Severe radiation-induced lymphopenia during concurrent chemoradiotherapy for stage III non-small cell lung cancer: external validation of two prediction models.

Background: Severe radiation-induced lymphopenia (RIL) in patients undergoing chemoradiotherapy (CRT) for non-small cell lung cancer (NSCLC) is associated with decreased immunotherapy efficacy and survival. At The Christie and MD Anderson Cancer Center (MDACC), prediction models for lymphopenia were developed in lung and esophageal cancer patients, respectively. The aim of this study was to externally validate both models in patients with stage III NSCLC. Methods: Patients who underwent concurrent CRT for stage III NSCLC in 2019-2021 were studied. Outcomes were grade ≥3 and grade 4 lymphopenia during CRT. The Christie model predictors for grade ≥3 lymphopenia included age, baseline lymphocyte count, radiotherapy duration, chemotherapy, mean heart and lung doses, and thoracic vertebrae V20Gy. MDACC predictors for grade 4 lymphopenia were age, baseline lymphocyte count, planning target volume (PTV), and BMI. The external performance of both models was assessed. Results: Among 100 patients, 78 patients (78%) developed grade ≥3 lymphopenia, with grade 4 lymphopenia in 17 (17%). For predicting grade ≥3 lymphopenia, the Christie and MDACC models yielded c-statistics of 0.77 and 0.79, respectively. For predicting grade 4 lymphopenia, c-statistics were 0.69 and 0.80, respectively. Calibration for the Christie and MDACC models demonstrated moderate and good agreement, respectively. Conclusion: The PTV-based MDACC prediction model for severe RIL demonstrated superior external performance in NSCLC patients compared to the dosimetry-based Christie model. As such, the MDACC model can aid in identifying patients at high risk for severe lymphopenia. However, to optimize radiotherapy planning, further improvement and external validation of dosimetry-based models is desired.

A DNA damage response-like phenotype defines a third of colon cancers at onset.

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow-up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR-associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T-lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non-senescent, non-apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non-DDR cases. DDR+ MMRd cases were preferentially retaining wild-type MLH1. The outcome after 5FU-based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.

Automated multimodal fluorescence microscopy for hyperplex spatial-proteomics: Coupling microfluidic-based immunofluorescence to high resolution, high sensitivity, three-dimensional analysis of histological slides.

In situ multiplexing analysis and in situ transcriptomics are now providing revolutionary tools to achieve the comprehension of the molecular basis of cancer and to progress towards personalized medicine to fight the disease. The complexity of these tasks requires a continuous interplay among different technologies during all the phases of the experimental procedures. New tools are thus needed and their characterization in terms of performances and limits is mandatory to reach the best resolution and sensitivity. We propose here a new experimental pipeline to obtain an optimized costs-to-benefits ratio thanks to the alternate employment of automated and manual procedures during all the phases of a multiplexing experiment from sample preparation to image collection and analysis. A comparison between ultra-fast and automated immunofluorescence staining and standard staining protocols has been carried out to compare the performances in terms of antigen saturation, background, signal-to-noise ratio and total duration. We then developed specific computational tools to collect data by automated analysis-driven fluorescence microscopy. Computer assisted selection of targeted areas with variable magnification and resolution allows employing confocal microscopy for a 3D high resolution analysis. Spatial resolution and sensitivity were thus maximized in a framework where the amount of stored data and the total requested time for the procedure were optimized and reduced with respect to a standard experimental approach.

Mapping the Immune Landscape in Metastatic Melanoma Reveals Localized Cell-Cell Interactions That Predict Immunotherapy Response.

While immune checkpoint-based immunotherapy (ICI) shows promising clinical results in patients with cancer, only a subset of patients responds favorably. Response to ICI is dictated by complex networks of cellular interactions between malignant and nonmalignant cells. Although insights into the mechanisms that modulate the pivotal antitumoral activity of cytotoxic T cells (Tcy) have recently been gained, much of what has been learned is based on single-cell analyses of dissociated tumor samples, resulting in a lack of critical information about the spatial distribution of relevant cell types. Here, we used multiplexed IHC to spatially characterize the immune landscape of metastatic melanoma from responders and nonresponders to ICI. Such high-dimensional pathology maps showed that Tcy gradually evolve toward an exhausted phenotype as they approach and infiltrate the tumor. Moreover, a key cellular interaction network functionally linked Tcy and PD-L1+ macrophages. Mapping the respective spatial distributions of these two cell populations predicted response to anti-PD-1 immunotherapy with high confidence. These results suggest that baseline measurements of the spatial context should be integrated in the design of predictive biomarkers to identify patients likely to benefit from ICI. SIGNIFICANCE: This study shows that spatial characterization can address the challenge of finding efficient biomarkers, revealing that localization of macrophages and T cells in melanoma predicts patient response to ICI. See related commentary by Smalley and Smalley, p. 3198.

The normal and fibrotic mouse lung classified by spatial proteomic analysis.

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

Bronchial Stenosis in Central Pulmonary Tumors Treated With Stereotactic Body Radiation Therapy.

PURPOSE: Stereotactic body radiation therapy (SBRT) in lung tumors has an excellent local control due to the high delivered dose. Proximity of the proximal bronchial tree (PBT) to the high dose area may result in pulmonary toxicity. Bronchial stenosis is an adverse event that can occur after high dose to the PBT. Literature on the risk of developing bronchial stenosis is limited. We therefore evaluated the risk of bronchial stenosis for tumors central to the PBT and correlated the dose to the bronchi. METHODS AND MATERIALS: Patients with a planning tumor volume (PTV) ≤2 cm from PBT receiving SBRT (8 × 7.5 Gy) between 2015 to 2019 were retrospectively reviewed. Main bronchi and lobar bronchi were manually delineated. Follow-up computed tomography scans were analyzed for bronchial stenosis and atelectasis. Bronchial stenosis was assessed using Common Terminology Criteria for Adverse Events Version 4.0 (CTCAEv4). Patient, tumor, dosimetric factors and survival were evaluated between patients with and without stenosis using uni- and multivariate and Kaplan-Meier analysis. RESULTS: Fifty-one patients were analyzed with a median age of 70 years and World Health Organization (WHO) performance status ≤1 in 92.2%. Median follow-up was 36 months (interquartile range [IQR], 19.6-45.4) and median overall survival 48 months (IQR 21.5-59.3). In 15 patients (29.4%) bronchial stenosis was observed on follow-up computed tomography scan. Grade 1 stenosis was seen in 21.6% (n = 11), grade 2 in 7.8% (n = 4). No grade ≥3 stenosis was observed. Median time to stenosis was 9.6 months (IQR 4.4-19.2). Patients who developed stenosis had significantly larger gross tumor volume with a median of 19 cm3(IQR 7.7-63.2) versus 5.2 cm3 (IQR 1.7-11.3, P <.01). Prognostic factors in multivariate analysis for stenosis were age (P = .03; odds ratio [OR] 1.1), baseline dyspnea (P = .02 OR 7.7), and the mean lobar bronchus dose (P = .01; OR 1.1). CONCLUSIONS: Low-grade (≤2) lobar bronchial stenosis is a complication in approximately one-third of patients after SBRT for lung tumors with a PTV ≤2 cm from PBT. Prognostic risk factors were age, baseline dyspnea and mean dose on a lobar bronchus.

DNA Damage in Circulating Hematopoietic Progenitor Stem Cells as Promising Biological Sensor of Frailty.

Frailty is an age-related syndrome that exposes individuals to increased vulnerability. Although it is potentially reversible, in most cases it leads to negative outcomes, including mortality. The different methods proposed identify frailty after the onset of clinical manifestations. An early diagnosis might make it possible to manage the frailty progression better. The frailty pathophysiology is still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated. A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress, described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation in aged cells including the adult stem cell compartment. Likely, this accumulation is implicated in frailty status. The oxidative status of our frail, pre-frail, and non-frail population was characterized. In addition, the DNA damage in hematopoietic cells was evidenced by analyzing the peripheral blood mononuclear cell and their T lymphocyte, monocyte, circulating hematopoietic progenitor stem cell (cHPSC) subpopulations. The phosphorylation of C-terminal of histone H2AX at amino acid Ser 139 (γ-H2AX), which occurs at the DNA double-strand break focus, was evaluated. In our frail population, increased oxidative stress and a high level of DNA damage in cHPSC were found. This study may have potential implications because the increment of DNA damage in cHPSC could be suggestive of an organism impairment preceding the evident frailty. In addition, it may open the possibility for attenuation of frailty progression throughout specific drugs acting on preventing DNA damage or removing damaged cells.

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage.

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time-dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins.

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

Antibodies validated for routinely processed tissues stain frozen sections unpredictably.

Background: Antibody validation for tissue staining is required for reproducibility; criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed (formalin-fixed, paraffin-embedded) tissue. Materials & methods: We applied to lightly fixed frozen sections a panel of 126 antibodies validated for formalin-fixed, paraffin-embedded tissue with extended criteria. Results: Less than 30% of the antibodies performed as expected with all fixations. 35% preferred one fixation over another, 13% gave nonspecific staining and 23% did not stain at all. Conclusion: Individual antibody variability of the paratope's fitness for the fixed antigen may be the cause. Revalidation of established antibody panels is required when they are applied to sections whose fixation and processing are different from the tissue where they were initially validated.

PKHhigh/CD133+/CD24- Renal Stem-Like Cells Isolated from Human Nephrospheres Exhibit In Vitro Multipotency.

The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24- cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.

The Adaptive and Innate Immune Cell Landscape of Uterine Leiomyosarcomas.

Reactivation of the anti-tumor response has shown substantial progress in aggressive tumors such as melanoma and lung cancer. Data on less common histotypes are scanty. Immune checkpoint inhibitor therapy has been applied to few cases of uterine leiomyosarcomas, of which the immune cell composition was not examined in detail. We analyzed the inflammatory infiltrate of 21 such cases in high-dimensional, single cell phenotyping on routinely processed tissue. T-lymphoid cells displayed a composite phenotype common to all tumors, suggestive of antigen-exposure, acute and chronic exhaustion. To the contrary, myelomonocytic cells had case-specific individual combinations of phenotypes and subsets. We identified five distinct monocyte-macrophage cell types, some not described before, bearing immunosuppressive molecules (TIM3, B7H3, VISTA, PD1, PDL1). Detailed in situ analysis of routinely processed tissue yields comprehensive information about the immune status of sarcomas. The method employed provides equivalent information to extractive single-cell technology, with spatial contexture and a modest investment.

Diagnostic accuracy of the CRS-R index in patients with disorders of consciousness.

Objective: To obtain a CRS-R index suitable for diagnosis of patients with disorders of consciousness (DOC) and compare it to other CRS-R based scores to evaluate its potential for clinics and research. Design: We evaluated the diagnostic accuracy of several CRS-R-based scores in 124 patients with DOC. ROC analysis of the CRS-R total score, the Rasch-based CRS-R score, CRS-R-MS and the CRS-R index evaluated the diagnostic accuracy for patients with the Unresponsive Wakefulness Syndrome (UWS) and Minimally Conscious State (MCS). Correlations were computed between the CRS-R-MS, CRS-R index, the Rasch-based score and the CRS-R total score. Results: Both the CRS-R-MS and CRS-R index ranged from 0 to 100, with a cut-off of 8.315 that perfectly distinguishes between patients with UWS and MCS. The CRS-R total score and Rasch-based score did not provide a cut-off score for patients with UWS and MCS. The proposed CRS-R index correlated with the CRS-R total score, Rasch-based score and the CRS-R-MS. Conclusion: The CRS-R index is reliable to diagnose patients with UWS and MCS and can be used in compliance with the CRS-R scoring guidelines. The obtained index offers the opportunity to improve the interpretation of clinical assessment and can be used in (longitudinal) research protocols. Abbreviations: CRS-R: Coma Recovery Scale-Revised; CRS-R-MS: Coma Recovery Scale-Revised Modified Score; DOC: Disorders of Consciousness; MCS: Minimally Conscious State; UWS: Unresponsive Wakefulness Syndrome; ROC: Receiver Operating Characteristic; AUC: Area Under the Curve; IRT: Item Response Theory.

Pre-hydration in cisplatin-based CCRT: Effects on tumour concentrations and treatment outcome.

AIMS: Pre-hydration is routinely applied to reduce nephrotoxicity in concurrent cisplatin-based chemo-radiotherapy (CCRT). However, pre-hydration may also have systemic effects, potentially leading to lower tumour cisplatin concentrations. We investigated the impact of pre-hydration on tumour cisplatin concentrations in mice, and on treatment outcomes in a clinical cohort study. MATERIALS AND METHODS: Four groups of 20 mice received either no pre-hydration prior to full-dose (6 mg/kg) or half-dose cisplatin, overnight dehydration prior to full-dose cisplatin (dehydration), or NaCl intraperitoneally prior to full-dose cisplatin (pre-hydration). Kidney function and tumour platinum concentration were measured. In patients, a retrospective study compared 2 historical NSCLC cohorts which received CCRT with daily cisplatin, with and without standard pre-hydration. Overall survival (OS) and progression free survival (PFS) were compared using Kaplan-Meier and cox-regression. RESULTS: Pre-hydration significantly decreased cisplatin tumour concentrations in mice, comparable to mice receiving half the dose. In 419 patients (211 without and 208 with pre-hydration) with median follow-up 22 months, there were no significant differences in PFS (18 vs. 15 months) or OS (23 vs. 23 months). CONCLUSION: Pre-hydration reduces cisplatin tumour concentrations in mice, but it does not compromise treatment outcomes in NSCLC patients treated with daily cisplatin and radiotherapy.

Heart dose associated with overall survival in locally advanced NSCLC patients treated with hypofractionated chemoradiotherapy.

Association of heart dose and overall survival was investigated in a cohort including 469 locally-advanced NSCLC patients receiving daily low-dose hypofractionated chemo-radiotherapy. Significant associations were found over a range of dose parameters. Multivariate analysis showed significant associations of heart_V2Gy:HR=1.007%-1 (95% CI:1.002-1.013; p=0.006), age:HR=1.026year-1 (1.011-1.042; p=0.001) and GTV volume:HR=1.001cc-1 (1.000-1.002; p=0.006) with overall survival.

Mitigating differential baseline shifts in locally advanced lung cancer patients using an average anatomy model.

BACKGROUND AND PURPOSE: Differential baseline shifts between primary tumor and involved lymph nodes in locally advanced lung cancer patients compromise the accuracy of radiotherapy. The purpose of this study was to evaluate the performance of an average anatomy model (AAM) derived from repeat imaging and deformable registration to reduce these geometrical uncertainties. METHODS AND MATERIALS: An in-house implementation of a B-Spline deformable image registration (DIR) algorithm was first validated using three different validation approaches: (a) a circle method to test the consistency of the DIR, (b) fiducial marker target registration error, and (c) the recovery of a known deformation vector field (DVF). Subsequently, AAM was generated by first averaging five DVFs resulting from cone beam CT (CBCT) to planning CT (pCT) DIR and second by applying the inverse of the average DVF to the pCT. The proposed method was evaluated on 15 locally advanced lung cancer patients receiving daily motion compensated CBCT and a repeat CT (rCT) for adaptive radiotherapy. Reduction of systematic baseline shifts of the primary tumor were quantified for the fractions used to build the AAM as well as over the whole treatment and compared to the performance of the rCT. RESULTS: The deformable registration accuracy was ≤ 2 mm vector length for the first two validation methods and about 3 mm for the third method. The systematic baseline shifts over the five fractions prior to the rCT used to build the AAM reduced from 5.9 mm vector length relative to the pCT to 2.3 and 4.2 mm relative to the AAM and rCT, respectively. The overall systematic errors in the left-right, cranio-caudal, and anterior-posterior directions were [3.4, 3.8, 3.3] mm, [2.3, 2.9, 2.6] mm, and [2.3, 3.1, 2.7] mm for the pCT, AAM, and rCT, respectively. CONCLUSIONS: The AAM mitigates systematic errors occurring during treatment due to differential baseline shifts between the primary tumor and involved lymph nodes similar to (or even better than) rCT. The superior performance of the AAM in terms of the systematic error derived from the initial fractions indicates that further analysis of the optimum intervention time is required. This model has the potential to be used as an efficient and accurate alternative for rCT in adaptive radiotherapy of locally advanced lung cancer patients, obviating the need for rescanning and recontouring.

Dose-effect analysis of radiation induced rib fractures after thoracic SBRT.

BACKGROUND AND PURPOSE: To determine a dose-effect relation for radiation induced rib fractures after stereotactic body radiation therapy (SBRT) in early stage non-small cell lung cancer (NSCLC). Automatic rib delineation has enabled the analysis of a large patient group. MATERIAL AND METHODS: Four-hundred and sixty-six patients with stage I/II NSCLC received SBRT with a median of 54Gy in 3 fractions. The optimal EQD2-corrected dose parameter to predict (a)symptomatic fractures was found using Cox regression. Three normal tissue complication probability (NTCP) models based on this optimal parameter were constructed: (1) at a median follow up (FU) of 26months, (2) for all data, with time to toxicity taken into account and (3) at a FU of 26months, excluding low dose ribs. RESULTS: The median time to fracture was 22 (range 5-51) months. Maximum rib dose best predicted fractures. The TD50 (dose with 50% complication) of the second NTCP model was 375Gy. The TD50 was significantly higher for the other models indicating an under-estimation of the dose effect at the median follow-up time and/or when excluding low dose ribs. CONCLUSIONS: The risk of symptomatic rib fractures after SBRT was significantly correlated to dose, and was <5% at 26months when Dmax<225Gy.

Intrafraction Motion in Stereotactic Body Radiation Therapy for Non-Small Cell Lung Cancer: Intensity Modulated Radiation Therapy Versus Volumetric Modulated Arc Therapy.

PURPOSE: Stereotactic body radiation therapy (SBRT) for early-stage inoperable non-small cell lung cancer (NSCLC) patients delivers high doses that require high-precision treatment. Typically, image guidance is used to minimize day-to-day target displacement, but intrafraction position variability is often not corrected. Currently, volumetric modulated arc therapy (VMAT) is replacing intensity modulated radiation therapy (IMRT) in many departments because of its shorter delivery time. This study aimed to evaluate whether intrafraction variation in VMAT patients is reduced in comparison with patients treated with IMRT. METHODS AND MATERIALS: NSCLC patients (197 IMRT and 112 VMAT) treated with a frameless SBRT technique to a prescribed dose of 3 × 18 Gy were evaluated. Image guidance for both techniques was identical: pretreatment cone beam computed tomography (CBCT) (CBCTprecorr) for setup correction followed immediately before treatment by postcorrection CBCT (CBCTpostcorr) for verification. Then, after either a noncoplanar IMRT technique or a VMAT technique, a posttreatment (CBCTpostRT) scan was acquired. The CBCTpostRT and CBCTpostcorr scans were then used to evaluate intrafraction motion. Treatment delivery times, systematic (Σ) and random (σ) intrafraction variations, and associated planning target volume (PTV) margins were calculated. RESULTS: The median treatment delivery time was significantly reduced by 20 minutes (range, 32-12 minutes) using VMAT compared with noncoplanar IMRT. Intrafraction tumor motion was significantly larger for IMRT in all directions up to 0.5 mm systematic (Σ) and 0.7 mm random (σ). The required PTV margins for IMRT and VMAT differed by less than 0.3 mm. CONCLUSION: VMAT-based SBRT for NSCLC was associated with significantly shorter delivery times and correspondingly smaller intrafraction motion compared with noncoplanar IMRT. However, the impact on the required PTV margin was small.