Hi,

I am not a hemato-oncologist myself, but I think the HLA changes in leukemia normally are of phenotypic nature, due to some translocations (look for genetic changes in leukemia, there are heaps of papers in Pubmed).

In my oppinion, translocations somewhere will not change the HLA genotype of a patient.

HLA genotyping is carried out with genomic DNA, and in this case changes in HLA genes are not expected.

At any case the HLA genotyping is performed in the remission phase, when the blast cells are not majoritary in the blood sample.  

So,its better to do HLA-typing when patients have normal count in WBC after having some chemotherapy.

LOH (loss of heterocygosity) is a well known phenomenon in all leukemias.

I think you have HLA-typed on of the leukemia clones!

Insteed of WBC DNA, use buccal swap DNA of the patient for HLA-typing!

Of course, the typing through  buccal swap DNA is a good choice, but if the HLA typing kit requires a high DNA amount (such as some SSP kits for example), a HLA typing with 2 methods (SSP and SSO), and 2 genomic DNA sources (blood and oral) becomes necessary. 

Dear Sir, Genc Sulcebe,

In my lab we do HLA-typing by SSP method. I normally keep 60 ng/μl for HLA-B typing and  30 ng/μl for HLA-A,DR typing. From the experience i got to know that such concentration give reproducible result. So, Will i get good quality DNA from buccal swap ?.

Dear Sir, Thomas M.C. Binder,

As you know that patient is undergoing chemotherapy so her oral mucus membrane is probably not good as healthy people.So,In such case,Will this treatment may affect WBC count in oral cavity ? 

I have did HLA-typing of this patient. She is eager to bone marrow transplant.So,in search of donor, doctor has recommended for HLA typing of his 3 siblings(2 brothers & 1 sister) .

From that report i have observed that patient's HLA matching is less than 30% with all of her siblings but  siblings has more than 80% matching with one another.

Dear Dr Sikder Nahidul Islam Rabbi,

chemotherapy did not  effect the number of fibroblasts in the oral cavity very much.

Sure it is a little bit diffecult to get enough of DNA from buccal swaps. Perhaps you must use two or more buccal swaps for DNA isolation. Maybe, after DNA isolation you must concentrate the DNA.

Is it possible to HLA-type the parents of patient, too?

Could you send me the typing results of the patient and of all family members you have HLA-typed, so far? Than I could look for the HLA haplotypes in this family and perhaps in the patient, too. 

Dear Sir, Thomas M.C. Binder,

Thank you for your information. In this situation i think patient  will do HLA-typing again.I have one limitation that i am not used to with DNA concentrated method. Would you mind giving me a link , so that  i can know about the DNA concentrated method.

As you said so , probably i can send you the report by tomorrow. Thank you for your kind cooperation.

Dear Dr Sikder Nahidul Islam Rabbi,

Here are some informations about  DNA precipitation ('DNA concentration'):

https://en.wikipedia.org/wiki/Ethanol_precipitation

Please send me your report tomorrow.

If possible, please HLA-type the parents of the patient, too!

Are all siblings are children of the same mother and the same father?

Dear Sir, Thomas M.C. Binder

Thank you for your information regarding DNA concentrated method. Hope it will help me a lot. Yes Sir i will send you the report tomorrow .

Sir, parent of the patients didn't do HLA-typing so far. i have only patient's and her three siblings reports.

Yes, Sir all the siblings are children of same mother and father.

I would recommend to HLA-type the parents, too.

Okay Sir, Thomas M.C. Binder.

I will consult with the parents of the patient's in doing HLA-typing.

Fine, thank you!

In my experience, LMA doesn't affect HLA type if you use genomic DNA. We usually type leukemia patients at diagnosis, no matter the status of their disease, and type parents and siblings at the same time to save time. 

We also use SSP for both and high/intermediate resoluciton. My experience is that SSP is quite stringent in DNA quality and quantity, so if you try to type a bad sample, with few leukocytes, or just after QT, your results will be difficult to interpret unless you can compare with the rest of the family. It is known that leukemic blasts can lose HLA, but if you are thinking of a transplant this will not be your problem.

If you wait for remission after therapy: a) perhaps you can't achieve remission with the induction therapy, so you are were you started b) perhaps recovery from aplasia takes too long c) you can't achieve a complete recovery and you still have leucopenia. Some groups wait to type untill they have all prognostic makers (cariotype, molecular, phenotype...) then it can happen that the leukemia doesn't respond as expected, so you are where you started, and wasted a month.

Bucal swaps will not provide more or better DNA for HLA typing than blood. We use them when patients are aplastic.

I suggest that you perform the SSP tests strictly as specified by the manufacturer, improve your DNA extraction method and type the available first degree relatives from the start. The number of blasts in peripheral blood is your least problem.

I should add, if your HLA typing costs are not an issue.

This will depend on your resources, the size of the patient's family and their availability. If  relatives live far away, better get hold of them as soon as you can!.

If you need to reduce costs, you start typing the patient and relatives  serologically for locus A and B, and then type by molecular techniques the patient, serologically  identical siblings and parents. 

If your initial HLA type is complex (homozigosity, technical problems, type not clear by serology) then you can extend your molecular type to other siblings. 

If the family is ample (i.e. more than 4 siblings) we type first male siblings  and if no identical sibling is found, women. 

Dear Sir, Iñigo Romon ,

As you said that leukemia blasts normally lose HLA, but i have observed a different type of genotyping variation in between patient's and her siblings,where siblings shared more than 80% genotyping similarity with one another .

Moreover, I have got to know that Doctor said , as patient's has lower HLA matching with her aspect donors. So,here he is not interested to do bone marrow transplant from those donors.

Dear Nahid,

Here are pictures of the family tree with the 4 HLA haplotypes.

All seems to be correct.

This is nothing, you could publish in journal.

Best regards,

Thomas

Dear Sir, Thomas M.C. Binder

Thank you so much for the identification of the family tree with the 4 HLA haplotypes. Sir, according to your thinking, can  patient (keya) is able to take bone marrow from any of her siblings ?. 

Or what do you recommend ?. Will she need to search for another Donor from her family relatives ?

Dear Nahid,

In Germany we would say no and would look for a full match donor in a stem cell registry.

I do not know, if in Bangladesh they make haplo-identical bone marrow or stem cell transplantations. If yes, sister Salma (or one of her parents) could be a haplo-identical donor.

Best regards,

Thomas

Dear Sir, Thomas M.C. Binder,

Thank you so much for information. Patient's family also was in tension about that. Now they will find a better way towards treatment.

Sir,so far i have known they will go to TATA memorial hospital for the AML treatment for the patient. In Bangladesh Stem cell transplant it is in research scale only ,no hospital or institutions offering its as a treatment so far. 

Thank you so much Sir,for your kind effort towards information related to such case.I hope this information will help them for better transplant.

Best regards,

Nahid.

Did the patient more as these 3 brothers and sisters you presented to me?

If yes, than HLA-type the other siblings, too!

Perhaps, you could look for relatives (cousins) in the families of the brother and sisters of the patient parents, too.

Yes Sir, Thomas M.C. Binder

Patient have one more sister,who didn't do HLA typing so far. As you said ,So i will let them know to do the HLA typing of that sister also.

Bone marrow transplantation is good option, too!

The stem cell donor registries have also bone marrow donors.

Dear Colleage,

As  Dr Binder said now you have to possibilities: to look for a non related donor and, if the patient's HLA type is infrequent and you can't find a suitable donor in an acceptable time frame, then you still have a sibling or a parent in reserve for an haploidentical transplant. 

Sadly, most donor registries are located in USA/Europe and have recruited donors of European descent. I hope the Bangladeshy registry grows as needed by your patient population. 

Best regards

Iñigo 

Dear Sir, Thomas M.C. Binder & Iñigo Romon

Sir, in Bangladesh we have no such stem cell donor registries, as because treatment with stem cell it not yet established here. Now It is in research scale, hope it will be available for treatment soon.

Cancer cells are known to have loss of heterozygosity in its genome. For hematological malignancies, the incidence of loss of heterozygosity although rare, does occur in the HLA region whereby genes/alleles are lost especially when it occurs on short arm of chromosome 6. One way of monitoring, is by screening the blood counts for blasts and confirmation by cytogenetics. To avoid doing this, the best way is to extract DNA from buccal swab or saliva from the patient and type for HLA-A, -B, and DR loci at High resolution and search for matches from the registries, if there is no matching family member. You can search The Anthony Nolan Bone Marrow Registry in England that has many donors from the Indian sub-continent or in the US, National Marrow Donor Program.

Maybe transplantation of two units of cord blood could be an option - you do not need to be as precise in matching as with adult donors.

Together, we found a HLA identical donor in the patient family!

We will have a bone marrow transplantation in the next weeks.

Good news for the patient. Do you know where the Tx will be done, as it was stated that this treatment cannot be done in Bangladesh?

They will do the bone marrow transplantation in India (or Bangladesh).

Transplantation with PBSC is in the experimental stage, in Bangladesh.

One of the patients sisters, is HLA identical.

(There were some Little problems to identify the 4 HLA haplotypes in this Family.)

Dear Julian Strobel,

Tx will be done at TATA memorial hospital so far i have known. Patient already arrived there.Specials Thanks to Sir, Thomas M.C. Binder for his kind co-operation, suggestion and probability of getting a complete matching within her family.When he known that one of patient's siblings didn't do her HLA-typing.

It comes true, when the other sibling did her HLA-typing ( A complete matching with the patient's ) . Who didn't do HLA-typing at first time.

Best regards,

Nahid islam