Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress
, Annette K. Brenner 1, Maria Hernandez-Valladares 2, Even Birkeland 2, Håkon Reikvam 1,3, Frode Selheim 2, Frode S. Berven 2 and Øystein Bruserud 1,3,*Round 1
Reviewer 1 Report
The manuscript discusses the viability and proliferation capacity in 41 cryopreserved primary AML specimens collected from peripheral blood. The authors report a high level of variation. Studying factors impacting the viability of cryopreserved samples is of importance to the field as primary samples are a crucial resource for research. The authors further analyze protein expression using mass spectrometry to identify proteins associated with viability.
The data the authors report on viability and proliferation capacity of cryopreserved primary AML cells after 48 h culture is of high interest. Summary statistics of the distribution of viability percentage and proliferation index should be provided.
The statistical analysis approach used by the authors is based on dichotomizing the data into two groups using an arbitrary cell viability percentage threshold followed by an analysis of differences between the two groups. Transforming a continuous variable (viability %) into a categorical variable (high/low viability) leads to a substantial loss of power to detect correlations. Regression-based methods and statistical methods developed to identify correlations between categorical-continuous variables would be more powerful in identifying novel correlations in these data. The impact of the current paper would be substantially improved by the inclusion of proteomic data in a format that would enable further analysis.
Of high interest would any results indicating what factors enable cells to better survive freeze-thaw and to discuss can such results be used to devise interventions to improve the viability of AML in vitro.
Specific comments:
Introduction:
In the introduction, the authors should clarify what exactly they mean by "spontaneous apoptosis".
Cells undergo severe damage and cell death as a consequence of biopsy followed by freeze-thaw which is likely the primary factor inducing apoptosis in this setting.
Page 2, line 58:
“It will probably be important for our understanding and interpretation of results from experimental in vitro AML studies to characterize the molecular mechanisms behind this spontaneous and probably stress-induced in vitro apoptosis and how pro-and anti-apoptotic molecular mechanisms differ between patients and thereby contribute to patient heterogeneity.”
The word “probably” is used twice but can be omitted from this sentence.
Page 2, lines 183-185:
“The two groups differed significantly with regard to patients with detectable spontaneous or autocrine in vitro proliferation (14 out of the 20 with high viability versus 7 out of 21 with low viability; Fisher’s exact test, p=0.0244), “
Since the authors are comparing two continuous variables (viability and proliferation), Spearman or Pearson correlation should be reported.
Page 4, line 167:
For clarity, the authors should restate here that the cells were cultured after cryopreservation.
Page 5, Figure 1:
Figure 1 shows a high level of variability in the viability of cryopreserved AML. These data are of high interest to the field. Further characterization of the distribution should be provided. Please include summary statistics (at least minimum, maximum, mean, median).
Page 9, The BCL-2 family
Results of regression analysis of viability % and BCL2/BAX ratio should be provided.
Page 9, lines 332-334:
These sentences look like they belong in the figure legend.
Page 10. Figure 3:
The figure shows an outlier with protein levels lower than other samples. What is causing this outlier?
Discussion
The authors should discuss whether or not their analysis identifies factors that may allow cryopreserved AML to survive freeze-thaw better? For instance, could ER stress inhibitors improve viability?
Page 13, line 420:
The authors speculate that in vitro viability could have prognostic significance. However, a major limitation is a technical variation in sample collection and processing in the clinic that will impact viability. How has potential variation in sample handling been addressed in this study?
Page 14, line 517:
The potential role of cryopreservation and thawing-induced damage on ER stress induction should be discussed here.
Author Response
REVIEWER 1
The manuscript discusses the viability and proliferation capacity in 41 cryopreserved primary AML specimens collected from peripheral blood. The authors report a high level of variation. Studying factors impacting the viability of cryopreserved samples is of importance to the field as primary samples are a crucial resource for research. The authors further analyze protein expression using mass spectrometry to identify proteins associated with viability.
The data the authors report on viability and proliferation capacity of cryopreserved primary AML cells after 48 h culture is of high interest. Summary statistics of the distribution of viability percentage and proliferation index should be provided.
Response: We are very grateful for this general comment. Summary statistics about viability and proliferation for the two patient subsets included in the proteomic comparisons studies are now included (page 5). To allow the reader to judge about the quality of our data we present the proliferation data as raw data (i.e. counts per minute, median of triplicate cultures) and not as a stimulatory index; we hope this is acceptable.
The statistical analysis approach used by the authors is based on dichotomizing the data into two groups using an arbitrary cell viability percentage threshold followed by an analysis of differences between the two groups. Transforming a continuous variable (viability %) into a categorical variable (high/low viability) leads to a substantial loss of power to detect correlations. Regression-based methods and statistical methods developed to identify correlations between categorical-continuous variables would be more powerful in identifying novel correlations in these data. The impact of the current paper would be substantially improved by the inclusion of proteomic data in a format that would enable further analysis.
Of high interest would any results indicating what factors enable cells to better survive freeze-thaw and to discuss can such results be used to devise interventions to improve the viability of AML in vitro.
Response: We have included a new chapter at the end of the discussion section where we discuss possible alternative for improving the viability of cryopreserved cells (page 16). New relevant references have been included. Correlation analyses are now included as requested in specific points below.
We have also added new statistical analyses as will be described below. For the bioinformatical analyses we have maintained our comparison of two patient groups. This analytical is simple, is easy to use to reproduce our results, the analytical methods are generally accepted as relevant and are commonly used for bioinformatical analyses. We hope this can be accepted.
Introduction:
In the introduction, the authors should clarify what exactly they mean by "spontaneous apoptosis".
Cells undergo severe damage and cell death as a consequence of biopsy followed by freeze-thaw which is likely the primary factor inducing apoptosis in this setting.
Response: The term “spontaneous apoptosis” is now explained in detail in the third chapter of the Introduction section (page 2).
Page 2, line 58:
“It will probably be important for our understanding and interpretation of results from experimental in vitro AML studies to characterize the molecular mechanisms behind this spontaneous and probably stress-induced in vitro apoptosis and how pro-and anti-apoptotic molecular mechanisms differ between patients and thereby contribute to patient heterogeneity.”
The word “probably” is used twice but can be omitted from this sentence.
Response: This has been corrected as suggested by the reviewer (page 2; first sentence in the last chapter of the Introduction section).
Page 2, lines 183-185:
“The two groups differed significantly with regard to patients with detectable spontaneous or autocrine in vitro proliferation (14 out of the 20 with high viability versus 7 out of 21 with low viability; Fisher’s exact test, p=0.0244), “
Since the authors are comparing two continuous variables (viability and proliferation), Spearman or Pearson correlation should be reported.
Response: The results from an additional analysis of continuous data using the Pearson’s correlation test is now included in the text (page 2; first sentence in the last chapter of the Introduction section).
Page 4, line 167:
For clarity, the authors should restate here that the cells were cultured after cryopreservation.
Response: The text is modified as suggested by the reviewer (page 2).
Page 5, Figure 1:
Figure 1 shows a high level of variability in the viability of cryopreserved AML. These data are of high interest to the field. Further characterization of the distribution should be provided. Please include summary statistics (at least minimum, maximum, mean, median).
Response: The figure legend has also been extended based on the comment by reviewer 2. We have now in addition included summary of the statistics as suggested by the reviewer (page 5).
Page 9, The BCL-2 family
Results of regression analysis of viability % and BCL2/BAX ratio should be provided.
Response: We are very grateful for this comment and have analyzed the impact of the Bax:Bcl2 ratio more in detail:
- Correlation analyses between the ratio and both proliferation and viability have been performed; no evidence for correlations was observed (page 2).
- We have a new section 3.6 (pages 12-13) where we describe where patients with low Bax:Bcl2 ratio cluster together in Figure 3; we conclude that a low Bax:Bcl2 ration (favoring antiapoptotic signaling) is associated with high viability (i.e. low apoptosis) only for a subset of these patients,
- A new Table S5 is now included to clearly show how patients with a low ratio cluster together.
We hope our presentation of these new analyses can be accepted.
Page 9, lines 332-334:
These sentences look like they belong in the figure legend.
Response: We have now included this detailed information in the Figure legend (Figure 3, page 10); the text in the first chapter of section 3.5 has been rewritten (page 11). However, the text on page 9 is still relatively detailed; this has been done to make it easier for the reader to refer to the figure while reading the text. We hope our solutions can be accepted.
Page 10. Figure 3:
The figure shows an outlier with protein levels lower than other samples. What is causing this outlier?
Response: This patient is now commented in the text (page 11).
Discussion
The authors should discuss whether or not their analysis identifies factors that may allow cryopreserved AML to survive freeze-thaw better? For instance, could ER stress inhibitors improve viability?
Response: The possible improvement of cryopreservation methods have been discussed in a new last chapter in the Discussion section (page 16).
Page 13, line 420:
The authors speculate that in vitro viability could have prognostic significance. However, a major limitation is a technical variation in sample collection and processing in the clinic that will impact viability. How has potential variation in sample handling been addressed in this study?
Response: It is now stated that all AML cell samples were collected during the patients stay at our hospital (page 2), and preparation/cryopreservation could therefore be done without delay as commented in a new chapter of the Discussion section (page 13).
Page 14, line 517:
The potential role of cryopreservation and thawing-induced damage on ER stress induction should be discussed here.
Response: As suggested by the reviewer a new chapter is added in the Discussion section (page 15).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dense paper. Have very little corrections, suggestions or just thought I had as I read the paper. I also ask maybe a proteomic expert be part of the next round of reviewers to do it justice.
My suggestions
Line 29-30 Please clarify this sentence :
cellular stress, and this difference seems to be related to differences with regard to induction of and/or response to the unfolded protein stress response…
Line 42-47 is too long, a run-on sentence
Such experimental studies have been important for characterization of the hierarchically organized AML cell populations and for identification of new molecular target [PUT A PERIOD /FULLSTOP HERE THEN START A NEW SENTENCE]. Both clonogenic assays and suspension cultures have then been used to characterize AML cell proliferation/survival, the leukemic cell communication with non-leukemic bone marrow stromal cells and the effects of experimental interventions on the AML cells [4,5]
Figure 1- Could you describe/explain it a lot more
Maybe it was just me, but I did not understand line 190-192, especially this “and the 31 patients included in the proteomic studies represented an unselected subset. The left-out patients were four patients with low and five patients with high viability”
Line 293-295 "and for in vitro cultured AML cells this stress response is associated with increased apoptosis and decreased leukemic cell viability after cryopreservation/thawing/in vitro culture".
This cannot really be correlated to what happens in patients because patient don’t get cryopreserved, but could it reflect what happens after chemotherapy treatment?? Just something I was thinking about.
Author Response
Dense paper. Have very little corrections, suggestions or just thought I had as I read the paper. I also ask maybe a proteomic expert be part of the next round of reviewers to do it justice.
Response: We are very grateful for these general comments.
Line 29-30 Please clarify this sentence :
cellular stress, and this difference seems to be related to differences with regard to induction of and/or response to the unfolded protein stress response…
Response: This last part of the abstract has been rewritten (page 1).
Line 42-47 is too long, a run-on sentence
Such experimental studies have been important for characterization of the hierarchically organized AML cell populations and for identification of new molecular target [PUT A PERIOD /FULLSTOP HERE THEN START A NEW SENTENCE]. Both clonogenic assays and suspension cultures have then been used to characterize AML cell proliferation/survival, the leukemic cell communication with non-leukemic bone marrow stromal cells and the effects of experimental interventions on the AML cells [4,5]
Response: This sentence has been rewritten, the suggested alteration has been made (pages1-2 ).
Figure 1- Could you describe/explain it a lot more
Response: The figure legend has been rewritten and extended (page 5).
Maybe it was just me, but I did not understand line 190-192, especially this “and the 31 patients included in the proteomic studies represented an unselected subset. The left-out patients were four patients with low and five patients with high viability”
Response: This part of the text has been rewritten (page 5). There was a mistake in the original text stating 31 instead of 32 patients included in the proteomic studies. This has now been corrected, we apologize for this mistake.
Line 293-295 "and for in vitro cultured AML cells this stress response is associated with increased apoptosis and decreased leukemic cell viability after cryopreservation/thawing/in vitro culture".
This cannot really be correlated to what happens in patients because patient don’t get cryopreserved, but could it reflect what happens after chemotherapy treatment?? Just something I was thinking about.
Response: We have rewritten and extended this comment (page 9). The suggestion is also commented in a new chapter in the Discussion section (page 16). We hope our solution can be accepted.
Author Response File: Author Response.pdf
