Ajit Johnson Nirmal

• Doctor of Philosophy
• Professor (Assistant) at Harvard University

Computational pathology foundation models (CPathFMs) have emerged as a powerful approach for analyzing histopathological data, leveraging self-supervised learning to extract robust feature representations from unlabeled whole-slide images. These models, categorized into uni-modal and multi-modal frameworks, have demonstrated promise in automating c...

Background A subset of melanoma patients treated with immune checkpoint blockade(ICB) have long-term radiographic persistence of disease despite durable progression-free survival, termed ‘long-term stable disease’. Our recent study of seven patients with long-term stable disease after ICB showed no viable melanoma cells in biopsies of residual lesi...

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identifie...

PI3K-δ inhibitors have shown impressive activity in lymphoid malignancies but have been hampered by autoimmune and infectious toxicities, leading to market withdrawals. We previously demonstrated activity of the PI3K-δγ inhibitor duvelisib in T cell lymphomas (TCLs) that was associated with inflammatory adverse events. As reported here, we conducte...

Multiplexed imaging data are revolutionizing our understanding of the composition and organization of tissues and tumors. A critical aspect of such tissue profiling is quantifying the spatial relationship relationships among cells at different scales from the interaction of neighboring cells to recurrent communities of cells of multiple types. This...

Cancer is a complex cellular ecosystem where malignant cells coexist and interact with immune, stromal and other cells within the tumor microenvironment (TME). Recent technological advancements in spatially resolved multiplexed imaging at single-cell resolution have led to the generation of large-scale and high-dimensional datasets from biological...

Primary cutaneous melanomas develop in stages from melanoma precursor cells that may progress to melanoma in situ (MIS) and eventually to invasive disease. Molecular changes, such as tumor cell state switches from melanocytic to undifferentiated and neural-crest-like phenotypes, have been associated with the development of melanoma metastases and t...

To uncover the intricate, chemotherapy-induced spatiotemporal remodeling of the tumor microenvironment, we conducted integrative spatial and molecular characterization of 97 high-grade serous ovarian cancer (HGSC) samples collected before and after chemotherapy. Using single-cell and spatial analyses, we identify increasingly versatile immune cell...

Our goal was to characterize the effects of chemotherapy on the tumor microenvironment of high-grade serous ovarian cancer (HGSC) and suggest effective combination treatment strategies. We employed comprehensive multiomic spatial and single-cell profiling techniques, including tissue cyclic immunofluorescence t-CycIF, scRNA sequencing, and spatial...

Highly multiplexed tissue imaging and in situ spatial profiling aim to extract single-cell data from specimens containing closely packed cells of diverse morphology. This is challenging due to the difficulty of accurately assigning boundaries between cells (segmentation) and then generating per-cell staining intensities. Existing methods use gating...

Homeostasis of normal tissues and the emergence of diseases such as cancer are controlled by changes in the proportions and states of diverse cell types, cell-cell interactions, and acellular components of the tissue microenvironment. Spatial omics using highly multiplexed tissue profiling makes it possible to study these processes in situ, usually...

Background: Cancer is a complex cellular ecosystem where malignant cells coexist and interact with immune, stromal, and other cells within the tumor microenvironment. Recent technological advancements in spatially resolved multiplexed imaging at single-cell resolution have led to the generation of large-scale and high-dimensional datasets from biol...

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are es...

In this study, we demonstrate the utility of whole-slide CyCIF (tissue-based cyclic immunofluorescence) imaging for characterizing immune cell infiltrates in immune checkpoint inhibitor (ICI)-induced dermatologic adverse events (dAEs). We analyzed six cases of ICI-induced dAEs, including lichenoid, bullous pemphigoid, psoriasis, and eczematous erup...

Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas. Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending...

Cancer immunotherapies that activate cytotoxic T cell responses against tumors have been remarkably effective in some patients, but the determinants of response remain unclear. To investigate how spatial organization of T cells might influence immunotherapy response, we developed a tissue-based cyclic immunofluorescence (t-CyCIF) platform to charac...

Tumor-infiltrating lymphocyte (TIL) density has been identified as a prognostic and predictive biomarker in select tumors treated with defined therapies. These observations suggest that TILs may be general markers of patient outcomes, but evidence in support of this hypothesis has been limited by small cohorts. We validated ImmunoPROFILE, a multipl...

Neoadjuvant Platinum-based combination chemotherapy (NACT) is commonly offered to patients with high-grade serous ovarian cancer (HGSC) harboring unresectable disease in the primary setting. In order to develop effective immunotherapeutic and combinatorial treatment strategies for HGSC, a deeper knowledge on the effects of NACT on the spatial tumor...

Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras/Trp53-mutant mouse...

In cancer, complex ecosystems of interacting cell types play fundamental roles in tumor development, progression, and response to therapy. However, the cellular organization, community structure, and spatially defined microenvironments of human tumors remain poorly understood. With the emergence of new technologies for high-throughput spatial profi...

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (...

New highly-multiplexed imaging technologies have enabled the study of tissues in unprecedented detail. These methods are increasingly being applied to understand how cancer cells and immune response change during tumor development, progression, and metastasis, as well as following treatment. Yet, existing analysis approaches focus on investigating...

Highly multiplexed tissue imaging (MTI) is accomplished by applying powerful antibody-based spatial proteomics technologies that characterize tissues in situ at single-cell and potentially subcellular resolution and enable the creation of two-dimensional tissue maps. Example MTI methods include cyclic immunofluorescence (CyCIF), multiplex immunohis...

Lymphocytes play a key role in immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. Here, we used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of tumor-bearing lung tissues fro...

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (...

Cutaneous melanoma is a highly immunogenic malignancy, surgically curable at early stages, but life-threatening when metastatic. The spatial organization of the tumor ecosystem during early-stage melanoma is not well understood. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics t...

ThiNew multiplexed tissue imaging technologies have enabled the study of normal and diseased tissues in unprecedented detail. These methods are increasingly being applied to understand how cancer cells and immune response change during tumor development, progression, and metastasis as well as following treatment. Yet, existing analysis approaches f...

Cutaneous melanoma is a highly immunogenic malignancy that is surgically curable at early stages but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially resolved microregion transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular n...

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed...

The imminent release of tissue atlases combining multichannel microscopy with single-cell sequencing and other omics data from normal and diseased specimens creates an urgent need for data and metadata standards to guide data deposition, curation and release. We describe a Minimum Information about Highly Multiplexed Tissue Imaging (MITI) standard...

Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here...

Cutaneous melanoma is a highly immunogenic disease, surgically curable at early stages, but life-threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborh...

Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology. Recent advances in spatial transcriptomics rely on fresh frozen specimens making years of archived tissues samples unusable. Here we describe a method, called Pick-Seq, for spatial transcriptional profiling of formalin fixed histology specimens ba...

Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens...

Crucial transitions in cancer—including tumor initiation, local expansion, metastasis, and therapeutic resistance—involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecede...

Introduction Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is >70. Approximately 5% of ATLL patients in Japan present at age <50. Here, we hypothesized that AT...

Tail amputation by tail docking or as an extreme consequence of tail biting in commercial pig production potentially has serious implications for animal welfare. Tail amputation causes peripheral nerve injury that might be associated with lasting chronic pain. The aim of this study was to investigate the short- and long-term effects of tail amputat...

The immune composition of the tumor microenvironment regulates processes including angiogenesis, metastasis, and the response to drugs or immunotherapy. To facilitate the characterization of the immune component of tumors from transcriptomics data, a number of immune cell transcriptome signatures have been reported that are made up of lists of mark...

Aim of Investigations: The aim of this study was to conduct a transcriptome analysis of gene expression patterns in the caudal dorsal root ganglia of pigs subjected to tail amputation at two different stages of development consistent with tail docking age (neonate) and tail biting outbreaks (juvenile) to develop a better understanding of the short...

The immune response to a given cancer can profoundly influence a tumour’s trajectory and response to treatment, but the ability to analyse this component of the microenvironment is still limited. To this end, a number of immune marker gene signatures have been reported which were designed to enable the profiling of the immune system from transcript...

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could sup...

Signaling via the colony-stimulating factor 1 receptor (CSF1R) controls the survival, differentiation, and proliferation of macrophages. Mutations in CSF1 or CSF1R in mice and rats have pleiotropic effects on postnatal somatic growth. We tested the possible application of pig CSF1-Fc fusion protein as a therapy for low birth weight (LBW) at term, u...

Supplementary figure and table legends

Table S1 Details on the datasets used in this study The subject details, experimental design, data source, and comparisons for all datasets included in this study are listed in Table S1. This includes the three primary data datasets, 22 validation datasets for skin conditions, three psoriasis datasets for integrating the keratinocyte differentiatio...

Table S3 Gene ontology enrichment and example genes for each signature In addition to highlighting a number of key genes and significant gene ontology (GO) enrichment result for each SkinSig, the supplementary file also includes the full set results from gene ontology enrichment.

Table S4 Comparison between SkinSig and genes found to be significantly altered during ageing Genes reported to change in their expression during ageing are compared with SkinSig, showing a high percentage of the sebaceous gland and hair signatures being reported to alter with ageing by Glass et al [19].

Supplementary discussion. Further discussion on other gene clusters of interest and justifications for the assignation of annotation of co‐expression signatures without a significant or relevant gene ontology term

Figure S1 Sample–sample correlation and signal comparisons between the RNA‐seq and microarray datasets. (A) Sample–sample correlation plots of data used in these studies, using the maximum Pearson correlation coefficient threshold that still retained all samples for the RNA‐seq (r ≥ 0.93) and (B) the microarray (r ≥ 0.97) datasets. There is minimal...

Table S2 SkinSig and co‐expression signatures derived from the analysis of different datasets derived from normal human skin The supplementary file includes the co‐expression signatures for all network analysis, including the individual full original datasets, gene‐symbol restricted datasets, and SkinSig.

Figure S2 Pearson correlation thresholds in randomized and original data. (A) When expression values for each gene were randomized across the samples of the RNA‐seq dataset, only 95 pairing (edges) were observed at a threshold used in this analysis (r ≥ 0.73), whilst the untransformed data yielded 87 121 edges. Taking into account that a total of 1...

Numerous studies have explored the altered transcriptional landscape associated with skin diseases to understand the nature of these disorders. However, data interpretation represents a significant challenge due to a lack of good maker sets for many of the specialised cell types that make up this tissue, whose composition may fundamentally alter du...

The outcome of many diseases is commonly correlated with the immune response at the site of pathology. The ability to monitor the status of the immune system in situ provides a mechanistic understanding of disease progression, a prognostic assessment and a guide for therapeutic intervention. Global transcriptomic data can be deconvoluted to provide...

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genom...

Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limit...

Transcriptomics data is a valuable resource for understanding a system’s response to disease or other perturbation. Frequently, pathological states are associated with the recruitment of immune cells to the site of disease and/or changes in the relative numbers or activation state of other cell types. Identifying these alterations are key to the co...

Micronuclei (MN) formation is generally attributed to error in DNA synthesis or mitosis, which are represented by the S or G(2)/M phase respectively, in the cell-cycle histogram. Interestingly, many of the known anticancer drugs target these cell-cycle phases to elicit cytotoxicity. Here, we attempted to identify whether any correlation exists betw...

Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limit...