Clinical value of HLA-matched donors

The first allogeneic haematopoietic stem cell transplantation (HSCT), pioneered by Donnall Thomas, was reported in 1957, and the momentous and successful transplantation of the one millionth-patient was achieved in December 2012 . Today more than 70 diseases are treated with blood stem cell transplantation for the replacement of abnormal haematopoiesis and reconstitution of the bone marrow (BM), such as acute and chronic leukaemia, lymphoma, aplastic anaemia, Fanconi anaemia, severe congenital immunodeficiencies and haemoglobinopathies, including sickle cell anaemia and thalassaemia major .

Stem cells for transplantation are usually obtained from the BM, peripheral blood or umbilical cord blood (UCB) of an allogeneic (non-self) donor with matched human leukocyte antigen (HLA) tissue type, the latter so that the donor material is not recognized as foreign by the recipient’s immune system. Umbilical cord blood transplantation (UCBT) from an HLA-identical sibling was first introduced for haematopoietic reconstitution in a patient with Fanconi anaemia in 1989 . UCBT has been associated with a lower incidence of complications, although for particular diseases, it has been recently proven to be as effective as bone marrow transplantation (BMT) . Cord blood collection, however, may not be able to collect an adequate number of cells for cryopreservation and subsequent transplantation. Overall, the major constraint for the HSCT is the availability of an HLA-matched donor. Within a family, if there is only one sibling, the probability of an affected child having an HLA-matched sibling is 25%, whereas if there are two siblings, this chance increases to 43.7%. It is generally mentioned that only 30% of patients can find a suitable donor within their family, while a recent study demonstrated that the likelihood of having a sibling match ranges from 13% to 51%, depending on patient age and ethnicity . In the absence of a matched sibling, a matched unrelated donor may be searched for in national or international donor registries. There are now more than 29 million voluntary stem cell donors worldwide, but finding an unrelated matched donor is very difficult, especially for patients with rare HLA allotypes and haplotypes.

HSCT from matched related donors shows superior outcomes with fewer complications (risk of rejection and graft-versus-host disease, GvHD) and higher overall survival than HSCT from matched unrelated donors, despite considerable progress towards improving outcome for both related and unrelated donor transplants, achieved through advances in HLA typing and high resolution allelic matching, modifications of transplant conditioning regimen and improved supportive care treatment.

When a patient fails to find a matched donor, alternative sources include the use of a related haploidentical donor or mismatched unrelated donor. However, these options cannot guarantee equivalent success compared to transplantation with a complete HLA match .

Apart from the stem cell source, donor category and degree of histocompatibility, additional factors affecting the HSCT outcome include the pre-transplant clinical status and the recipient age at transplantation . Planning for HSCT should, therefore, consider the urgency to transplant and the likelihood of a clinically beneficial outcome.

Clinical value of HLA typing through PGD

In the past, failure to find an HLA-matched donor to cure an affected child often led parents to try natural conception for an HLA-matched baby; there are some reports of performing prenatal diagnosis to identify the HLA status of the unborn baby .

In 2001, an HLA-matched pregnancy was achieved using new expertise known as pre-implantation genetic diagnosis (PGD). PGD, first reported in 1990, requires the use of assisted reproduction technology (ART) to create several pre-implantation stage embryos, followed by biopsy of embryonic cells for genetic testing and transfer of selected embryos to the womb to establish a pregnancy . Pre-implantation HLA typing can be offered as a sole indication when the affected child requires transplantation to treat an acquired disease or in combination with PGD to concurrently avoid the risk of producing another affected child. In some countries, for ethical reasons, pre-implantation HLA typing is only considered acceptable when it is combined with PGD. In the first PGD-HLA case, four clinical ART/PGD cycles and testing of 33 embryos eventually led to the birth of Adam Nash, unaffected by Fanconi anaemia and HLA-matched to his affected 6-year-old sister, Molly. Molly was successfully transplanted and cured using stem cells from the UCB of her healthy matched brother .

Through the years, different biopsy approaches have been attempted for PGD: biopsy of first and second polar body from a fertilized oocyte, single blastomere biopsy from a cleavage stage embryo (day 3) and biopsy of 4–5 trophectoderm cells from a blastocyst (day 5). The advantages and disadvantages of each biopsy stage have been evaluated, and the general trend is to select the biopsy stage on a patient-specific basis and optimize the outcome for the cycle overall .

The European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium has retrospectively collected data on PGD cycles performed by its centre members since 1997 .

On the basis of their 2012 report, referring to data from 10 years of data collection, the majority of PGD-HLA cycles (75.5%) have been performed for beta-haemoglobinopathies. HSCT is the only curative treatment for beta-haemoglobinopathies, the success of which was first reported in 1982, and results are currently very favourable for children with HLA-identical familial donors . Table 1 presents a general list of conditions for which PGD-HLA has been offered, based on published data.

Varied opinions and clinical uncertainty among health care professionals regarding the role and usage of both general PGD and PGD-HLA technology have been demonstrated in many studies. Evidently, many patients in need for related HSCT are unaware of the PGD-HLA procedure, and in a recent survey conducted by Zierhut et al. (2013), less than 35% of parents of children with Fanconi anaemia had been offered this option . This highlights the need for practice guidelines to clarify when, how and by whom the procedure should be offered . The ultimate aim should be to promote the best practice among professionals and encourage patient autonomy and informed decision-making.

Requirements

All specialists involved (haematologists, gynaecologists, embryologists, geneticists, genetic counsellors, nurses and radiotherapists) must work together to support and counsel parents on all steps of the procedure. Along with focusing on practical steps, emphasis should also be given to educating parents about psychosocial and emotional responses before, during and long after the transplant procedure. Centres should provide psychological support for all involved, at any stage required, considering that the procedure is associated with varying and conflicting levels of stress, hope and ethical concerns (see below).

It is generally agreed that before embarking on the PGD-HLA procedure, parents should undergo the following:

• a)
  

  Evaluation of disease status in the affected child by the appropriate treating clinician. A clinical report should be provided, confirming that the disease status justifies undertaking the PGD-HLA procedure and, based on the severity of the disease, that there is sufficient time available for HSCT to be completed. The report should also confirm that HSCT is the recommended therapeutic approach for treating the disease in question, that alternative treatment options are not available and that a matched donor has not been found. Reference should be made to any additional supporting evidence that related-HSCT results in a significantly better clinical outcome over unrelated or non-matched HSCT . Finally, additional details relevant to the HSCT procedure (i.e. at what donor–recipient age will the HSCT procedure be performed and what stem cell source may be used) and the expected outcome, i.e. the chance of HSCT success, should be discussed.

  
  
• b)
  

  Careful assessment of the couple’s reproductive status by an ART specialist. All treatment aspects should be optimized to increase the likelihood of producing an adequate number of oocytes and embryos for analysis because of the low chance of identifying genetically suitable embryos (see below). Geneticists should also evaluate couples with regard to genetic risk for the familial disease and other population-specific diseases, if relevant (e.g. haemoglobinopathies, cystic fibrosis and spinal muscular atrophy).

  
  
• c)
  

  Genetic testing and assurance by the PGD laboratory that an accurate, robust and sensitive diagnostic protocol is available for clinical application on biopsied cells during PGD (see below). If PGD-HLA is not available locally or nationally, collaboration with another centre should be established .

Overall, any centre initiating an ART/PGD procedure should follow the best practice guidelines, published by ESHRE, with regard to the ART and genetic work-up before and during the PGD cycle .

Family counselling

All potential ART/PGD limitations should be communicated to the couple, including possible risks and complications of ART and PGD biopsy, technical limitations of PGD, possibility of failure of diagnosis or misdiagnosis, success rate and need for invasive prenatal testing to confirm PGD results. It is critical that disposition of any remaining non-compatible embryos or surplus compatible embryos is discussed. Options include embryo cryopreservation for potential future embryo transfer or embryo donation.

Emphasis should be given to the unpredictability of the overall outcome, which depends on the woman’s response to hormonal stimulation, the number and quality of embryos produced by ART, the genetic chance of finding a transferable embryo and the fact that not all embryo transfers result in pregnancy. With regard to genetic chance, in cases of pre-implantation HLA typing only, it is expected that 25% of fertilized embryos will be matched to the affected sibling, and in the case of PGD-HLA, the chance of identifying matched unaffected embryos varies depending on the mode of inheritance of the single gene disorder. When concurrently excluding a recessive autosomal or X-linked monogenic disease, it falls to 18.8%, and when excluding a dominant disease, it falls to 12.5% .

More complex cases such as HLA typing along with PGD for two different single gene disorders have also been reported . Couples should also receive information on the experience and pregnancy rates of their ART clinic of choice. Knowledge of success rates is important for families to not only be psychologically prepared for the difficult task they embark on but also enable financial planning as the cost of both the ART and PGD procedures can be extremely high .

With regard to timing, it must be emphasized that initial evaluation, genetic set-up for PGD (taking a minimum of 4–8 weeks) and completion of an ART/PGD cycle may overall require a minimum of 2–3 months. Even if the procedure is successful in the first attempt, it will be followed by 9 months of pregnancy before cord blood can be collected. Therefore, overall, the minimum time before possible HSCT is approximately 1 year in the best of scenarios. In reality, many patients undergo two or more ART-PGD attempts before they deliver an HLA-matched child. In a study investigating the transplantation outcome of patients with diseases whose siblings were conceived through ART/PGD, the median time between the beginning of the first cycle and transplantation was 3.7 (1–9) years . The timing becomes even more critical when considering that the majority of mothers requesting pre-implantation HLA typing are of advanced reproductive age, a factor that limits success in an ART cycle because of the correlation of increasing maternal age with the likelihood of embryo aneuploidy and low ovarian reserve. In some PGD-HLA cases, advanced maternal age is almost unavoidable. For example, in cases of Fanconi anaemia, the mean age of disease onset is 7 years, and the majority of patients are diagnosed months to years after onset . Furthermore, with regard to length of time until HSCT is possible, cord blood transplantation may not suffice for the successful HSCT; therefore, a second transplantation involving BM mixed with the remaining portion of the cord blood sample is performed after the sibling child has gained sufficient weight to donate his or her BM cells .

With the length of time required for a successful outcome of PGD-HLA, clinicians and geneticists should aim to minimize time between initial diagnosis of an affected child to the couple’s decision to initiate this procedure. Kahraman et al. (2014) found that for patients with leukaemia, the average time from diagnosis to first consultation with the ART–PGD centre was 1.5 years . Unfortunately, with delays in many steps of the procedure, some cases fail to be completed in time, and therefore, several children have died either before or immediately after their HLA-matched sibling was born ( Fig. 1 ).

Flow-chart of the PGD-HLA procedure in preparation for HSCT. ART: assisted reproductive technology, UCB: umbilical cord blood, UCBT: umbilical cord blood transplantation, BMT: bone marrow transplantation.

Technical aspects of the genotyping procedures—past/current trends and future prospects

The development of a reliable test for HLA typing on a single cell from a pre-implantation embryo has to address the large size of the HLA region (>3.6 Mb on chromosome 6p21), its highly polymorphic nature and the high frequency of recombination (near 5%, mostly maternal) observed within the HLA locus .

In addition, there are PCR problems when amplifying from a single genome compared to highly concentrated samples used for routine DNA diagnostics. These include amplification failure, allele dropout (ADO) or contamination and can result in either failure to make a diagnosis or, worse, in misdiagnosis. The current recommended approaches to overcome limitations of single-cell PCR involve cautious single-cell manipulation and the use of carefully designed and optimized highly multiplexed PCR protocols, performed either directly on single cells or following whole genome amplification (WGA) under stringent conditions. Prior to application of the PCR protocol, biopsied single cells are lysed by the alkaline lysis or Proteinase K lysis method .

Because of the high frequency of recombination, it is recommended that during the preclinical PGD workup, any available first-degree family members are also tested, aside from parents and the affected child, to exclude the possibility of genetic recombination having occurred in the affected child. In such case, PGD may identify only a relatively close match for transfer. The appropriateness of this approach for HSCT should be confirmed by the haematologist and BMT experts.

The earliest pre-implantation HLA-typing protocols involved direct genotyping of single nucleotide variations associated with important HLA-specific genes, which required design and optimization of unique protocols for each family . A more generic approach for single-cell HLA typing was reported in 2004, involving linkage analysis of polymorphic short tandem repeat (STR) markers located throughout the HLA complex region to identify matching HLA haplotypes between the tested embryos and the affected sibling . This strategy has subsequently become the preferred methodology for the PGD-HLA matching. To date, there are specific guidelines indicating the minimum number of informative STRs that should be used and the HLA regions that should be covered (HLA-A, HLA-B, HLA-C, HLA-DR and HLA-DQ regions) . The robustness of the STR linkage approach is strongly correlated with the number of STR markers used for HLA haplotyping; therefore, for optimal clinical utility, protocols have to be highly multiplexed .

The use of multiple STRs across the HLA region also minimizes misdiagnosis because of preferential amplification and ADO and enables detection of potential recombination, aneuploidy or uniparental disomy of the analysed chromosome 6, which may also affect the diagnostic accuracy of HLA typing of the embryo. If there is a need to concurrently exclude a familial hereditary condition, then multiplex PCR must be standardized to amplify, in addition to the HLA STRs, the mutated region and mutation-linked STRs, enabling direct and indirect genotyping for the familial disease . It has been supported that for accurate diagnosis, at least three informative markers should be analysed for each HLA class regions I, II and III and four markers (two upstream and two downstream) that are close to the disease-related mutation site.

Several earlier PGD-HLA protocols involved a combination of embryo biopsy and genetic analysis at different stages, i.e. polar body biopsy for the detection of the maternal causative SGD mutations, followed by embryo biopsy for subsequent HLA typing in disease-free embryos. The largest studies to date on the clinical application of PGD-HLA have reported a single biopsy step, followed by a multistep protocol, involving an initial multiplex PCR and several nested-PCRs and downstream analytical steps. However, other simpler one-step protocols have also been successfully applied .

In the case of WGA of the biopsy material, individual PCRs follow to separately amplify all loci of interest. WGA potentially provides more DNA from the limited initial amount of embryonic genome and thereby facilitates the analysis of the biopsied cell without requiring the development and optimization of a specific single cell multiplex test. However, the markedly increased ADO following WGA (ranging from 10 to 39%, in contrast to the <10% acceptable rate for PGD protocols) severely restricts the diagnostic utility of WGA .

More recent technologies, such as single-nucleotide polymorphism arrays (SNP-arrays, e.g. karyomapping) and next-generation sequencing (NGS), have been employed in biopsied embryonic cells and applied in a few PGD cases, including PGD-HLA cases . These technologies provide a high throughput approach and overcome certain limitations of multiplex PCR. However, they have other disadvantages. A recent study indicated that karyomapping could not be applied in 23.7% of families referred for PGD and in 5.3% of families requesting pre-implantation HLA typing because of unavailability of relatives, the presence of a de novo disease-causing mutation or identical parental mutations, or when recombination was detected in the affected child . Compared to karyomapping, NGS offers a technological advantage as it provides the greatest sensitivity and accuracy for analysing the embryonic genome and enables the direct detection of family-specific mutations. Its technical limitations mainly involve the risk of misdiagnosis owing to the mistaken analysis of pseudogenes, the failure to detect ADO, which is higher after the (mandatory) WGA step, and the high cost. The latter may be reduced by testing many samples simultaneously, while recent reports have attempted to overcome this by reducing sequencing breadth across the genome and targeting a specific genomic region to selectively increase sequencing depth. NGS analysis necessitates embryo cryopreservation to allow time for completion of diagnosis, followed by embryo transfer in a subsequent vitrified-warmed cycle.

A strategy that has the potential to optimize reproductive outcomes of ART and ART/PGD, especially for couples of advanced reproductive age, involves screening for embryo aneuploidy to avoid the transfer of chromosomally abnormal embryos (known as pre-implantation genetic screening, PGS, or aneuploidy screening). The hypothesis is that chromosomally abnormal embryos may be lost either before or after implantation or may lead to a chromosomally abnormal pregnancy/baby . The value of the combined PGD/PGS approach was recently investigated in a retrospective study by Goldman et al., where only 26% of blastocysts tested for a range of autosomal dominant, autosomal recessive and X-linked inheritance diseases, were both unaffected and euploid (in comparison to the 55% of genetically transferable blastocysts from the PGD-only group of cycles) . Overall, 50% of blastocysts were aneuploid, further supporting previously reported findings . Similarly, Rechitsky et al. demonstrated a higher pregnancy rate (68.4% vs. 45.4%) and a lower miscarriage rate (5.5% vs. 15%) in the PGD with the aneuploidy testing (PGS) group, versus the PGD group, despite transferring a lower number of embryos. This benefit was also demonstrated for PGD-HLA vs. PGS-HLA-PGS cycles and for HLA-only vs. HLA-PGS cycles; however, the number of cycles included was small . It must be noted that these data include embryos biopsied at both cleavage and blastocyst stage and embryo transfers occurring in both fresh and frozen cycles, variables known to affect pregnancy outcomes.

Requirements

All specialists involved (haematologists, gynaecologists, embryologists, geneticists, genetic counsellors, nurses and radiotherapists) must work together to support and counsel parents on all steps of the procedure. Along with focusing on practical steps, emphasis should also be given to educating parents about psychosocial and emotional responses before, during and long after the transplant procedure. Centres should provide psychological support for all involved, at any stage required, considering that the procedure is associated with varying and conflicting levels of stress, hope and ethical concerns (see below).

It is generally agreed that before embarking on the PGD-HLA procedure, parents should undergo the following:

• a)
  

  Evaluation of disease status in the affected child by the appropriate treating clinician. A clinical report should be provided, confirming that the disease status justifies undertaking the PGD-HLA procedure and, based on the severity of the disease, that there is sufficient time available for HSCT to be completed. The report should also confirm that HSCT is the recommended therapeutic approach for treating the disease in question, that alternative treatment options are not available and that a matched donor has not been found. Reference should be made to any additional supporting evidence that related-HSCT results in a significantly better clinical outcome over unrelated or non-matched HSCT . Finally, additional details relevant to the HSCT procedure (i.e. at what donor–recipient age will the HSCT procedure be performed and what stem cell source may be used) and the expected outcome, i.e. the chance of HSCT success, should be discussed.

  
  
• b)
  

  Careful assessment of the couple’s reproductive status by an ART specialist. All treatment aspects should be optimized to increase the likelihood of producing an adequate number of oocytes and embryos for analysis because of the low chance of identifying genetically suitable embryos (see below). Geneticists should also evaluate couples with regard to genetic risk for the familial disease and other population-specific diseases, if relevant (e.g. haemoglobinopathies, cystic fibrosis and spinal muscular atrophy).

  
  
• c)
  

  Genetic testing and assurance by the PGD laboratory that an accurate, robust and sensitive diagnostic protocol is available for clinical application on biopsied cells during PGD (see below). If PGD-HLA is not available locally or nationally, collaboration with another centre should be established .

Overall, any centre initiating an ART/PGD procedure should follow the best practice guidelines, published by ESHRE, with regard to the ART and genetic work-up before and during the PGD cycle .

Family counselling

All potential ART/PGD limitations should be communicated to the couple, including possible risks and complications of ART and PGD biopsy, technical limitations of PGD, possibility of failure of diagnosis or misdiagnosis, success rate and need for invasive prenatal testing to confirm PGD results. It is critical that disposition of any remaining non-compatible embryos or surplus compatible embryos is discussed. Options include embryo cryopreservation for potential future embryo transfer or embryo donation.

Emphasis should be given to the unpredictability of the overall outcome, which depends on the woman’s response to hormonal stimulation, the number and quality of embryos produced by ART, the genetic chance of finding a transferable embryo and the fact that not all embryo transfers result in pregnancy. With regard to genetic chance, in cases of pre-implantation HLA typing only, it is expected that 25% of fertilized embryos will be matched to the affected sibling, and in the case of PGD-HLA, the chance of identifying matched unaffected embryos varies depending on the mode of inheritance of the single gene disorder. When concurrently excluding a recessive autosomal or X-linked monogenic disease, it falls to 18.8%, and when excluding a dominant disease, it falls to 12.5% .

More complex cases such as HLA typing along with PGD for two different single gene disorders have also been reported . Couples should also receive information on the experience and pregnancy rates of their ART clinic of choice. Knowledge of success rates is important for families to not only be psychologically prepared for the difficult task they embark on but also enable financial planning as the cost of both the ART and PGD procedures can be extremely high .

With regard to timing, it must be emphasized that initial evaluation, genetic set-up for PGD (taking a minimum of 4–8 weeks) and completion of an ART/PGD cycle may overall require a minimum of 2–3 months. Even if the procedure is successful in the first attempt, it will be followed by 9 months of pregnancy before cord blood can be collected. Therefore, overall, the minimum time before possible HSCT is approximately 1 year in the best of scenarios. In reality, many patients undergo two or more ART-PGD attempts before they deliver an HLA-matched child. In a study investigating the transplantation outcome of patients with diseases whose siblings were conceived through ART/PGD, the median time between the beginning of the first cycle and transplantation was 3.7 (1–9) years . The timing becomes even more critical when considering that the majority of mothers requesting pre-implantation HLA typing are of advanced reproductive age, a factor that limits success in an ART cycle because of the correlation of increasing maternal age with the likelihood of embryo aneuploidy and low ovarian reserve. In some PGD-HLA cases, advanced maternal age is almost unavoidable. For example, in cases of Fanconi anaemia, the mean age of disease onset is 7 years, and the majority of patients are diagnosed months to years after onset . Furthermore, with regard to length of time until HSCT is possible, cord blood transplantation may not suffice for the successful HSCT; therefore, a second transplantation involving BM mixed with the remaining portion of the cord blood sample is performed after the sibling child has gained sufficient weight to donate his or her BM cells .

With the length of time required for a successful outcome of PGD-HLA, clinicians and geneticists should aim to minimize time between initial diagnosis of an affected child to the couple’s decision to initiate this procedure. Kahraman et al. (2014) found that for patients with leukaemia, the average time from diagnosis to first consultation with the ART–PGD centre was 1.5 years . Unfortunately, with delays in many steps of the procedure, some cases fail to be completed in time, and therefore, several children have died either before or immediately after their HLA-matched sibling was born ( Fig. 1 ).

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