[suzierose]

Metaphase cytogenetics vs. FISH cytogenetics

by suzierose on Thu Dec 01, 2011 3:48 pm

I read an article (actually editorial) which stated:

"In patients with active myeloma, more than 90% have abnormal cytogenetics by FISH if tested for hyperdiploidy and for the common translocations involving 14q32. In contrast, using metaphase cytogenetics, only one-third of patients will show an abnormal karyotype, which is usually complex and on an average 11 chromosomes are involved. The other two-thirds will have normal metaphase cytogenetics. These normal metaphases are not derived from the myeloma cells, but from the remaining normal hematopoietic cells.

The reason for failure to have informative mitoses is the low proliferative capacity of the myeloma cells. It is the failure to obtain informative cytogenetics in the majority of patients that has led to a shift away from metaphase cytogenetics to interphase FISH. Deletion of chromosome 13 and hypodiploidy by metaphase cytogenetics are associated with a poor outcome.

Based on these findings, it has been assumed that deletion of chromosome 13 by FISH would have a similar poor prognosis as the metaphase abnormality. In fact, in the IFM 99 study, poor prognosis was defined as a combination of deletion 13 by FISH and elevated B2 microglobulin level.

Unfortunately, mixing results of metaphase cytogenetics with those obtained by FISH has resulted in confusion and statements that deletion of chromosome 13 (as determined by FISH) was not a poor prognostic factor. Patients with inferior outcome as analyzed by FISH were those with t(4;14), t(14;16) and gene deletion of p53.

Also, it was claimed that on metaphase cytogenetics not deletion 13, but hypodiploidy was associated with poor outcome. It is clear that often there is an association between hypodiploidy and deletion of chromosome 13. However, our work has shown that both hypodiploidy without deletion of chromosome 13 and deletion of chromosome 13 without hypodiploidy are associated with a poor prognosis.

When analyzing outcome of patients with or without FISH deletion 13, those with deletion 13 (approximately 50% of all patients) as a group have an inferior outcome (Figure 1), but if we divide patients with deletion 13 by FISH into those with normal versus abnormal metaphase karyotypes, it becomes evident that the inferior prognosis of patients with FISH deletion 13 is entirely due to the one-third of those patients (17% of the overall patient population) with abnormal metaphase cytogenetics, whereas those with normal metaphase cytogenetics and deletion 13 by FISH have an outcome similar to those without FISH deletion 13 (Figure 2)."
http://www.nature.com/leu/journal/v20/n9/full/2404330a.html
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How do you tell from your FISH report if it is metaphase or internuclei cytogentics that was performed? Is there the same confusion around TP53 and 14q32?

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The editorial goes on to say:

"Their findings confirm our data that abnormal metaphase cytogenetics are associated with a poor outcome and that patients with FISH deletion 13 but without abnormal metaphase cytogenetics had similar outcomes to those with no FISH deletion 13. Addition of ploidy status to deletion 13 by FISH did not make a difference in survival prediction. Deletion of chromosome 13 by FISH was much more frequent in patients with non-hyperdiploidy, t(4;14) and deletion of p53.

The new and interesting findings of this paper relate to the fact that patients with 'poor prognostic' FISH markers such as t(4;14) or deletion p53 without abnormal metaphase cytogenetics had outcomes comparable to those without these poor prognostic markers and those with normal metaphase cytogenetics. "
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If your FISH report says you have deletion p53 how do you determine if you also LACK abnormal metaphase cytogenetics and will still have outcomes similiar to those withOUT those poor prognostic factors.?

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Finally, the authors pose this query:

"Why are the results of metaphase cytogenetics so much more informative than FISH cytogenetics? Metaphase cytogenetic analysis is a biological test. If myeloma cells are stroma-dependent, as is the case in early myeloma, taking these myeloma cells out of their supportive microenvironment will result in apoptosis of myeloma cells, and therefore no informative mitoses.

If myeloma cells have become stroma-independent in the advanced stages of the disease, taking the myeloma cell out of the microenvironment will not result in their cell death and the myeloma cells will not only survive, they will proliferate and give rise to abnormal mitoses. Finding abnormal mitoses is still the best surrogate marker we have available at this time for stroma-independent and therefore advanced myeloma...snip....There is no problem with performing additional FISH cytogenetics, but it should not replace metaphase cytogenetics because it has an inferior prognostic significance."



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Which brings up an additional 2 questions.

Is interphase nuclei FISH inferior prognostically to metaphase as authors state? If so, how do you go about getting a metaphase (for better prognostication) if you have an interphase nuclei FISH

and lastly,

How does stroma-independent multiple myeloma cells impact therapeutic options?

Does the phrase "taking the myeloma cell out of the microenvironment will not result in cell death but they will survive and proliferate" refer to research (in-vitro) or is this how the disease pathogeneticity is advancing in our own tissues (in-vivo)?

[Dr. Peter Voorhees]

Re: Metaphase cytogenetics vs. FISH cytogenetics

by Dr. Peter Voorhees on Sat Dec 03, 2011 7:48 am

Dear Suzierose,

I cannot believe you are making me think this hard -- on a Saturday morning, no less! You have brought up a lot of important issues and I will do my best to address them.

The editorial and the paper it is addressing look at two very different approaches to identifying chromosome abnormalities in myeloma cells: conventional cytogenetics, which is not a FISH-based test, and FISH.

With conventional cytogenetic testing, bone marrow cells taken at the time of the bone marrow aspiration are processed and grown in culture. Only those cells that are proliferating and dividing (i.e. only cells in the metaphase of the cell cycle), are evaluated. 20 cells in metaphase are typically reviewed. A normal result would be 46 XX or 46 XY in all 20 cells analyzed.

Conventional cytogenetics is not an analysis restricted to the myeloma cells. All bone marrow cells are analyzed. So, many of the cells that are evaluated wind up not being plasma cells. If you have a highly proliferative, aggressive myeloma (more myeloma cells actively growing and dividing), you have a much higher likelihood of picking up a chromosome abnormality in the myeloma cells by conventional cytogenetics. If the burden of disease is low and proliferating at a slow rate (i.e. if there are very few myeloma cells in the metaphase of the cell cycle), conventional cytogenetics are likely to be normal.

FISH allows you to analyze chromosomes regardless of the phase of the cell cycle. This allows you to detect abnormalities even in non dividing (i.e interphase) cells.

The statement in the editorial that cases where conventional cytogeentics are abnormal are stroma independent is somewhat of a leap. Growth factors are used in vitro to try and get the myeloma cells to divide (go into metaphase, thus be evaluable for the test). Plus, the cells are grown in culture for a short time -- in most cases, they would not be able to continue to grow indefinitely outside of the bone marrow. However, I do agree that in cases where conventional cytogenetics are abnormal, the cells may be less reliant on the marrow microenvironment to prosper (at least in many instances).

The fact that these authors found that the 13q deletion was only an adverse prognostic factor when identified by conventional cytogenetics means that the 13q deletion was only of significance in cases where the myeloma cells were highly proliferative so the 13q deletion could be seen by conventional cytogenetics. This is a well established finding.

I am surprised that these authors found the same thing for the 4;14 translocation and the 17p deletion. In other words, the paper did not see an adverse prognosis when the 4;14 translocation or 17p deletion was picked up by FISH but the conventional cytogenetics were normal. One of the reasons that they may not have seen an impact with FISH only is that the numbers of patients in the study who had 4;14 translocations or 17p deletions was small. The same group has subsequently shown that FISH detection of the 4;14 translocation or 17p deletion is of prognostic value.

The take home point should be that no one individual prognostic marker should be viewed as a death sentence. However, the more adverse factors that a myeloma has, the worse the prognosis and the greater need for a comprehensive approach to the disease in those instances.

Most of the cells evaluated by FISH are in interphase. You may have had conventional cytogenetics performed on your original bone marrow test. I would inquire about this. I do agree with the authors of the editorial that having both tests is more useful than FISH alone.

I hope this helps! I hope all is well with you.

Pete V.

[Lisa B.]

Re: Metaphase cytogenetics vs. FISH cytogenetics

by Lisa B. on Sat Dec 03, 2011 12:18 pm

Dear Dr. Vorhees,

I was very interested in the response to Suzierose's questions as well.

Your explanation/opinion completely cleared it up for me, and I doubt I'm half as smart as she is, so I'm sure she'll be quite satisfied too!!

Thank you for "engaging your brain" early on a Saturday morning for the benefit of myeloma patients and caregivers (although I seriously doubt your brain is EVER "disengaged"!!!! ).

Blessings,

Lisa

[suzierose]

Re: Metaphase cytogenetics vs. FISH cytogenetics

by suzierose on Sat Dec 03, 2011 1:21 pm

Good Morning Dr. V!!

Thanks for being so kind as to do some heavy duty 'gray matter' lifting early on a Saturday morning.As the complexity had overwhelmed me it was nice to know it was no 'instant breakfast' . And I greatly admire your skill to create a digestible bite size meal out of this stuff!!

You write: "Only those cells that are proliferating and dividing (i.e. only cells in the metaphase of the cell cycle), are evaluated. 20 cells in metaphase are typically reviewed. A normal result would be 46 XX or 46 XY in all 20 cells analyzed. Conventional cytogenetics is not an analysis restricted to the myeloma cells. All bone marrow cells are analyzed. So, many of the cells that are evaluated wind up not being plasma cells. If you have a highly proliferative, aggressive myeloma (more myeloma cells actively growing and dividing), you have a much higher likelihood of picking up a chromosome abnormality in the myeloma cells by conventional cytogenetics. If the burden of disease is low and proliferating at a slow rate (i.e. if there are very few myeloma cells in the metaphase of the cell cycle), conventional cytogenetics are likely to be normal. FISH allows you to analyze chromosomes regardless of the phase of the cell cycle. This allows you to detect abnormalities even in non dividing (i.e interphase) cells."
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AHA!!!!!!!!!.....so this explains why my test says: No clonal abnormalities were identified in this study and identified my normalcy by citing 46,XX (20)!! ...soooo, it is possible to deduce that I did not have (in the overall BM,) a high rate of proliferation and/or multiple myeloma cells in metaphase!! Got it!!

But then when they looked ONLY at the cells in the interphase, they found chromosomal changes on the FISH. Which means I have had both metaphase and FISH testing on my plasma cells and that when I read conventional cytogenetics, think metaphase, not FISH...it is so nice when light bulbs come on!
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In addition you write:
"The fact that these authors found that the 13q deletion was only an adverse prognostic factor when identified by conventional cytogenetics means that the 13q deletion was only of significance in cases where the myeloma cells were highly proliferative so the 13q deletion could be seen by conventional cytogenetics. This is a well established finding."

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OK, Can you help me put this into clinical perspective?

Does that mean one can deduce that cells are not "highly proliferative" or disease is not "aggressive" when the conventional cytogenetics come back stating normal, even if there are FISH abnormalities detected?

This is confounding as several articles on the pathogenesis of multiple myeloma state that the chromosomal abnormalities, identified as bad prognosticators, (del 1p/gain1q,TP53 etc) as seen on FISH are the sequela of tumor aggressiveness. IOW's the chromosomal changes are due to advanced stage disease and only seen in late stage disease. For example, is the level of of aggressiveness determined by the number of cells in interphase exhibiting those changes. let's say, (4-11%) vs. (>60%) but that the disease itself is advanced simply by virtue of those FISH negative prognostic factors?

You go on to say:
"I am surprised that these authors found the same thing for the 4;14 translocation and the 17p deletion"

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Why would the negative conventional cytogenetics with positive FISH be well established for del 13q but the corollary not be true for 4:14 and 17p, given positive conventional cytogenetics is indicative of high proliferation? I think this question leads back to the percent of cells in the proliferative metaphase vs. FISH interphase, being able to detect changes independent of proliferative rate, no? I think I am somewhat confused on this? Perhaps, it is as simple as multiple myeloma is not a highly proliferative disease (low metabolics) overall, and as such by the time you do pick up interphase changes on FISH it is advanced???.. ...heeelp...but then even if you do have negative FISH if your conventional cytogenetics are normal, your outcomes (CR but not OS) are equivalent to those withOUT the FISH abnormalities?...is that the take home message..?
http://bloodjournal.hematologylibrary.org/content/106/10/3553/F1.large.jpg


Dr. Voorhees
Thank you for helping me to grasp a crucial difference between metaphase and interphase as well as know that perhaps there is no death sentence for one negative prognostic factor....and I REALLLY appreciate you doing it on a Saturday morning!!!...hopefully it made the rest of the day, downhill...

[Dr. Peter Voorhees]

Re: Metaphase cytogenetics vs. FISH cytogenetics

by Dr. Peter Voorhees on Mon Dec 05, 2011 4:10 am

Dear Suzierose,

Yes, it would appear that you have had conventional cytogenetics performed on your bone marrow biopsy.

You asked: "Does that mean one can deduce that cells are not "highly proliferative" or disease is not "aggressive" when the conventional cytogenetics come back stating normal, even if there are FISH abnormalities detected?"

Not necessarily in all cases. For the 13q deletion, yes. If it is detectable by FISH only, it is not felt to be a marker of aggressive/bad prognosis disease. For the 4;14 translocation and the 17p deletion, studies done looking at these abnormalities by FISH only have shown they are high risk features. My guess (and what has been my experience anecdotally) is that a 17p deletion that can be detected by conventional cytogenetics and FISH is worse than FISH alone, but I think both scenarios carry prognostic weight.

There are other ways of determining the aggressiveness of myeloma beyond conventional cytogenetics and FISH (high stage, high LDH, high plasma cell labeling index, gene expression profiling, etc).

Once again, I think it is important to look at the totality of the data in any given case when determining risk and the optimal treatment approach.

I hope this helps. Take care!

Pete V.