Thrombocytopenia and platelet transfusions in ICU patients: an international inception cohort study (PLOT-ICU)

Purpose Thrombocytopenia (platelet count < 150 × 109/L) is common in intensive care unit (ICU) patients and is likely associated with worse outcomes. In this study we present international contemporary data on thrombocytopenia in ICU patients. Methods We conducted a prospective cohort study in adult ICU patients in 52 ICUs across 10 countries. We assessed frequencies of thrombocytopenia, use of platelet transfusions and clinical outcomes including mortality. We evaluated pre-selected potential risk factors for the development of thrombocytopenia during ICU stay and associations between thrombocytopenia at ICU admission and 90-day mortality using pre-specified logistic regression analyses. Results We analysed 1166 ICU patients; the median age was 63 years and 39.5% were female. Overall, 43.2% (95% confidence interval (CI) 40.4–46.1) had thrombocytopenia; 23.4% (20–26) had thrombocytopenia at ICU admission, and 19.8% (17.6–22.2) developed thrombocytopenia during their ICU stay. Non-AIDS-, non-cancer-related immune deficiency, liver failure, male sex, septic shock, and bleeding at ICU admission were associated with the development of thrombocytopenia during ICU stay. Among patients with thrombocytopenia, 22.6% received platelet transfusion(s), and 64.3% of in-ICU transfusions were prophylactic. Patients with thrombocytopenia had higher occurrences of bleeding and death, fewer days alive without the use of life-support, and fewer days alive and out of hospital. Thrombocytopenia at ICU admission was associated with 90-day mortality (adjusted odds ratio 1.7; 95% CI 1.19–2.42). Conclusion Thrombocytopenia occurred in 43% of critically ill patients and was associated with worse outcomes including increased mortality. Platelet transfusions were given to 23% of patients with thrombocytopenia and most were prophylactic.publishedVersio

Repurposing 18F-FMISO as a PET tracer for translational imaging of nitroreductase-based gene directed enzyme prodrug therapy

Nitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, 18F-FMISO, as substrate for the NTR enzyme. Methods: The efficacy with which 18F-FMISO could be used to report NfsB NTR activity in vivo was investigated using the MDA-MB-231 mammary carcinoma xenograft model. For validation, subcutaneous xenografts of cells constitutively expressing NTR were imaged using 18F-FMISO PET/CT and fluorescence imaging with CytoCy5S, a validated fluorescent NTR substrate. Further, examination of the non-invasive functionality of 18F-FMISO PET/CT in reporting NfsB NTR activity in vivo was assessed in metastatic orthotopic NfsB NTR expressing xenografts and metastasis confirmed by bioluminescence imaging. 18F-FMISO biodistribution was acquired ex vivo by an automatic gamma counter measuring radiotracer retention to confirm in vivo results. To assess the functional imaging of NTR-based GDEPT with 18F-FMISO, PET/CT was performed to assess both gene transduction and cytotoxicity effects of prodrug therapy (CB1954) in subcutaneous models. Results: 18F-FMISO retention was detected in NTR+ subcutaneous xenografts, displaying significantly higher PET contrast than NTR- xenografts (p < 0.0001). Substantial 18F-FMISO retention was evident in metastases of orthotopic xenografts (p < 0.05). Accordingly, higher 18F-FMISO biodistribution was prevalent ex vivo in NTR+ xenografts. 18F-FMISO NfsB NTR PET/CT imaging proved useful for monitoring in vivo NTR transduction and the cytotoxic effect of prodrug therapy. Conclusions: 18F-FMISO NfsB NTR PET/CT imaging offered significant contrast between NTR+ and NTR- tumours and effective resolution of metastatic progression. Furthermore, 18F-FMISO NfsB NTR PET/CT imaging proved efficient in monitoring the two steps of GDEPT, in vivo NfsB NTR transduction and response to CB1954 prodrug therapy. These results support the repurposing of 18F-FMISO as a readily implementable PET imaging probe to be employed as companion diagnostic test for NTR-based GDEPT systems

Repurposing 18F-FMISO as a PET tracer for translational imaging of nitroreductase-based gene directed enzyme prodrug therapy

Nitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, 18F-FMISO, as substrate for the NTR enzyme. Methods: The efficacy with which 18F-FMISO could be used to report NfsB NTR activity in vivo was investigated using the MDA-MB-231 mammary carcinoma xenograft model. For validation, subcutaneous xenografts of cells constitutively expressing NTR were imaged using 18F-FMISO PET/CT and fluorescence imaging with CytoCy5S, a validated fluorescent NTR substrate. Further, examination of the non-invasive functionality of 18F-FMISO PET/CT in reporting NfsB NTR activity in vivo was assessed in metastatic orthotopic NfsB NTR expressing xenografts and metastasis confirmed by bioluminescence imaging. 18F-FMISO biodistribution was acquired ex vivo by an automatic gamma counter measuring radiotracer retention to confirm in vivo results. To assess the functional imaging of NTR-based GDEPT with 18F-FMISO, PET/CT was performed to assess both gene transduction and cytotoxicity effects of prodrug therapy (CB1954) in subcutaneous models. Results: 18F-FMISO retention was detected in NTR+ subcutaneous xenografts, displaying significantly higher PET contrast than NTR- xenografts (p < 0.0001). Substantial 18F-FMISO retention was evident in metastases of orthotopic xenografts (p < 0.05). Accordingly, higher 18F-FMISO biodistribution was prevalent ex vivo in NTR+ xenografts. 18F-FMISO NfsB NTR PET/CT imaging proved useful for monitoring in vivo NTR transduction and the cytotoxic effect of prodrug therapy. Conclusions: 18F-FMISO NfsB NTR PET/CT imaging offered significant contrast between NTR+ and NTR- tumours and effective resolution of metastatic progression. Furthermore, 18F-FMISO NfsB NTR PET/CT imaging proved efficient in monitoring the two steps of GDEPT, in vivo NfsB NTR transduction and response to CB1954 prodrug therapy. These results support the repurposing of 18F-FMISO as a readily implementable PET imaging probe to be employed as companion diagnostic test for NTR-based GDEPT systems

Histological evaluations of tumour characteristics and spread of disease.

<p>Organs were fixed in formaldehyde, sectioned and stained with HE to confirm presence of tumour tissue and for histological characterization of tumour. Sections from a representative mouse depict a large tumour mass in the left uterine horn (A) with necrotic tissue in the centre. Normal uterine morphology is seen in the right uterine horn with endometrial glands and normal stroma and myometrium. Detail of tumour in the left uterine horn (B) reveals solid growing tumour, resembling a grade 3 endometrioid endometrial cancer. Solid tumour masses were also detected in ovaries (C). Inguinal lymph node, macroscopically suspected to be metastatic, was confirmed to represent a metastasis (D), however, without visible surrounding lymphoid tissue. Solid tumour components are depicted in the pancreas (E) with tumour tissue infiltrating surrounding fat tissue. Metastasis is observed on the outer surface of the liver (F), and tumour tissue is also detected in blood vessels of the lung (G), the latter indicating hematogenous spread.</p

Multimodal imaging of the same mouse by MRI, ¹⁸F-FDG PET, ¹⁸F-FLT PET and BLI.

<p>MRI three weeks presacrificed (A) depicting large uterine tumour tissue in the left uterine horn (thin arrows) with intrauterine fluid cranial of the tumour (filled large arrow) and small amounts of free intraperitoneal fluid cranial to the right kidney (K) (small arrows). The tumour tissue is moderately enhancing on T1-weighted series after contrast and the tumour exhibits restricted diffusion with hyperintensity on high b-value DWI with corresponding low apparent diffusion coefficient (ADC) value (1.11 x 10⁻³ mm²/s) on the ADC map (A). BLI 4 to 1 weeks presacrificed (B) shows increasing BLI signal corresponding to the tumour of the left uterine horn; the corresponding tumour tissue was evident macroscopically and confirmed microscopically at necropsy (B). ¹⁸F-FDG PET-CT two weeks presacrificed (C) depicts a large ¹⁸F-FDG-avid tumour in the left uterine horn (arrows) with estimated metabolic tumour volume of 33 ml. ¹⁸F-FLT PET-CT one week presacrificed (D) depicts large ¹⁸F-FLT-avid tumour in the left uterine horn (arrows) with estimated metabolic tumour volume of 44 ml. ¹⁸F-FDG/¹⁸F-FLT-avidity in a VOI in the nuchal muscular tissue (C and D; small arrows) was used as reference tissue to define a threshold for likely tumour tissue (activity of x2 and of x6 for ¹⁸F-FLT and ¹⁸F-FDG, respectively) to be included in the estimated metabolic tumour volume. B: bladder; H: heart.</p

Tumour growth monitored by PET-CT.

<p>Tumour growth in the left uterine horn (A) and growth of abdominal metastasis (B) measured by ¹⁸F-FLT PET-CT at 5, 6 and 7 weeks after inoculation of cells; and by ¹⁸F-FDG PET-CT 8 weeks after inoculation (A and B) in the same mouse. Estimated metabolic tumour volume increased from 5 to 7 weeks after inoculation based on ¹⁸F-FLT PET-CT but was stable or slightly decreased from 7 to 8 weeks after inoculation based on ¹⁸F-FLT PET-CT (week 7) and ¹⁸F-FDG PET-CT (week 8) (C/D, G/H). The estimated ¹⁸F-FLT-SUV_mean x MTV steadily increased from 5 to 7 weeks after inoculation in both the primary tumour (E) and in the metastasis (I). Panel F and J show Total Lesion Glycolysis (¹⁸F-FDG-SUV_mean x MTV) for primary tumor and metastasis, respectively. Histologic examination of the uterus (K) and the pancreas (L) validated presence of malignant tissue (asterisks) as detected with PET-CT.</p

Tumour development and metastasis dissemination in Ishikawa^Luc model.

<p><i>NA</i>: <i>Not applicable</i>.</p><p><i>Total number of mice with organs affected by disease</i>, <i>defined by positive BLI signal and presence of cancer cells in histologic sections</i>.</p

Orthotopic injection of Ishikawa^Luc cells results in weight loss and reduced survival.

<p>Mice injected with Ishikawa^Luc cells were monitored weekly for signs of disease development. Weight loss (A) was detected as an early sign of disease. Mice developing symptoms of severe disease were sacrificed and the overall survival is visualized in a Kaplan-Meier survival plot (B).</p

Tumour growth monitored by Bioluminescence Imaging (BLI).

<p>Tumour growth was monitored weekly by <i>in vivo</i> BLI and an increase in the net bioluminescence versus time was observed (A, B). Organs were also examined by BLI post-mortem to visualize metastatic spread (C). Strong BLI signals were detected at site of injection (left uterine horn; luh), right ovary (o), connective tissue surrounding the uterine horn (ct), pancreas (p) and metastatic node (mn). Spot signals were detected in the liver (l), spleen (s), kidneys (k), heart (h) and lung (lu). No signal was detected in adrenal gland (a).</p