In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13 perivascular and CXCL12LTB and PD-L1 epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Furthermore, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar, but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are coadministered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naïve LSCC patients treated with corticosteroids before surgery, compared with untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment. Corticosteroid treatment during chemotherapy negatively affects the development of tertiary lymphoid structures and abrogates their prognostic value in patients with lung cancer. .

Antibody-drug conjugates (ADCs) represent a promising class of biopharmaceuticals with the potential to localize at the tumor site and improve the therapeutic index of cytotoxic drugs. While it is generally believed that ADCs need to be internalized into tumor cells in order to display optimal therapeutic activity, it has recently been shown that non-internalizing antibodies can efficiently liberate disulfide-linked drugs at the extracellular tumor site, leading to potent anti-cancer activity in preclinical animal models. Here, we show that engineered variants of the F16 antibody, specific to a splice isoform of tenascin-C, selectively localize to the subendothelial tumor extracellular matrix in three mouse models of human cancer (U87, A431, MDA-MB-231). A site-specific coupling of F16 in IgG format with a monomethyl auristatin E (MMAE) derivative, featuring a valine-citrulline dipeptide linker equipped with a self-immolative spacer, yielded an ADC product, which cured tumor-bearing mice at a dose of 7 mg/Kg. The observation of an efficient extracellular proteolytic cleavage of the valine-citrulline linker was surprising, as it has generally been assumed that this peptidic structure would be selectively cleaved by cathepsin B in intracellular compartments. The products described in this article may be useful for the treatment of human malignancies, as their cognate antigen is strongly expressed in the majority of human solid tumors, lymphomas and aggressive leukemias, while being virtually undetectable in most normal adult tissues.

Assessment of tumor response after chemotherapy using 18F-FDG PET metrics is gaining acceptance. Several studies have suggested that the parameters metabolically active tumor volume (MTV) and total lesion glycolysis (TLG) are superior to SUVmax for measuring tumor burden. However, the measurement of MTV and TLG is still controversial; the most common method uses an absolute threshold of 42% of SUVmax. Recently, we implemented a background-adaptive method to determine the background-subtracted lesion activity (BSL) and the background-subtracted volume (BSV). In this study, we investigated the correlation between such PET metrics and histopathologic response in non–small cell lung carcinoma (NSCLC). Methods Forty-four NSCLC patients were retrospectively identified. Their PET/CT data on both types of scan before and after neoadjuvant chemotherapy were analyzed regarding SUVmax, MTV, TLG, BSL, and BSV, as well as the relative changes in these parameters. The tumor regression score as an indicator of histopathologic response was scored on hematoxylin- and eosin-stained sections of the surgical specimens using a 4-tiered scale (scores 1–4). The correlation between score and the absolute and relative PET metrics after chemotherapy was analyzed using Spearman rank correlation tests. Results Tumors that demonstrated a good response after neoadjuvant chemotherapy had significantly lower 18F-FDG activity than non-responding tumors (scores 3 and 4: SUVmax, 4.2 [range, 1.8–7.9] vs. scores 1 and 2: SUVmax, 8.1 [range, 1.4–40.4]; P = 0.001). The same was found for change in SUVmax and score (P = 0.001). PET volume metrics based on a 42% fixed threshold for SUVmax did not correlate with score (TLG, P = 0.505; MTV, P = 0.386). However, both background activity–based PET volume metrics—BSL and BSV—significantly correlated with score (P < 0.001 each). Conclusion PET volume metrics based on background-adaptive methods correlate better with histopathologic score in NSCLC patients under neoadjuvant chemotherapy than algorithms and methods using a fixed threshold (42% SUVmax).

Recent technological advances allow profiling of tumor samples to an unparalleled level with respect to molecular and spatial composition as well as treatment response. We describe a prospective, observational clinical study performed within the Tumor Profiler (TuPro) Consortium that aims to show the extent to which such comprehensive information leads to advanced mechanistic insights of a patient's tumor, enables prognostic and predictive biomarker discovery, and has the potential to support clinical decision making. For this study of melanoma, ovarian carcinoma, and acute myeloid leukemia tumors, in addition to the emerging standard diagnostic approaches of targeted NGS panel sequencing and digital pathology, we perform extensive characterization using the following exploratory technologies: single-cell genomics and transcriptomics, proteotyping, CyTOF, imaging CyTOF, pharmacoscopy, and 4i drug response profiling (4i DRP). In this work, we outline the aims of the TuPro study and present preliminary results on the feasibility of using these technologies in clinical practice showcasing the power of an integrative multi-modal and functional approach for understanding a tumor's underlying biology and for clinical decision support.

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