spectrum, thereby linking molecular fingerprints directly to genomic aberrations. This integrative spectroscopic strategy captures disease heterogeneity and informs therapeutic strategies. Taken together, our proof-of-concept findings highlight RS as a powerful, noninvasive tool for quantifying chromosomal alterations and metabolic phenotypes, adding crucial insights into the complex biology of HD B-ALL and paving the way for broader applications in precision medicine.

1

Comparison of the spectra of normal B cells (purple, n _p = 8, n _s = 397) with HD B-ALL lymphoblasts (aqua, n _p = 16, n _s = 381). (a) Score plot of PCA along the first two PCs (PC-1 and PC-2). (b) Loading plots of PC-1 and PC-2 are presented in a color scale. Only bands for which the PC-1 and PC-2 had the highest values (PC-1: >0.05 and <−0.05, PC-2: >0.07 and <−0.07) were included. The PC-2 loading values were multiplied by (−1) to maintain the color scale. PCA analysis was performed in the spectral range of 1800–600 cm^–1.

2

Comparison of spectra of HD lymphoblasts (marked in aqua) and a mixture of other subtypes of B-ALL studied: TCF3-PBX1 (marked in light pink, n _p = 12, n _s = 175), KMT2A-r (dark pink, n _p = 12, n _s = 176), BCR-ABL1 (navy, n _p = 11, n _s = 139), ZNF384 (blue, n _p = 7, n _s = 96), and TEL-AML1 (light blue, n _p = 13, n _s = 162). (a) Score plot of principal component factors PC-2 and PC-3. (b) Loading plot for the PC-2 component presented on a color scale. Only bands for which the PC-2 loading had the highest values (greater than 0.06 and less than −0.06) were included. PCA analysis was performed in the spectral range of 1800–600 cm^–1. (c) Hierarchical cluster analysis of the average spectra of the studied subtypes.

3

O-PLS-R model that examines the relationship between the Raman signal and the number of chromosomes in clinical samples calculated on the whole-cell spectra. (a) Score plot of latent variables LV-1 and LV-2 for the training data set. In total, six LVs were used. (b) Model calibration result. (c) Score plot of the latent variables LV-1 and LV-2 for the test data set, which was not included in model training. (d) Prediction results of the model in test samples. (e) Plot of the regression vector of the model is presented on a color scale. Only bands for which the variable importance in projection (VIP) scores had the highest values (>1) were included. (f) Plot of the LV-1 loading of the model is presented on a color scale. Only bands for which the LV-1 loading had the highest values (greater than 0.06 and less than −0.06) were included. O-PLS-R analysis was performed in the spectral range of 1800–600 cm^–1. (g) Graphical representation of the integral intensity ratios of selected characteristic bands of whole-cell spectra. The samples were colored according to the number of chromosomes designated according to panel legend (a).