A streamlined mass spectrometry-based proteomics workflow for large scale FFPE tissue analysis

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner

The 25th Anniversary of the National Transplantation Pregnancy Registry.

The National Transplantation Pregnancy Registry (NTPR) is a unique resource for comprehensive information about parenthood after transplantation. To date, 1461 female solid organ transplant recipients with 2609 pregnancies and 879 male recipients who fathered 1358 pregnancies have participated in the NTPR. Over the first 25 years of the NTPR, pregnancy after transplantation has progressed from a situation where termination was once advised, to a topic of pre-transplant counselling with likelihood for success if established criteria are met. Pregnancy after transplantation remains high-risk; it should be carefully considered, planned, and monitored by a multidisciplinary health care team. Pregnancy and maternal outcomes vary based on multiple factors, especially on the type of organ transplanted and the pre-pregnancy graft function. As an open-ended condition-based study, the NTPR accumulates a vast amount of data that is used for comparisons that measure the reliability and benefits of treatments and for developing state-of-the-art management guidelines based on a review of current practices at participating transplant centers. NTPR data analyses have contributed to quantifying issues surrounding post-transplant parenthood such as location of the transplanted organ in proximity to the developing fetus, the safety of various immunosuppressive regimens for pregnancy and fatherhood, the teratogenicity of maternal exposure to mycophenolate during pregnancy, the advisability and timing of planning a posttransplant pregnancy, the dosing of medications during pregnancy, the incidence and treatment of comorbidities during pregnancy, and the effect of in utero or breast milk exposure to immunosuppressants on the developing child. As the face of transplantation evolves, the NTPR will continue to collect and disseminate information to assist recipients and their healthcare providers in making informed decisions about the advisability of pregnancy and care for those who choose to become parents after a solid organ transplant. To insure the continued success of our study, all transplant centers and recipients are encouraged to contact the NTPR to report any post-transplant pregnancy

Breast-Feeding After Transplantation

Transplantation affords recipients the potential for a full life and, for some, parenthood. Female transplant recipients must continue to take immunosuppression during pregnancy and breast-feeding. This article reviews case and series reports regarding breast-feeding in those taking transplant medications. Avoidance of breast-feeding has been the customary advice because of the potential adverse effects of immunosuppressive exposure on the infant. Subsequent studies have demonstrated that not all medication exposure translates to risk for the infant, that the exposure in utero is greater than via breast milk and that no lingering effects due to breast-feeding have been found to date in infants who were breast-fed while their mothers were taking prednisone, azathioprine, cyclosporine, and/or tacrolimus. Thus, except for those medications where clinical information is inadequate (mycophenolic acid products, sirolimus, everolimus, and belatacept), the recommendation for transplant recipients regarding breast-feeding has evolved into one that is cautiously optimistic

Pregnancy Outcomes with Exposure to the Mycophenolic Acid Products (MPA)

Conclusions: Reports to NTPR to date continue to reveal an increased incidence of birth defects in transplant recipients maintained on MPA products during gestation compared to those not maintained on these agents. Structural birth defects consisting of microtia (ear deformity) and facial defects suggest a pattern of malformations. Evaluation of the higher incidence of non-viable outcomes requires further study in this cohort. Centers are encouraged to report all pregnancy exposures in transplant recipients to the NTPR

Immunosuppressive Drugs And Fetal Outcome

Successful pregnancies have been reported in all types of solid-organ transplant recipients on a variety of immunosuppressive regimens. Immunosuppression is essential to maintain the transplanted organ and maternal health, thus the safety of these medications continues to be studied. This article reviews information in the literature and data from the National Transplantation Pregnancy Registry (NTPR) in the United States related to immunosuppressive medication and pregnancy. Although most maintenance immunosuppressive regimens have not been shown to affect the outcome of posttransplant pregnancies, mycophenolic acid products are associated with an increased incidence of spontaneous abortion and an increase in the incidence and a specific pattern of birth defects. When counseling transplant recipients about the prospect and safety of pregnancy, the health of the mother, her graft, and the developing fetus must all be taken into account

Pregnancy Outcomes in Female Lung Transplant Recipients

Conclusions: Female lung recipients appear to face higher risks related to pregnancy when compared to other solid organ transplant recipients. Although successful pregnancies have been reported in female lung recipients, analyses of larger numbers of cases may help to identify pre-pregnancy factors predictive of adverse outcomes. Centers are encouraged to report all pregnancies in transplant recipients to the NTPR

Solid-Organ Transplant Recipients and Breastfeeding While on Immunosuppression

Conclusions: The relatively small amount of drug transferred in breast milk and the lack of reported adverse effects along with the known benefits of breastfeeding may outweigh the risk of drug exposures in the transplant population. The threshold for determining the level of exposure of an immunosuppressive agent that is acceptable is not known at present. Continued study and follow-up of all breast-fed transplant recipient offspring is warranted. Centers are encouraged to report all pregnancies in transplant recipients to the NTRR

Pregnancy Outcomes in Pancreas-Kidney vs. Kidney-Alone Diabetic Female Transplant Recipients

Conclusions: The high incidences of hypertension, preeclampsia, and infection during pregnancy underscore the high-risk nature of pregnancy in both pancreas-kidney and kidney alone recipients. Although overall offspring outcomes are similar in both PK and K groups, pregnancy in PK recipients may be associated with higher incidences of maternal infections and overall graft loss. Centers are encouraged to report all pregnancies in transplant recipients to the NTPR

Pregnancy outcomes in heart transplant recipients.

BACKGROUND: The population of female heart transplant recipients of reproductive age is growing, and counseling regarding reproductive decisions is important. We describe maternal and fetal outcomes of pregnancy in the Transplant Pregnancy Registry International. METHODS: Data regarding pregnancies between 1987 and 2016 were collected via questionnaires, phone interviews, and medical records review. Demographics, comorbidities, changes in immunosuppressive regimens, rejection episodes during pregnancy, data on maternal retransplants, and deaths were recorded. RESULTS: A total of 91 patients reported 157 pregnancies. Mean maternal age at conception was 27 ± 5.6 years. The most common indications for transplant were congenital heart disease (22%) and viral myocarditis (18%). Average transplant to conception interval was 7 ± 6.1 years. Immunosuppression was calcineurin inhibitor-based in almost all patients, with 20% of recipients taking mycophenolic acid (MPA) while pregnant. Complications during pregnancy included pre-eclampsia (23%) and infections (14%). Rejection was reported during 9% of pregnancies and within 3 months postpartum in 7%. Livebirths occurred in 69%, with no neonatal deaths. Miscarriages occurred in 26% of pregnancies, 49% of which had MPA exposure. Mean follow-up post pregnancy was 8.9 ± 6.5 years. At last follow-up, 30 recipients had died, an average of 9.4 ± 6.2 years after pregnancy. The most common causes included allograft vasculopathy and rejection. CONCLUSIONS: This is the largest reported series of pregnancies in heart transplant recipients and demonstrates that two thirds of pregnancies reported are successful. MPA exposure is associated with increased risk of teratogenicity and miscarriage. Pre-pregnancy counseling should include discussions of risk of MPA exposure, rejection, graft dysfunction, and maternal survival