Given that adults don’t have a functional thymus, how do T cells reconstitute after a bone marrow transplant?

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The Thymus – Wikipedia is where T cell – Wikipedia develop. Progenitor cells from the bone marrow enter the thymus to engage in a complicated developmental process and the ones who make it past various bottlenecks leave the thymus as functional, mature CD4 and CD8 T cells.

In the mouse, the thymus dramatically shrinks with age, a process called Involution (medicine) – Wikipedia that severely curtails its output of new, naive, i.e., antigen-inexperienced, T cells, and for long researchers simply extrapolated from mouse that thymic involution must be similarly consequential in humans as well. Important to remember here that the mouse is the most common experimental model for basic immunological research. Hence the assumption ‘Given that adults don’t have a functional thymus‘.

However, over time, the weight of experimental data informs us that the mouse isn’t simply a mini-human, albeit a four-legged, nocturnal, short-lived (maximum lifespan ~2 years) rodent version but rather a species unto itself whose physiological attributes cannot be directly extrapolated to the human (please do imagine my exaggerated eye roll here that it ever even got to the point that something so self-evident even needed saying). Specifically, extrapolating mouse thymus function to human is quite flawed.

This answer describes

  • Key differences between mouse and human thymic function.
  • Effects of thymectomy on T cell numbers and function in humans.
  • Effects of transplant-related thymus impairment in humans.

Key Differences Between Mouse & Human Thymic Function

That a similar process of fewer new T cells coming out of the thymus with age had similar relevance in humans as it does in the mouse was long assumed. However, it turns out naive T cell maintenance is quite a different process in humans, one that makes it less dependent on the thymus over time.

  • Circulating naive T cells in humans self-renew (1), a key difference in kind from naive mouse T cells. Specifically, this groundbreaking study (n=45) showed that the circulating naive T cell pool in elderly humans comprised 10% recent thymic emigrants and 90% self-proliferating (homeostatic proliferation), proportions that were exactly reversed in aged mice. Other studies (2) by other groups including an in silico modeling approach (3) have since corroborated that self-renewal following their emergence from the thymus is a major feature of human naive T cells. Such renewal depends on local levels of IL-7 (Interleukin 7 – Wikipedia) and some other cytokines.
  • While short-lived in mice (1), naive T cells are very long lived in humans (4).
  • Human thymic output may be already less relevant by the 20s since there is little evidence of increasing turnover of circulating naive T cells between 20 and 70 years of age (5). This implies that already by the 20s, the human thymus has pushed out T cell specificities most biologically relevant for an individual to maintain their immunity during their lifetime.
  • Though human thymus begins to involute (reduces its output) early in life, it still remains active until beyond the 60s, only abruptly crashing in the 80s (6). Thus, blunt extrapolation from mouse thymus involution rates to humans is not only inaccurate but also inherently flawed because even the aged human thymus retains measurable capacity to generate new T cells (see below from 7, emphasis mine).

‘As an individual ages, the thymus involutes and the output of new T cells falls significantly [38–40]. In 1985, Steinman et al elegantly demonstrated that thymic function gradually starts decreasing from year one of life [38,39]. The observation of dual components of the human thymus, the true thymic epithelial space, in which thymopoiesis occurs, and the non-epithelial non thymopoietic perivascular space [38,39], was critical to the current understanding of thymic atrophy. The expansion of the perivascular space (adipocytes, peripheral lymphocytes, stroma) with age results in a shift in the ratio of true thymic epithelial space to perivascular space. The thymic epithelial space shrinks to less than 10% of the total thymus tissue by 70 years of age. When extrapolated, Steinman’s data suggest that the thymus would cease to produce new T cells at approximately 105 years of age (Figure 1) [41]…We and others have also demonstrated that while the thymopoietic area of the human thymus decreases with age, the thymopoietic potential per cell, as measured by sjTRECs [47] or by TCR ligation-mediated polymerase chain reaction [3], remains constant at least until approximately 50 years of age [43,45,47–50].’

Such data help understand what had long remained a conundrum, why the naive human T cell population only shrank modestly with age (8, 9, 10, 11), a reduction not in line with the much more rapid pace and extent of thymic involution. Since circulating naive human T cells appear to both hang around longer and be capable of proliferating (homeostatic proliferation) to maintain themselves, in practical terms, this means with age, human T cell repertoire depends less on a functioning thymus, relying instead on maintaining what’s already emerged from the thymus, a difference in kind from the mouse that makes the human T cell system more resilient to withstand damage to the T cell generating capabilities of the thymus. Repertoire refers to the antigen specificities of individual T cell TCRs (T-cell receptor – Wikipedia), with a broader diversity reflecting better anticipatory preparedness. After all, being prepared to specifically engage with antigens not previously encountered is the calling card of the Adaptive immune system – Wikipedia.

  • Homeostatic proliferation is more important for CD4 rather than CD8 T cell maintenance (12), which may be why even the very old have a measurable naive CD4 T cell pool (11) and why impact of aging is greater for human CD8 T cells.
  • None of this negates the importance of the thymus in introducing T cells with new specificities, i.e., T cells with TCRs capable of recognizing new antigens (epitopes). After all homeostatic proliferation can only maintain, not expand, the existing T cell repertoire, a function that’s the sole purview of the thymus. To quote from 1 (emphasis mine),

‘Although these data show that in terms of naive T cell numbers created per day, peripheral T cell proliferation by far exceeds thymic output in human adults, the thymus may still have an essential role – if only because new T cell specificities can only be created by the thymus.’

Effects of Thymectomy on T cell Numbers & Function in Humans

At 1 in 100 newborns, congenital heart disease is among the most common of birth defects (13). Over the past 30 years, having become safer and thus routine, open-heart surgery is increasingly used to correct these defects. However, this also often requires complete or partial thymectomy (thymus removal) since the thymus blocks surgical access to the heart and large vessels, especially in newborns. Researching immune function in such thymectomized individuals in turn provides valuable information on the consequences of such thymectomy.

  • One study (14) on neonatally thymectomized children examined impact in either the short- (within 1 to 5 years, n=17 and 19 healthy controls) or longer-term (at least 10 years later, n=26 and 11 healthy controls) and found it drastically affects T cell diversity (measured as range of TCRs) in the short-term but that the thymus at such a young age is capable of some degree of regeneration since diversity is restored in later life.
  • Another study (15) found rates of autoimmunity and allergy among the neonatally thymectomized (n=7) to be similar to healthy controls (n=7).
  • Though both degree (partial versus total) (16) and age (17) at thymectomy influence outcome on T cell number and function, they don’t affect general health on the whole apart from a tendency for exaggerated responses to cytomegalovirus (CMV) (18). How to explain this? One plausible explanation could be that thymectomy provides impetus for increased plasma IL-7 levels (19), which in turn could support T cell homeostatic proliferation (20, 21).
  • >20 years post-thymectomy, patients infected with CMV had fewer circulating naive T cells and reduced TCR diversity (18) and delayed primary antibody response to tick-borne encephalitis vaccination (22, 23).

Since routine neonatal open-heart surgery and its attendant thymectomy are at 30 years or so of recent vintage, lifelong impact on immunity and health is still work in progress. However, these and other studies suggest impact is individual, hard to predict and highly influenced by chronic infections, especially CMV.

Effects of Transplant-related Thymus Function or Impairment in Humans

Preparing the body for transplant (transplant conditioning regimens) entails the kinds of preparatory treatments (irradiation, chemotherapy, steroids) that severely damage the thymus, impairing its capacity to push out new T cells.

  • Adequate thymic recovery was observed after autologous stem cell transplant
    • Even in those with severe autoimmune diseases (n=10, age range 16 to 49 years old, n=21 controls, age range 20 to 55 years old) (24).
    • In Multiple Sclerosis (MS) patients (n=7, age range 28 to 53) (25).
  • Adequate thymic recovery was observed after heterologous kidney transplant (n=48 patients, 39 controls) (26).
  • One study (27) of 32 adult breast cancer patients treated with autologous peripheral blood stem cell transplant (age range 30 to 69) found thymic recovery to be strictly a function of age, with measurable thymus enlargement (sign of recovery) post-transplant in
    • 4 of 5 of those 30 to 39 years old
    • 6 of 13 in those 40 to 49 years old.
    • Only 2 of 14 in those >50 years old.

Given its capacity for some regeneration as well as its ability to continue to push out new T cells even later in life, albeit at markedly lower rates, no surprise that the human thymus can bounce back after neonatal thymectomy as well as from transplant-related damage. Degree of recovery is also unsurprisingly a function of age and environment. As well, unlike their mouse counterparts, human naive T cell capacity for self-propagation helps maintain them even into old age.

Bibliography

1. den Braber, Ineke, et al. “Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans.” Immunity 36.2 (2012): 288-297. https://ac.els-cdn.com/S10747613…

2. Thome, Joseph JC, et al. “Longterm maintenance of human naive T cells through in situ homeostasis in lymphoid tissue sites.” Science immunology 1.6 (2016). https://www.ncbi.nlm.nih.gov/pmc…

3. Johnson, Philip LF, et al. “Peripheral selection rather than thymic involution explains sudden contraction in naive CD4 T-cell diversity with age.” Proceedings of the National Academy of Sciences 109.52 (2012): 21432-21437. http://www.pnas.org/content/109/…

4. Vrisekoop, Nienke, et al. “Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool.” Proceedings of the National Academy of Sciences 105.16 (2008): 6115-6120. http://www.pnas.org/content/105/…

5. Naylor, Keith, et al. “The influence of age on T cell generation and TCR diversity.” The Journal of Immunology 174.11 (2005): 7446-7452. https://www.researchgate.net/pro…

6. Czesnikiewicz-Guzik, Marta, et al. “T cell subset-specific susceptibility to aging.” Clinical Immunology 127.1 (2008): 107-118. https://www.researchgate.net/pro…

7. Gruver, A. L., L. L. Hudson, and G. D. Sempowski. “Immunosenescence of ageing.” The Journal of pathology 211.2 (2007): 144-156. http://onlinelibrary.wiley.com/d…

8. Bertho, Jean-Marc, et al. “Phenotypic and immunohistological analyses of the human adult thymus: evidence for an active thymus during adult life.” Cellular immunology 179.1 (1997): 30-40.

9. Douek, Daniel C., et al. “Changes in thymic function with age and during the treatment of HIV infection.” Nature 396.6712 (1998): 690.

10. Jamieson, Beth D., et al. “Generation of functional thymocytes in the human adult.” Immunity 10.5 (1999): 569-575. http://www.cell.com/immunity/pdf…

11. Wertheimer, Anne M., et al. “Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans.” The Journal of Immunology 192.5 (2014): 2143-2155. http://www.jimmunol.org/content/…

12. Goronzy, Jörg J., et al. “Naive T cell maintenance and function in human aging.” The Journal of Immunology 194.9 (2015): 4073-4080. http://www.jimmunol.org/content/…

13. Hoffman, Julien IE, and Samuel Kaplan. “The incidence of congenital heart disease.” Journal of the American college of cardiology 39.12 (2002): 1890-1900. https://ac.els-cdn.com/S07351097…

14. Van Den Broek, Theo, et al. “Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.” The Journal of clinical investigation 126.3 (2016): 1126. https://www.ncbi.nlm.nih.gov/pmc…

15. Silva, Susana L., et al. “Autoimmunity and allergy control in adults submitted to complete thymectomy early in infancy.” PloS one 12.7 (2017): e0180385. http://journals.plos.org/plosone…

16. Halnon, Nancy J., et al. “Thymic function and impaired maintenance of peripheral T cell populations in children with congenital heart disease and surgical thymectomy.” Pediatric research 57.1 (2005): 42-48. https://www.researchgate.net/pro…

17. Prelog, Martina, et al. “Thymectomy in early childhood: significant alterations of the CD4+ CD45RA+ CD62L+ T cell compartment in later life.” Clinical immunology 130.2 (2009): 123-132. http://www.musiklexikon.ac.at:80…

18. Sauce, Delphine, et al. “Evidence of premature immune aging in patients thymectomized during early childhood.” The Journal of clinical investigation 119.10 (2009): 3070. Evidence of premature immune aging in patients thymectomized during early childhood

19. Mancebo, E., et al. “Longitudinal analysis of immune function in the first 3 years of life in thymectomized neonates during cardiac surgery.” Clinical & Experimental Immunology 154.3 (2008): 375-383. http://onlinelibrary.wiley.com/d…

20. Fry, Terry J., and Crystal L. Mackall. “The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance.” The Journal of Immunology 174.11 (2005): 6571-6576. http://www.jimmunol.org/content/…

21. Silva, Susana L., et al. “Human naive regulatory T-cells feature high steady-state turnover and are maintained by IL-7.” Oncotarget 7.11 (2016): 12163. https://pdfs.semanticscholar.org…

22. Prelog, Martina, et al. “Diminished response to tick-borne encephalitis vaccination in thymectomized children.” Vaccine 26.5 (2008): 595-600.

23. Zlamy, Manuela, et al. “Antibody dynamics after tick-borne encephalitis and measles–mumps–rubella vaccination in children post early thymectomy.” Vaccine 28.51 (2010): 8053-8060.

24. Thiel, Andreas, et al. “Direct assessment of thymic reactivation after autologous stem cell transplantation.” Acta haematologica 119.1 (2008): 22-27.

25. Muraro, Paolo A., et al. “Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients.” Journal of Experimental Medicine 201.5 (2005): 805-816. http://jem.rupress.org/content/j…

26. Nickel, Peter, et al. “CD31+ Naïve Th Cells Are Stable during Six Months Following Kidney Transplantation: Implications for Post‐transplant Thymic Function.” American journal of transplantation 5.7 (2005): 1764-1771. http://onlinelibrary.wiley.com/d…

27. Hakim, Frances T., et al. “Age-dependent incidence, time course, and consequences of thymic renewal in adults.” Journal of Clinical Investigation 115.4 (2005): 930. http://content-assets.jci.org/ma…

https://www.quora.com/Given-that-adults-dont-have-a-functional-thymus-how-do-T-cells-reconstitute-after-a-bone-marrow-transplant/answer/Tirumalai-Kamala

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Are there any Chimeric Antigen Receptors (CAR-T) that can demonstrate a therapeutic response against solid tumors?

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A few case reports here and there show Chimeric Antigen Receptor-T (CAR-T) therapeutic response against solid tumors.

  • Currently one CAR-T case report shows it successfully targeting and eliminating a patient’s solid tumors. One of the earliest CAR-T approaches against Glioblastoma – Wikipedia (GBM) targeted IL13RA2 – Wikipedia which is often over-expressed by such tumors.
    • A small pilot first-in-human clinical trial used such CAR-Ts in 3 GBM patients and reported the Rx generated measurable anti-tumor immune responses with manageable side-effects.
    • In one patient (1), tumors had not only proven resistant to surgery, radiation and chemotherapy but also spread to the spine. For this reason, the researchers decided to infuse anti-IL-13Ra2 CAR-Ts not just in one but in two locations. This approach apparently eliminated their tumors, with the patient reported to be tumor-free as of February 2017.
  • In two case reports (2), CAR-Ts that target Mesothelin – Wikipedia positive tumor MPM (malignant pleural Mesothelioma – Wikipedia) were found to infiltrate tumors with minimal on-target, off-tumor toxicity. However, though completed, no final trial results have yet been posted (3).
  • CAR-Ts targeting the GD2 antigen were effective against pediatric neuroblastoma in 3 of 11 patients (4). No other results have been reported from this trial which is still listed as ongoing (5).

Most CAR-T clinical trials target blood cancer, specifically B cell cancers expressing the cell-surface molecule CD19 (see below from 6, 7).

Practical Obstacles to CAR-Ts Targeting Solid Tumors

The process of making CAR-Ts capable of targeting solid tumors entails

  • Identifying antigens specifically expressed by solid cancer cells on their cell surface and validating them.
    • Targeting tumor-associated antigens (TAAs) rather than tumor-specific antigens (TSAs) increases risk for on-target, off-tumor toxicity which can even be life-threatening. On-target, off-tumor toxicity is a serious problem even with anti-CD19 CAR-Ts thought to only target B cells. Potential for toxicity is theoretically even higher when targeting solid tumors.
    • Germline cancer antigens are a third type of cancer antigen. Such antigens are usually expressed by germ (sperm, eggs) cells but not by somatic cells. Germ cells typically don’t express MHC molecules. This reduces the likelihood they could directly present such antigens to CAR-T cells that could make them a target of their immune responses. This inherent safety feature makes germline cancer antigens an attractive immunotherapeutic target.
  • Generating antibodies against such antigens and then deriving mAbs out of such antibodies.
  • Validating such mAbs to ensure their safety and using their single-chain variable fragment (scFv) to genetically engineer CARs.
    • Apart from identifying and validating tumor-specific antigens, immunologically targeting a tumor using a patient’s own cells requires identifying and isolating tumor-specific T cells from cancer patients. In the case of CAR-T cells, the need to isolate and identify tumor-specific T cells is rendered moot by taking the patient’s T cells out and genetically engineering them in vitro to express tumor-specific CAR.
    • Such CARs are hybrid molecules whose extracellular piece consist of a monoclonal antibody’s (mAb) scFv. Thus, regardless an individual T cell’s antigenic specificity, once it’s genetically engineered to be a CAR-T, it can target cells that express that CAR’s antigen.
  • Successfully targeting solid tumors also requires many more time-consuming genetic engineering steps.
    • For one, CAR-Ts need to be able to efficiently penetrate solid tumors. Ability to kill tumor cells is of little use if CAR-Ts can’t enter the tumor. This requires genetically engineering additional pieces to enable CAR-Ts to do so.
    • Safeguards may also need to be engineered to minimize toxicity, for example, by engineering CAR-Ts to target two TAAs at a time rather than one to increase the chance that the CAR-T in question only got activated by a tumor that expressed both of them rather than by normal tissue that would more likely express one or the other but not both simultaneously.

Each of these steps constitutes several years’ worth of research.

The CARs in CAR-Ts currently being tested started not from scratch but rather by engineering into T cells scFvs from mAbs already approved for clinical anti-cancer use by the FDA or other regulatory agencies. Problem with such an approach is these old-generation mAbs target TAAs rather than TSAs.

For example, skin (keratinocytes) as well as heart muscle cells (cardiac myocytes) express Epidermal growth factor receptor – Wikipedia (EGFR) and HER2/neu – Wikipedia (ERBB2), which is why mAbs targeting these receptors can trigger skin- and heart-related toxicities (8). GD2 is expressed not just by neuroblastoma and sarcoma but also by peripheral sensory nerve fibers and neurons. This is why anti-GD2 mAbs can trigger neuropathic pain (9), which could presumably occur with CAR-Ts that expressed scFv derived from such mAbs.

CAR-T therapies targeting TAAs or cancer germline antigens expressed by solid tumors are at various stages of development and testing (see below from 7). Not many results have yet been published, even though some of these trials are now completed.

Bibliography

1. Brown, Christine E., et al. “Regression of glioblastoma after chimeric antigen receptor T-cell therapy.” New England Journal of Medicine 375.26 (2016): 2561-2569. http://www.nejm.org/doi/pdf/10.1…

2. Beatty, Gregory L., et al. “Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce antitumor activity in solid malignancies.” Cancer immunology research 2.2 (2014): 112-120. http://cancerimmunolres.aacrjour…

3. Autologous Redirected RNA Meso-CIR T Cells – No Study Results Posted – ClinicalTrials.gov

4. Louis, Chrystal U., et al. “Antitumor activity and long-term fate of chimeric antigen receptor–positive T cells in patients with neuroblastoma.” Blood 118.23 (2011): 6050-6056. http://www.bloodjournal.org/cont…

5. Blood T-Cells and EBV Specific CTLs Expressing GD-2 Specific Chimeric T Cell Receptors to Neuroblastoma Patients – Full Text View – ClinicalTrials.gov

6. Klebanoff, Christopher A., Steven A. Rosenberg, and Nicholas P. Restifo. “Prospects for gene-engineered T cell immunotherapy for solid cancers.” Nature medicine 22.1 (2016): 26.

7. Yu, Shengnan, et al. “Chimeric antigen receptor T cells: a novel therapy for solid tumors.” Journal of hematology & oncology 10.1 (2017): 78. https://pdfs.semanticscholar.org…

8. Crone, Steven A., et al. “ErbB2 is essential in the prevention of dilated cardiomyopathy.” Nature medicine 8.5 (2002): 459-465. http://www.salk.edu/pdf/otmd/Art…

9. Yu, Alice L., et al. “Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma.” New England Journal of Medicine 363.14 (2010): 1324-1334. http://www.nejm.org/doi/pdf/10.1…

https://www.quora.com/Are-there-any-Chimeric-Antigen-Receptors-CAR-T-that-can-demonstrate-a-therapeutic-response-against-solid-tumors/answer/Tirumalai-Kamala

Why are some tumors more susceptible to bacterial infection than normal tissue?

Refers to article: http://science.sciencemag.org/content/357/6356/1156.full

This answer

Data in the paper, Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine

Gemcitabine – Wikipedia is a Nucleoside analogue – Wikipedia used to treat cancers such as breast, bladder, lung and pancreas.

The authors (1) first explored how some tumor cell lines became resistant to gemcitabine, finding it to be associated with some kind of filter- and antibiotic-susceptible entity, identifying that entity to be Mycoplasma hyorhinis – Wikipedia. Next, the authors found not just Mycoplasma but 13 of 27 different bacterial species tested to be capable of mediating gemcitabine resistance in cancer cell lines, with this capacity mostly but not always matching capacity to express the long form of the Cytidine deaminase – Wikipedia (CDD).

Having established a connection between presence of bacteria and capacity for gemcitabine resistance, the authors then examined whether there were bacteria in human tumors, choosing to focus on Pancreatic cancer – Wikipedia (PDAC) since gemcitabine is a critical Rx for it.

Quantitative Real-time polymerase chain reaction – Wikipedia to detect bacterial 16S ribosomal RNA – Wikipedia detected bacterial rDNA in 86 of 113 (76%) PDAC and 3 of 20 (15%) normal pancreas. Authors confirmed these results with additional assays such as Fluorescence in situ hybridization – Wikipedia (FISH) targeting 16S rRNA as well as Immunohistochemistry – Wikipedia using antibody to detect bacterial Lipopolysaccharide – Wikipedia (LPS).

52% of the bacteria were Gammaproteobacteria – Wikipedia, most belonging to Enterobacteriaceae – Wikipedia and Pseudomonadaceae – Wikipedia families.

Finally, authors cultured bacteria from 15 fresh human PDACs and found 14 of 15 capable of making the RKO and HCT116 human colon carcinoma cells fully resistant to gemcitabine.

Article’s main message is selective association of particular bacteria with the human tumor, PDAC, with such association being capable of conferring tumor resistance to the anti-cancer drug, gemcitabine. More than one other group (2, 3) has previously reported such bacterial (Mycoplasma) ability to alter anti-cancer nucleoside function.

Caveats to the study are mainly technical and don’t negate its main message.

  • What is by now a standard refrain in so many biomedical research papers (even those published in high impact factor journals such as Science), shoddy use of statistics to present data, especially from in vivo mouse models, as cleaner and much more dichotomous than they actually are. For example, a lack of consistency in the statistical tools used to summarize the data, being standard deviation in one figure, standard error of the mean in another, as well as the interchangeable use of the terms technical and biological replicates.
  • Having gone to some lengths to emphasize that bacterial expression of the long form of CDD was required for gemcitabine resistance, authors provide no explanation for mechanism of gemcitabine resistance in M. hyorhinis, which the authors themselves point out expresses the short, not long, form of CDD. Clearly, different bacteria likely have more than one mechanism for mediating gemcitabine resistance.

How Bacteria Might Home To & Grow Within Cancers

Growth within solid tumors typically entails destructive features. Though Neovascularization – Wikipedia, growth of new, disorganized and leaky blood vessels, crops up to support the ravenous oxygen and nutrient demand of such rapidly proliferating cancer cells, such demand more often than not outpaces supply with two major consequences.

Such leaky and disorganized vasculature could make it possible for circulating bacteria to gain access to tumor tissue while hypoxic tumor tissue, being a favorable growth environment for anaerobic bacteria (both obligate and facultative), becomes a magnet for such bacteria, which enter such regions and get settled there.

Further, more than usual number of cells dying in an unplanned manner within solid tumors typically overwhelms the local scavenging capacity of clean-up cells belonging to the local reticuloendothelial system (Mononuclear phagocyte system – Wikipedia) that would normally clear up the resulting cell carcasses and debris. This makes such areas pools of not only chemical messengers that could attract bacteria but also of nutrients that could sustain them (see below from 4).

Thus, not just one but several studies have reported presence of bacteria within solid tumors (see below from 4).

Link Between Mycoplasma & Solid Tumors

Among the smallest of bacteria and lacking a cell wall, association of Mycoplasma in particular with tumors is of an interesting piece within this sub-field of tumor biology, having been reported as far back as 1970 (5).

  • Cancer cell lines are notorious for becoming infected with Mycoplasma so much so numerous reviews outline how to prevent and eliminate such contamination (6, 7, 8, 9). Such cancer cell-Mycoplasma associations also have practical cost consequences. Since basic research can and often is sloppy enough that Mycoplasma contaminations may even pass unnoticed in some labs, which published responses pertain to cancer cell and which to the Mycoplasma associated with it is a real issue that remains unresolved (10, 11, 12).
  • A 1999 study (13) by researchers at the American Registry of Pathology at the Armed Forces Institute of Pathology showed chronic colonization of cell lines with Mycoplasma fermentans and M. penetrans to be associated with chromosomal instability and malignant transformation, i.e., potentially carcinogenic capacities.
  • A 2001 PCR-based study (14) found Mycoplasma consistently and preferentially associated in different types of tumor tissues (see below from 15 using data from 14).

Mycoplasma proclivity for cancer cell lines grown in lab cell cultures is itself an intriguing phenomenon deserving of basic research interest since it occurs in absence of the plausible factors proffered for such association in vivo, such as disorganized and leaky blood vessels messily sprouting up to supply ravenously growing tumor tissue, and the lack of attendant tumor hypoxia and necrosis, factors supposed to make solid tumors a magnet for bacterial (Mycoplasma) growth.

The Contentious History of Bacteria in Tumors

Presence of bacteria in tumors has a particularly contentious past in scientific research. After research by Louis Pasteur – Wikipedia and Robert Koch – Wikipedia, among many others, firmly established a scientific basis for the Germ theory of disease – Wikipedia, several prominent mid-20th century researchers tenaciously pursued presence of bacteria in tumors.

The focus of such research by scientists such as Virginia Livingston – Wikipedia, Eleanor Alexander-Jackson, Irene Diller, Florence B. Seibert – Wikipedia was nothing less than trying to uncover evidence that an infectious disease agent underlay any and all types of cancer, i.e., a germ theory of cancer.

However, while some viral, even bacterial and other microbial causes of cancer are certainly known and accepted, the germ theory of cancer became markedly unfashionable over the course of the latter part of the 20th century in the face of the considerable headwind gained by other mechanisms of Carcinogenesis – Wikipedia, which took increasing precedence, principally mechanisms involving the interplay between overexpression an/or mutations in Oncogene – Wikipedia and mutations in Tumor suppressor gene – Wikipedia.

Bacteria as sole cause of cancer is also difficult to reconcile with the fact that Germ-free animal – Wikipedia do develop cancers. Thus, today only one bacterium, Helicobacter pylori is recognized by the International Agency for Research on Cancer – Wikipedia (IARC) as an established human carcinogen (16).

However, consequence of such single-minded focus by one group of researchers trying to prove a germ theory of cancer and severe repudiation of the same by others that followed them may have been detrimental in the long run, detrimental since it seems to have firmly shut the door for at least several decades on a more open-minded exploration of the association between bacteria and cancer, and consequences thereof. After all, a more open-minded view suggests research and therapeutic utility, be such association cause or effect (see below from 17, emphasis mine),

‘Microbial profiling of cancer is agnostic as to whether bacteria are the cause or the effect, although in each scenario lies clinical utility. If the development of cancer is the underlying reason for shifts in the microbial community, then surveying bacteria becomes a new method for diagnosis. Microbial censuses could be used to diagnose malignancies and in surveillance for recurrence. Of clinical importance is that microbiome sampling, as opposed to blood sampling or tissue biopsies, is generally non-invasive. Such approaches are already revealing insights; for example, specific microbes in saliva have been associated with chronic pancreatitis and tumors of the pancreas [8]. On the other hand, if shifts in the microbial spectrum cause cancer, then not only does microbiome research become a new route into exploring pathogenesis but the microbiome itself becomes a new target for preventative strategies. Microbiomes could be sampled proactively for risk stratification in at-risk populations, and probiotics and microbiome transplants could be considered as strategies for cancer prevention.

Also intriguing is the idea that one might be able to categorize cancers that do or do not have a microbial influence. It has been demonstrated in head and neck cancers that microbes live deep within tumor tissue [9], thus contributing to the intracellular stromal environment. Unless their borders are breached by infection, organs such as the kidney and the pancreas have been thought to be completely sterile. With modern metagenomic profiling, which can identify fastidious and previously unculturable microorganisms, we might find that fewer organ systems are sterile than previously thought, and that the contribution of the microbial microenvironment might have a much more inward reaching influence than currently assumed. Classifying cancers into sterile and non-sterile, if sterile cancers truly exist, might garner increased biological understanding of tumorigenesis, perhaps providing new avenues for diagnosis and even novel treatment modalities for nonsterile cancers’

Bibliography

1. Geller, Leore T., et al. “Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.” Science 357.6356 (2017): 1156-1160. Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine

2. Voorde, Johan Vande, et al. “Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.” Journal of Biological Chemistry 289.19 (2014): http://www.jbc.org/content/289/1…

3. Lehouritis, Panos, et al. “Local bacteria affect the efficacy of chemotherapeutic drugs.” Scientific reports 5 (2015). https://www.ncbi.nlm.nih.gov/pmc…

4. Cummins, Joanne, and Mark Tangney. “Bacteria and tumours: causative agents or opportunistic inhabitants?.” Infectious agents and cancer 8.1 (2013): 11. https://infectagentscancer.biome…

5. Livingston, Virginia Wuerthele‐Caspe, and Eleanor Alexander‐Jackson. “A specific type of organism cultivated from malignancy: bacteriology and proposed classification.” Annals of the New York Academy of Sciences 174.1 (1970): 636-654.

6. Uphoff, Cord C., and Hans G. Drexler. “Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.” In Vitro Cellular & Developmental Biology-Animal 38.2 (2002): 86-89.

7. Drexler, Hans G., et al. “Mix-ups and mycoplasma: the enemies within.” Leukemia research 26.4 (2002): 329-333.

8. Drexler, Hans G., and Cord C. Uphoff. “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.” Cytotechnology 39.2 (2002): 75-90. https://www.ncbi.nlm.nih.gov/pmc…

9. Razin, Shmuel, and Leonard Hayflick. “Highlights of mycoplasma research—an historical perspective.” Biologicals 38.2 (2010): 183-190. https://www.researchgate.net/pro…

10. Callaway, Ewen. “Contamination hits cell work: Mycoplasma infestations are widespread and costing laboratories millions of dollars in lost research.” Nature 511.7511 (2014): 518-519. https://www.nature.com/polopoly_…

11. Heidegger, Simon, et al. “Mycoplasma hyorhinis-contaminated cell lines activate primary innate immune cells via a protease-sensitive factor.” PloS one 10.11 (2015): e0142523. http://journals.plos.org/plosone…

12. Gedye, Craig, et al. “Mycoplasma infection alters cancer stem cell properties in vitro.” Stem Cell Reviews and Reports 12.1 (2016): 156-161. https://www.researchgate.net/pro…

13. Feng, Shaw-Huey, et al. “Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells.” Molecular and cellular biology 19.12 (1999): 7995-8002. http://mcb.asm.org/content/19/12…

14. Huang, Su, et al. “Mycoplasma infections and different human carcinomas.” World journal of gastroenterology 7.2 (2001): 266. https://www.ncbi.nlm.nih.gov/pmc…

15. Burke, Jennie. “Changes in direction of cancer research over the 20th century: what prompted change: research results, economics, philosophy.” (2007). http://researchdirect.westernsyd…

16. http://monographs.iarc.fr/ENG/Cl…

17. Funchain, Pauline, and Charis Eng. “Hunting for cancer in the microbial jungle.” Genome medicine 5.5 (2013): 42. https://genomemedicine.biomedcen…

https://www.quora.com/Why-are-some-tumors-more-susceptible-to-bacterial-infection-than-normal-tissue/answer/Tirumalai-Kamala

What kind of analytics on clinicaltrials.gov data will be useful for physicians, CROs, sponsors and research sites?

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Lack of Results Reporting is a Major Problem in the Home – ClinicalTrials.gov database

With >200000 global trial registrations, Home – ClinicalTrials.gov is currently the largest global clinical trials database (1). Further, the 2007 Food and Drug Administration Amendments Act of 2007 – Wikipedia (FDAAA) legally (2)

mandates timely reporting of results of applicable clinical trials to Home – ClinicalTrials.gov.

This stipulation applies to clinical trials using FDA-regulated products and having at least one US research site.

However, since this ruling focused on clinical trials registered after 2007, it excluded a disproportionately large number of registered trials from results reporting oversight (3), ironically precisely those on which many treatments in use today are based (4). It also excluded all Phase I and small feasibility studies (4).

Dismayingly, the law (see below from 4, emphasis mine) also

‘did not establish an official auditing process to ensure compliance, and enforcement is lacking.’

Thus, where Home – ClinicalTrials.gov and both government (NIH, etc) and non-government (pharma, etc) funders are concerned, having a policy and adhering to it remain egregiously and stubbornly disconnected.

Responsibility for enforcing adherence are shared by the FDA and the NIH. Trial results not reported within 12 months of trial completion entail penalty of up to US $10000 per day ($11833 per day from May 24, 2017) by the FDA as well as withholding of future grants by the NIH (3). However, the FDA has till date not imposed any penalties (4) and neither has the NIH withheld grant funding on even a single entity that conducted, finished but didn’t report results on a clinical trial within the stipulated one year of completion (5).

Crucially, even the latest tweaks to the FDAAA, the so-called Final Rule (6), effective as of January 18, 2017 (7), are unlikely to improve this situation since (see below from 8, emphasis mine),

‘the consequence of non-reporting remains unspecified…Despite ethical obligations to participants, the values espoused by academic centers, and in some instances statutory requirements, there is no effective enforcement mechanism and no repercussions to academic institutions or individual investigators for failing to meet them’

As another analysis (9) elaborates,

‘But the final rule doesn’t go far enough, mainly because FDA lacks the staff and the political will to adequately enforce it. As STAT reported in December 2015, the FDA had never levied a single fine for clinical trial reporting violations. Representatives from the FDA cite legal complexities and lack of employees, yet critics have also pointed out the FDA is effectively on the pharmaceutical industry’s payroll. Under the Prescription Drug User Fee Act, the FDA supplements its budget by charging pharmaceutical companies drug application fees that totaled $855 million in fiscal year 2015.

The current FDA commissioner, Dr. Robert Califf, has said that the FDA will not be adding staff to enforce the final rule. That’s a mistake. How else can we expect the rule to be enforced? I work in a research group that conducts more than a dozen clinical trials and know firsthand that researchers don’t have the impetus to report their trials unless there are strong incentives to do so — like enforcement and the threat of fines.’

The NIH, Major Clinical Trials Funder as well as FDAAA Rules Enforcer, Itself Fails to Adhere to its Statutory Requirements

Such lack of enforcement is nowhere more glaringly clear than by the fact that the NIH itself largely fails to adhere to the statutory requirements it is itself mandated to enforce.

A 2012 study (10) found

  • 54% of NIH-funded trials remained unpublished even 30 months after trial completion.
  • Even among the published, median time to publication was 24 (interquartile range 14 to 36) months.

A 2013 study (11) of 244 clinical trials funded by the NHLBI and completed between January 1 2000 and December 31 2011 found that

  • Funding size mattered as 97% of trials with annual budgets >$500000 per year were eventually published.
  • However, 77, 43 and 25% of clinical trials remained unpublished within 12, 30 and 48 months, respectively, of trial completion. Trials funded by small, investigator-initiated grants were precisely the ones published slowly, if at all.

A 2016 analysis (8) specifically states (emphasis mine),

‘Notably, trials funded by the National Institutes of Health and other government or academic institutions were significantly less likely to adhere to the FDA Amendments Act mandate than were trials supported by industry.’

These conclusions are supported by two other independent studies from 2015 and 2016 that analyzed the publication records of completed clinical trials registered at Home – ClinicalTrials.gov (see below from 2, 12).

The NIH Needs to Better Adhere to the Rules They’re Required to Enforce

This begs the obvious question of who will police the policers. Clearly, shockingly poor compliance with FDAAA requirements and blatant disregard of the law is unsurprising, given the NIH itself has such a clear track record of not just non-enforcement but also non-adherence itself (2, 3, 8, 12, 13).

As a government agency tasked to enforce clinical trial results reporting and as the overseer of the Home – ClinicalTrials.gov database, the NIH should set its own house in order and put its money where its mouth is by ensuring that it adheres to the legal rules it is itself responsible for enforcing. After all, could an enforcer expect to have any credibility if they themselves fail to uphold the rules they are expected to enforce?

Laxity in this regard would be hugely consequential since selective publication and non-publication of clinical trial results are a discredit to the scientific enterprise, further erode public trust in biomedical research, commit a grave and unconscionable disservice to the millions of hopeful and well-intentioned patients and volunteers who choose to participate in clinical trials, and fail to safeguard the public need for safe and reliable medicines and medical interventions.

Thus, the most basic analytics necessary on Home – ClinicalTrials.gov are those that ensure that

  • Clinical trials funded by government agencies such as the NIH publish their results on the site within one year of trial completion.
  • As a major clinical trial sponsor itself, the NIH needs to ensure that any and all adverse events associated with the trials it itself funds are reported upon trial completion clearly, comprehensively and transparently.

Bibliography

1. Trends, Charts, and Maps

2. Anderson, Monique L., et al. “Compliance with results reporting at ClinicalTrials. gov.” New England Journal of Medicine 372.11 (2015): 1031-1039. http://www.nejm.org/doi/pdf/10.1…

3. Miller, Jennifer E., David Korn, and Joseph S. Ross. “Clinical trial registration, reporting, publication and FDAAA compliance: a cross-sectional analysis and ranking of new drugs approved by the FDA in 2012.” BMJ open 5.11 (2015): e009758. http://bmjopen.bmj.com/content/b…

4. Richardson, E. “Health Policy Brief: Transparency in Clinical Research [Internet].” Health Affairs (2016). http://healthaffairs.org/healthp…

5. STAT news, Charles Piller, February 17, 2016. STAT investigation sparked improved reporting of study results, NIH says

6. Zarin, Deborah A., et al. “Trial reporting in ClinicalTrials. gov—the final rule.” New England Journal of Medicine 375.20 (2016): 1998-2004. http://www.nejm.org/doi/pdf/10.1…

7. https://www.gpo.gov/fdsys/pkg/FR…

8. Chen, Ruijun, et al. “Publication and reporting of clinical trial results: cross sectional analysis across academic medical centers.” bmj 352 (2016): i637. http://www.bmj.com/content/bmj/3…

9. STAT news, Chris Cai, January 17, 2017. New rule on clinical trial reporting doesn’t go far enough

10. Ross, Joseph S., et al. “Publication of NIH funded trials registered in ClinicalTrials. gov: cross sectional analysis.” Bmj 344 (2012): d7292. http://www.bmj.com/content/bmj/3…

11. Gordon, David, et al. “Publication of trials funded by the National Heart, Lung, and Blood Institute.” New England journal of medicine 369.20 (2013): 1926-1934. http://www.nejm.org/doi/pdf/10.1…

12. Powell-Smith, Anna, and Ben Goldacre. “The TrialsTracker: automated ongoing monitoring of failure to share clinical trial results by all major companies and research institutions.” F1000Research 5 (2016). https://f1000researchdata.s3.ama…

13. Law, Michael R., Yuko Kawasumi, and Steven G. Morgan. “Despite law, fewer than one in eight completed studies of drugs and biologics are reported on time on ClinicalTrials. gov.” Health Affairs 30.12 (2011): 2338-2345. Despite Law, Fewer Than One In Eight Completed Studies Of Drugs And Biologics Are Reported On Time On ClinicalTrials.gov

https://www.quora.com/What-kind-of-analytics-on-clinicaltrials-gov-data-will-be-useful-for-physicians-CROs-sponsors-and-research-sites/answer/Tirumalai-Kamala

How does informed consent and privacy laws apply to the storage and retesting of biopsies and other biomedical specimens?

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Biopsies can be categorized as either diagnostic or research (non-diagnostic research). Genomics costs have decreased dramatically in recent years. Spurring the push for Big data – Wikipedia, commensurate efforts to mine minuscule tissue specimens for genomics information to identify Biomarker (medicine) – Wikipedia have also increased. This in turn has increased scope and frequency of research biopsies. This answer explicates some of the overarching issues pertaining to biopsies, specifically research biopsies,

  • Their risks and benefits for patients.
  • Whether patients fully understand their specific cost versus benefit, as in whether they are providing meaningful informed consent, and
  • Ownership of the ensuing data and safeguarding its privacy.

Risks and Benefits of Research Biopsies

Biopsies are usually used to perform diagnoses. Done and discarded. So far so good. However, the situation in terms of risk and benefits for patients changes, sometimes even drastically, where research biopsies are concerned. Research biopsies entail different purposes that constitute markedly different costs versus benefits for a given patient. For example, biopsies could be primarily for discovery or to help guide therapy, two very different purposes (see below from 1).

A patient may have a considerably different appreciation of cost versus benefit of a research biopsy depending on their understanding of whether or not they’re likely to benefit directly from its research utility (2). While a biopsy taken to guide the conduct of the trial itself, say, help calibrate drug or other Rx option, is obviously beneficial to the patient themselves (3), biopsies intended for performing correlative or purely exploratory research or even for use in some future as-yet undefined research clearly entail greater cost for a patient. Not only is a given patient unlikely to benefit directly from a research biopsy, multiple samples from the same patient increase potential risk.

Additionally, some types of biopsy are inherently risky. For example, lung biopsies entail risk of Pneumothorax – Wikipedia, collapsed lung in layman’s terms, (4) while multiple consecutive liver biopsies entail risk of increased bleeding (5). Needle-track seeding or potential to alter or spread a tumor during biopsy is another concern, with the risk being low, not absent (6, 7). For example, a systematic review of 57 clinical trials found 745 research biopsies performed on a total of 576 patients had overall and major complications of 5.2% (39 of 745 biopsies) and 0.8% (6 of 745 biopsies), respectively (8).

Do patients fully understand their specific cost versus benefit from research biopsies? Some do, many don’t

Does the process of taking biospcecimens entail meaningful informed consent? That is the nub of the ethical dilemma about research biopsies. With a bestselling book and now a mainstream film, the widely publicized travails regarding ownership of and profits from HeLa cells derived from Henrietta Lacks – Wikipedia have brought the issue of informed consent center-stage in the public domain and stoked layperson interest in the issues of autonomy, transparency and ethics pertaining to the collection of human biospecimens (9).

Where in the past the biomedical research community developed the informed consent process largely on its own behind the scenes without much public input or debate, clearly such a paternalistic approach is less likely to pass muster moving forward. However, despite such heightened awareness, data also suggest we are still in a transitional period since participants’ understanding of relative benefit and harm is highly variable across studies (10, 11). Since laws and regulations vary widely across countries, unsurprisingly a wide patchwork of rules and regulations apply to the issue of informed consent for human biological materials (see below from 9, 12).

Biopsies taken under different circumstances also entail different ethical dilemmas. For example, making biopsy mandatory to get enrolled into a clinical trial creates an obvious ethical morass (13). Those in favor of course argue that human biological materials are needed from every trial participant in order to understand not just who benefits from a drug but also why and that modern molecular medicine promises to do just this (14). Those against argue linking research biopsy to clinical trial enrollment could be easily coercive since patients could perceive their loss of access to potentially beneficial, even curative, experimental therapy as actual harm (15).

Indeed, one 2015 review (16) of 55 clinical trials (n = 636) found that requirement for mandatory biopsy, in this case lung biopsy for patients with advanced non-small cell lung cancer, was a barrier to trial enrollment, specifically that more patients (83 versus 55%, respectively) received study treatment and that too earlier (after 9 or 16 days, respectively) when the trial didn’t have a mandatory biopsy requirement. This dataset implies some patients indeed balk at paying the cost (a mandatory invasive biopsy) for perceived benefit (gaining access to experimental treatment).

As to the utility value of research biopsies, despite the fact that research biopsies are often a mandatory requirement for clinical trial enrollment, i.e., patient acquiesces to submitting research biopsies in return for participating in a clinical trial in order to gain to an experimental therapeutic, one 2017 review found that fully 61% (28 of 46 trials) did not report results from research biopsies (17).

Clearly the push for big data has become pervasive enough for the element of avarice to seep into the process so much so investigators seem to be collecting material just in case for some future unspecified use. Problem with such an approach is it elides not only the individual risk to patients but also sets up a much more contentious future reckoning since so many of the issues pertaining to data ownership, privacy and confidentiality from human biospecimens are far from settled and in fact are beginning to be debated only more vigorously in the wake of widespread publicizing of the Henrietta Lacks story.

Ownership, privacy and confidentiality of human biospecimen data

According to a 2015 treatise making the case for why patients should own their medical data (see below from 18),

‘the US legal framework is constructed in a manner to block individuals from accessing their own medical data—in 49 of the 50 states in America, these data are owned by doctors and hospitals.’

Contrast this to former president Obama saying at a February 2016 White House Summit on Precision Medicine (19),

‘I would like to think that if somebody does a test on me or my genes, that that’s mine, but that’s not always how we define these issues.’

The situation is especially fraught as it pertains to reporting research biopsy results to patients. For example, two reviews (20, 21) report that in the US, research biopsy results can only be given to patients if they were obtained by, or verified in, a Clinical Laboratory Improvement Amendments – Wikipedia (CLIA)-certified laboratory. While Institutional review board – Wikipedia (IRBs) govern the conduct of clinical research, a 2013 review (1) states reporting of results back to patients is determined by the hospitals concerned on a case-by-case basis, which merely serves to increase the burden on IRBs and individual researchers.

In the US, the Common Rule – Wikipedia mandates IRB approval for US government-funded biospecimen collection but this legal requirement doesn’t apply to industry-funded research (22). Donor consent isn’t even considered necessary for research use of anonymized samples. The issue gets even more complicated especially as it pertains to data privacy when archival material is newly mined for research purposes.

Depending on the situation and country, consent can be explicit, implied, presumed, not required at all or waived under different circumstances. Consider the situation in Denmark for example where one author noted that the opt-out system for routine tissue storage (23),

‘has created a strange system of double standards: no consent is needed for using a tissue sample for research if it is taken for diagnostic purposes and used for research only at a later stage; while samples taken specifically for research must be collected with consent.’

Given the prevalent imperative for big data and inexorable globalization of biomedical research, how secure is the anonymization process for human biospecimens? However, even that question cannot be fully answered since there isn’t even a consensus definition for anonymization (see below from 24).

Would be a mistake to relegate this discussion as an issue of dry, stultifying bureaucratese since it concerns nothing less than public trust in biomedical research. People’s willingness to share data and specimens turns on whether or not the process can guarantee their privacy and confidentiality. Confidentiality is defined as (25),

‘the respectful handling of information disclosed within relationships of trust, such as healthcare relationships, especially as regards further disclosure. Confidentiality serves privacy. Researchers invariably promise to respect data-subjects’ privacy, either by de-identifying the data to make them impersonal or by handling them securely.’

Consider then the shocking conclusion from a 2013 study (26, emphasis mine) that,

a combination of a surname with other types of metadata, such as age and state, can be used to triangulate the identity of the target. A key feature of this technique is that it entirely relies on free, publicly accessible Internet resources. We quantitatively analyze the probability of identification for U.S. males. We further demonstrate the feasibility of this technique by tracing back with high probability the identities of multiple participants in public sequencing projects.’

Informed consent about human biospecimens remains a fractious issue and if history is any guide, clarity will emerge one bestseller, one lawsuit at a time.

Bibliography

1. Basik, Mark, et al. “Biopsies: next-generation biospecimens for tailoring therapy.” Nature reviews Clinical oncology 10.8 (2013): 437-450.

2. Olson, Erin M., et al. “The ethical use of mandatory research biopsies.” Nature reviews Clinical oncology 8.10 (2011): 620-625. https://www.ncbi.nlm.nih.gov/pmc…

3. Peppercorn, Jeffrey. “Toward improved understanding of the ethical and clinical issues surrounding mandatory research biopsies.” (2012): 1-2. Toward Improved Understanding of the Ethical and Clinical Issues Surrounding Mandatory Research Biopsies

4. Tam, Alda L., et al. “Feasibility of image-guided transthoracic core-needle biopsy in the BATTLE lung trial.” Journal of Thoracic Oncology 8.4 (2013): 436-442. https://www.ncbi.nlm.nih.gov/pmc…

5. Grant, A., and James Neuberger. “Guidelines on the use of liver biopsy in clinical practice.” Gut 45.suppl 4 (1999): IV1-IV11. https://www.ncbi.nlm.nih.gov/pmc…

6. Silva, Michael A., et al. “Needle track seeding following biopsy of liver lesions in the diagnosis of hepatocellular cancer: a systematic review and meta-analysis.” Gut 57.11 (2008): 1592-1596. http://citeseerx.ist.psu.edu/vie…

7. Robertson, E. G., and G. Baxter. “Tumour seeding following percutaneous needle biopsy: the real story!.” Clinical radiology 66.11 (2011): 1007-1014.

8. Overman, Michael J., et al. “Use of research biopsies in clinical trials: are risks and benefits adequately discussed?.” Journal of Clinical Oncology 31.1 (2012): 17-22. Use of Research Biopsies in Clinical Trials: Are Risks and Benefits Adequately Discussed?

9. Beskow, Laura M. “Lessons from HeLa cells: the ethics and policy of biospecimens.” Annual review of genomics and human genetics 17 (2016): 395-417. https://pdfs.semanticscholar.org…

10. Kimmelman, Jonathan, Trudo Lemmens, and Scott Y. Kim. “Analysis of consent validity for invasive, nondiagnostic research procedures.” (2013). https://www.researchgate.net/pro…

11. D’Abramo, Flavio, Jan Schildmann, and Jochen Vollmann. “Research participants’ perceptions and views on consent for biobank research: a review of empirical data and ethical analysis.” BMC medical ethics 16.1 (2015): 60. https://bmcmedethics.biomedcentr…

12. Gefenas, Eugenijus, et al. “Research on human biological materials: What consent is needed, and when.” Biobanks and tissue research. Springer Netherlands, 2011. 95-110. http://dlib.bpums.ac.ir/multiMed…

13. Helft, Paul R., and Christopher K. Daugherty. “Are we taking without giving in return? The ethics of research-related biopsies and the benefits of clinical trial participation.” (2006): 4793-4795. Are We Taking Without Giving in Return? The Ethics of Research-Related Biopsies and the Benefits of Clinical Trial Participation

14. Olson, Erin M., et al. “The ethical use of mandatory research biopsies.” Nature reviews Clinical oncology 8.10 (2011): 620-625. https://www.ncbi.nlm.nih.gov/pmc…

15. Okie, Susan. “Access before approval—a right to take experimental drugs?.” New England Journal of Medicine 355.5 (2006): 437-440. http://www.nejm.org/doi/pdf/10.1…

16. Lim, Charles, et al. “Patients with advanced non–small cell lung cancer: are research biopsies a barrier to participation in clinical trials?.” Journal of Thoracic Oncology 11.1 (2016): 79-84. http://www.jto.org/article/S1556…

17. Parseghian, Christine, et al. “Under-reporting of Research Biopsies from Clinical Trials in Oncology.” Clinical Cancer Research (2017): clincanres-1449.

18. Kish, Leonard J., and Eric J. Topol. “Unpatients [mdash] why patients should own their medical data.” Nature biotechnology 33.9 (2015): 921-924. http://getmydata.org/assets/unpa…

19. President Weighs In on Data From Genes

20. Bredenoord, Annelien L., et al. “Disclosure of individual genetic data to research participants: the debate reconsidered.” Trends in Genetics 27.2 (2011): 41-47. https://pdfs.semanticscholar.org…

21. Wolf, Susan M., et al. “Managing incidental findings and research results in genomic research involving biobanks & archived datasets.” Genetics in medicine: official journal of the American College of Medical Genetics 14.4 (2012): 361. https://pdfs.semanticscholar.org…

22. Cooreman, Ann, et al. “Point of View: Traceability and Transparency Should be Mandatory for All Human Biospecimens.” (2017). http://trans-hit.com/static/uplo…

23. Hoeyer, Klaus. “An opt out system for tissue storage: lessons from Denmark.” Bmj-British Medical Journal-Clinical Research Edition (2008).

24. Wallace, Susan E. “What Does Anonymization Mean? DataSHIELD and the Need for Consensus on Anonymization Terminology.” Biopreservation and biobanking 14.3 (2016): 224-230.

25. Lowrance, William. “Learning from experience: privacy and the secondary use of data in health research.” Journal of Health Services Research & Policy 8.1_suppl (2003): 2-7.

26. Gymrek, Melissa, et al. “Identifying personal genomes by surname inference.” Science 339.6117 (2013): 321-324. https://pdfs.semanticscholar.org…

https://www.quora.com/How-does-informed-consent-and-privacy-laws-apply-to-the-storage-and-retesting-of-biopsies-and-other-biomedical-specimens/answer/Tirumalai-Kamala

Does aluminum really cause the disease of Alzheimer’s?

The question ‘Does aluminum really cause the disease of Alzheimer’s?‘ could be more accurately re-stated as ‘Could exposure to aluminum cause Alzheimer’s disease and could a causal link even be proven?‘ simply because aluminum is such a pervasive element in modern life, it’s practically impossible to pinpoint frequency, duration and dosage of exposure at the individual level, let alone establish a cause-and-effect linkage between this one element, aluminum, on the one hand, and a complex, obviously multi-factorial disease such as Alzheimer’s (AD) on the other hand. Simply, conclusive data’s lacking. Rather epidemiological support of link between cumulative aluminum exposure and correlative risk of developing AD is confusing and inconclusive.

This answer

  • Outlines some basic facts about aluminum as it pertains to degree and variety of biological exposure.
  • Summarizes conclusions from some recent meta-analyses, and systematic and umbrella reviews on the link between Aluminum and AD.

Aluminum’s Pervasive in Human Foods, Daily Use Products and Environment

Third only to oxygen and silicon in its prevalence, aluminum is estimated the most abundant metal in the Earth’s crust (1). Although there’s as yet no evidence it is metabolized or even metabolizable by living things, its exponential industrial use from mid-20th century onwards has likewise exponentially increased human exposure to it. Though since the early 1970s, the pervasive soda can is a poster child of such use, it’s far from the only one since aluminum has now become ubiquitous in not just human food and drinks but also in construction and the aircraft industry. After all, industrial aluminum use is pervasive, being used in everything from water treatment to generate drinking water to cosmetics, food, medical use and vaccines (see sequentially below from 1, 2),

‘The largest markets for aluminium metal and its alloys are in transportation, building and construction, packaging and in electrical equipment. Transportation uses are one of the fastest growing areas for aluminium use. Aluminium powders are used in pigments and paints, fuel additives, explosives and propellants. Aluminium oxides are used as food additives and in the manufacture of, for example, abrasives, refractories, ceramics, electrical insulators, catalysts, paper, spark plugs, light bulbs, artificial gems, alloys, glass and heat resistant fibres. Aluminium hydroxide is used widely in pharmaceutical and personal care products. Food related uses of aluminium compounds include preservatives, fillers, colouring agents, anti-caking agents, emulsifiers and baking powders; soybased infant formula can contain aluminium. Natural aluminium minerals especially bentonite and zeolite are used in water purification, sugar refining, brewing and paper industries.’

Source of ingested aluminum is thus either natural, or through food and drug additives and daily use products, which constitute both consumer as well as occupational exposure (in the form of work in aluminum production and user industries).

Natural: from its presence in foods grown in aluminum-containing soils. This can vary widely since aluminum compounds are more soluble in low pH soil, which is often the consequence of acid rain. This in turn increases aluminum content in plants animals and surface water (1, 3). Drinking water is another source since Flocculation – Wikipedia, a commonly used water treatment process, uses aluminum salts (1, 2), though the concentration is estimated low, <0.2mg/liter (4).

Food and drug additives: With regard to aluminum in foods, starting sometime in the late 19th century and progressively more so since the mid-20th century, large-scale industrial food production the world over has enabled the abrupt and dramatic switch from a largely unprocessed to processed diet, the so-called ‘Western’ diet. Doing so has only increased aluminum bioavailability, especially human oral exposure. Such additives are found in dairy (milk, processed cheese, yogurt), staples (cereals, flours, grains), sweets (sugar, jams, jellies, baking sodas, powdered or crystalline dessert products (1, 2, 4). Use in food thus ranges from anticaking agents to buffers, emulsifying agents, firming agents, leavening agents, neutralizing agents and texturizers (2, see below from 4).

While diet-based aluminum consumption is estimated to be ~10mg/day, over-the-counter drugs such as analgesics and antacids can increase this by several grams per day (5). Aluminum hydroxide for example is a common antacid ingredient that helps neutralize stomach acid while aluminum in antacids helps increase bioavailability of the active ingredient which is typically poorly soluble in the stomach’s acidic environment (4).

Daily use products: Cosmetics (perspirants, sunscreens, lotions, pigments), cookware, packaging are aluminum-containing daily use products. Aluminum’s heat conductivity explains its pervasive presence in cookware. Leaching from cookware and packaging is estimated to add 2 to 4mg of aluminum per day in food, representing ~20% of daily aluminum intake (6, 7, 8).

Estimated daily exposure between countries varies as much as 4-to 8-fold (see below from 4). This makes the task of estimating cumulative exposure in epidemiological studies attempting to discern a link between aluminum and AD or any other disease all the more challenging.

Aluminum and Alzheimer’s Disease (AD): Conclusions from Meta-analyses, & Systematic and Umbrella Reviews

Alzheimer’s disease is classified as either the less frequent familial (1 to 5%) or the far more prevalent late-onset AD (LOAD), which is presumed the outcome of complex genetic, epigenetic and environmental interactions. Since hereditary factors fail to explain most AD cases, environmental factors have become prime research focus.

Aluminum emerged as a candidate in the 1960s when a 1965 study observed neurofibrillary tangle (NFT)-like degeneration after directly injecting aluminum into rabbit brains (9), i.e., lesions similar but not identical to those considered a hallmark of AD. A 1973 study followed-up with the report of higher levels of Aluminum in post-mortem AD brain samples (10).

Numerous mechanistic studies in the succeeding decades have proven inconclusive. After all, brain tissue degeneration in AD may simply make it better suited to accumulate metals such as aluminum. How to prove cause and effect? In addition, AD brain increase in aluminum levels isn’t always accompanied by aluminum level increase in CSF (cerebrospinal fluid) with some studies suggesting it does and others not (11).

Thus, establishing a conclusive link between increased human bioavailability of Aluminum and Alzheimer’s disease remains elusive. For example, aluminum use in cosmetics such as antiperspirants became a focus of attention starting in the 1980s and yet, after decades of cumulative study, the FDA concluded (12),

‘The agency does not find the current evidence sufficient to conclude that aluminum from antiperspirant use results in Alzheimer’s disease.’

Epidemiological studies trying to establish link between aluminum exposure through food and risk for AD are extremely complicated since it’s present in such a wide variety of foods. Since AD’s assumed to require years if not decades to develop, such studies would have to monitor aluminum exposure not just long-term but also at great depth, examining large study populations so that subset numbers remain large even after stratification, all amounting to a prohibitively expensive proposition.

OTOH, epidemiological studies that tried to establish link, if any, between aluminum exposure through drinking water or occupational exposure and risk for AD have more promise since there’s less ambiguity about the degree of daily and cumulative exposure. One 2016 meta-analysis of 8 such studies (4 drinking water, 4 occupational) on a total population of 10567 individuals found a significant association between aluminum exposure and risk for AD (13). Specifically, this study established chronic exposure to aluminum increased AD risk by 71%, where chronic exposure was defined as >100µg/liter aluminum in drinking water or its equivalent occupational exposure.

A 2016 umbrella review of systematic reviews and meta-analyses (14), also concluded suggestive link between aluminum and AD. Other factors that also showed up as suggestive included factors as disparate as education, herepesviridae infection, low frequency electromagnetic fields and NSAIDs. OTOH, factors they concluded were highly suggestive included cancer, depression at any age, physical activity (high level being protective). However, the authors concluded cautiously (see below from 14, emphasis mine),

‘Several risk factors present substantial evidence for association with dementia and should be assessed as potential targets for interventions, but these associations may not necessarily be causal.’

Thus, as of 2017, there is no consensus on whether and how aluminum exposure, specifically its bioavailability, influences AD risk. This is because numerous meta-analyses and systematic reviews that examined the totality of aluminum exposure, not just through drinking water or occupational exposure but also through cosmetics, over-the-counter drugs, processed food, vaccines, found the evidence to be inconclusive. For example, following a massive 2007 systematic review (1) that concluded there was little unambiguous evidence that aluminum exposure increased AD risk, a 2014 systematic review examined in great detail a total of 469 peer-reviewed studies, delving into not just exposure sources but also routes, amounts and potential toxicity to different organ systems. Evaluating the data by comparing to existing standards and guidelines for aluminum, it too concluded that (see below from 15, emphasis mine),

‘The results of the present review demonstrate that health risks posed by exposure to inorganic Al depend on its physical and chemical forms and that the response varies with route of administration, magnitude, duration and frequency of exposure. These results support previous conclusions that there is little evidence that exposure to metallic Al, the Al oxides or its salts increases risk for AD, genetic damage or cancer

Bibliography

1. Krewski, Daniel, et al. “Human health risk assessment for aluminium, aluminium oxide, and aluminium hydroxide.” Journal of Toxicology and Environmental Health, Part B 10.S1 (2007): 1-269. https://www.researchgate.net/pro…

2. Yokel, Robert A. “Aluminum in food–the nature and contribution of food additives.” (2012): 203. http://uknowledge.uky.edu/cgi/vi…

3. http://www.who.int/ipcs/publicat…

4. Vignal, C., P. Desreumaux, and M. Body-Malapel. “Gut: An underestimated target organ for Aluminum.” Morphologie 100.329 (2016): 75-84. http://www.spritzer.com.my/wp-co…

5. Reinke, Claudia M., Jörg Breitkreutz, and Hans Leuenberger. “Aluminium in over-the-counter drugs.” Drug Safety 26.14 (2003): 1011-1025.

6. Jorhem, Lars, and Georg Haegglund. “Aluminium in foodstuffs and diets in Sweden.” Zeitschrift für Lebensmitteluntersuchung und-Forschung A 194.1 (1992): 38-42.

7. Wang, L., D. Z. Su, and Y. F. Wang. “Studies on the aluminium content in Chinese foods and the maximum permitted levels of aluminum in wheat flour products.” Biomedical and environmental sciences: BES 7.1 (1994): 91-99.

8. Al Juhaiman, Layla A. “Estimating Aluminum leaching from Aluminum cook wares in different meat extracts and milk.” Journal of Saudi Chemical Society 14.1 (2010): 131-137. http://ac.els-cdn.com/S131961030…

9. Klatzo, Igor, Henryk Wiśniewski, and Eugene Streicher. “Experimental production of neurofibrillary degeneration: I. Light microscopic observations.” Journal of Neuropathology & Experimental Neurology 24.2 (1965): 187-199.

10. Crapper, D. R., S. S. Krishnan, and A. J. Dalton. “Brain aluminum distribution in Alzheimer’s disease and experimental neurofibrillary degeneration.” Science 180.4085 (1973): 511-513.

11. Kapaki, Elisabeth N., et al. “Cerebrospinal fluid aluminum levels in Alzheimer’s disease.” Biological psychiatry 33.8 (1993): 679-681.

12. FR Doc 03-14140

13. Wang, Zengjin, et al. “Chronic exposure to aluminum and risk of Alzheimer’s disease: A meta-analysis.” Neuroscience letters 610 (2016): 200-206. http://ac.els-cdn.com/S030439401…

14. Bellou, Vanesa, et al. “Systematic evaluation of the associations between environmental risk factors and dementia: An umbrella review of systematic reviews and meta-analyses.” Alzheimer’s & Dementia 13.4 (2017): 406-418.

15. Willhite, Calvin C., et al. “Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts.” Critical reviews in toxicology 44.sup4 (2014): 1-80. https://www.researchgate.net/pro…

https://www.quora.com/Does-aluminum-really-cause-the-disease-of-Alzheimers

Is T-cell ALL more common in some ethnic groups than others and if so, which are the most vulnerable groups?

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Risk factors for ALL (Acute Lymphoblastic Leukemia) are still largely unknown. However, T-cell acute lymphoblastic leukemia – Wikipedia (T-cell ALL) is known to be rarer compared to B-cell ALL, comprising 10 to 15% of childhood and 20 to 25% of adult ALL in Europe, the US and Japan (1, 2), and has more than twice higher incidence among males (1, 3, 4). One possible explanation for such male preponderance is X-linked gene mutations/deletions such as that documented for the X-linked tumor suppressor gene, PHF6 (5).

Though ALL prevalence is higher among Whites and Hispanics among US children (6), more than one study has noted a higher predisposition for T-cell ALL among blacks (7, 8, 9), with one study identifying a particular SNP associated with this higher risk (10). Overall prognosis for T-cell ALL also remains poorer compared to B-cell ALL (1). Some of these differences are summarized in the figure below from 1.

Bibliography

1. Hunger, Stephen P., and Charles G. Mullighan. “Acute lymphoblastic leukemia in children.” New England Journal of Medicine 373.16 (2015): 1541-1552. https://www.researchgate.net/pro…

2. Belver, Laura, and Adolfo Ferrando. “The genetics and mechanisms of T cell acute lymphoblastic leukaemia.” Nature Reviews Cancer 16.8 (2016): 494-507.

3. Dores, Graça M., et al. “Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007.” Blood (2011): blood-2011. http://www.bloodjournal.org/cont…

4. Goldberg, John M., et al. “Childhood T-cell acute lymphoblastic leukemia: the Dana-Farber Cancer Institute acute lymphoblastic leukemia consortium experience.” Journal of Clinical Oncology 21.19 (2003): 3616-3622.

5. Van Vlierberghe, Pieter, et al. “PHF6 mutations in T-cell acute lymphoblastic leukemia.” Nature genetics 42.4 (2010): 338-342. https://pdfs.semanticscholar.org…

6. Ward, Elizabeth, et al. “Childhood and adolescent cancer statistics, 2014.” CA: a cancer journal for clinicians 64.2 (2014): 83-103. http://onlinelibrary.wiley.com/d…

7. Bhatia, Smita, et al. “Racial and ethnic differences in survival of children with acute lymphoblastic leukemia.” Blood 100.6 (2002): 1957-1964. https://pdfs.semanticscholar.org…

8. Kadan-Lottick, Nina S., et al. “Survival variability by race and ethnicity in childhood acute lymphoblastic leukemia.” Jama 290.15 (2003): 2008-2014. https://pdfs.semanticscholar.org…

9. Pui, Ching-Hon, et al. “Results of therapy for acute lymphoblastic leukemia in black and white children.” Jama 290.15 (2003): 2001-2007. https://www.researchgate.net/pro…

10. Yang, Wenjian, et al. “ARID5B SNP rs10821936 is associated with risk of childhood acute lymphoblastic leukemia in blacks and contributes to racial differences in leukemia incidence.” Leukemia 24.4 (2010): 894. https://www.nature.com/leu/journ…

https://www.quora.com/Is-T-cell-ALL-more-common-in-some-ethnic-groups-than-others-and-if-so-which-are-the-most-vulnerable-groups/answer/Tirumalai-Kamala

How effective is the Hepatitis B vaccine? If my wife is infected and I am vaccinated can I still get it?

Given the variables including but not only age, gender, health status, HLA (Human leukocyte antigen – Wikipedia) polymorphism, the fact that humans are an outbred species as compared to the inbred nature of most experimental animal models, and many others, no vaccine could give a 100% guarantee against future infection. This is especially so for an infection such as Hepatitis B virus (HBV), which can be transmitted not just sexually or through the bloodstream (injection drug use, transfusion) but also vertically from mother to child, an exposure route that entails profoundly different risk.

A vaccine’s track record can, however, give an estimate of risk of future infection. In that regard, cumulative data support long-term protection for immunocompetent individuals who have completed a HBV vaccine schedule (1).

There are two ways to assess effectiveness of any vaccine,

  • How effective it is in preventing getting infected, preexposure vaccine efficacy, and
  • How effective it is in preventing disease in those already infected, postexposure vaccine efficacy.

The Hepatitis B vaccine – Wikipedia stimulates immune response against the HBsAg – Wikipedia (HBV surface antigen), specifically antibody responses to the ‘a determinant’ located between amino acids 100 to 160 of the HBsAg (2). Cumulative data show it to be capable of providing robust protection (3).

  • Preexposure efficacy ranges from 80 to 100%.
  • Postexposure efficacy ranges from from 85 to 95%.

Circulating anti-HBs (anti-HBaAg) antibody concentrations of >/= 10 mIU/ml (mIU = milli International Unit, International unit – Wikipedia) is commonly considered protective, being empirically found to be important since anti-HBs antibody persistence, a measure of how long protection lasts, correlates with peak antibody level immediately post-vaccination (4).

Typically, circulating levels of anti-HBs antibodies decline rapidly within the 1st year post-vaccination and more slowly from thereon, specifically, levels decline to <10 mIU/ml circulating anti-HBs antibody concentrations in ~ 7 to 50% within 5 years post-vaccination and in ~30 to 60% within 9 to 11 years post-vaccination (3).

However, the critical issue for an infected person and their partner is duration of such protection and several studies that followed up for anywhere from 15 to 23 years found that declining circulating anti-HBs antibody levels aren’t a concern since primary HBV vaccination can prevent infection for >20 years, regardless these levels (3).

Robustness of HBV vaccine protection can be surmised from several studies around the world on populations ranging from infants to adolescents to young adults. In these studies, various groups who had been vaccinated, usually starting at birth, were re-vaccinated (booster dose).

Since blood was pulled at the time of booster dose as well as 1 or more times afterwards, it was possible to compare post-boost circulating anti-HBs antibody response to the baseline pre-boost level (see table below from 1). Doing so showed that even as much as 15 to 23 years later, 62 to 76% of those previously vaccinated had a 4-fold increase in circulating anti-HBV antibody response 1, 2 or 4 weeks post-boost. This implies the HBV vaccine is capable of driving strong immune memory in diverse populations with different levels of exposure to the virus.

However, strength of recall response against HBV is only one side of the equation. How effective is the HBV vaccine in preventing infection in someone already vaccinated?

While HBV endemicity (Endemic (epidemiology) – Wikipedia) is different in different parts of the world, studies across regions with vastly varying endemicity show breakthrough infections, i.e., HBV infections in those previously vaccinated (see below from 1)

  • Is low (1 to 13.8%) in the general population.
  • Risk is higher (1.7 to 33.3%) among those born to carrier mothers.
  • Clinically significant infections are few and far between even among those with breakthrough infections, meaning that prior vaccination substantially lowers disease risk even in those who do develop breakthrough infections.
  • Risk for hepatocellular carcinoma is 70 to 80% lower among vaccinated and <50 years of age compared to historical controls (5).

Bibliography

1. Leuridan, Elke, and Pierre Van Damme. “Hepatitis B and the need for a booster dose.” Clinical Infectious Diseases 53.1 (2011): 68-75. https://www.researchgate.net/pro…

2. Coleman, Paul F. “Detecting hepatitis B surface antigen mutants.” Emerging infectious diseases 12.2 (2006): 198. https://www.ncbi.nlm.nih.gov/pmc…

3. Van Damme, Pierre, et al. “Hepatitis B vaccines.” Vaccines, 6th edn, Philadelphia, PA, USA: Elsevier (2012): 205-234.

4. Floreani, Annarosa, et al. “Long-term persistence of anti-HBs after vaccination against HBV: an 18 year experience in health care workers.” Vaccine 22.5 (2004): 607-610.

5. Viviani, Simonetta, et al. “20 years into the Gambia Hepatitis Intervention Study: assessment of initial hypotheses and prospects for evaluation of protective effectiveness against liver cancer.” Cancer Epidemiology and Prevention Biomarkers 17.11 (2008): 3216-3223. http://cebp.aacrjournals.org/con…

https://www.quora.com/How-effective-is-the-Hepatitis-B-vaccine-If-my-wife-is-infected-and-I-am-vaccinated-can-I-still-get-it/answer/Tirumalai-Kamala

Why didn’t the anti-CD19 CAR-T immunotherapies from Novartis or Kite suffer from neurotoxicity adverse events as the ones from Juno did?

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Brief Background on Anti-CD19 CAR-T Immunotherapy

So far, CAR-T immunotherapy has been most successful against CD19-positive tumors such as Acute lymphoblastic leukemia – Wikipedia (specifically B-ALL). CD19 is a cell surface molecule mainly expressed by mature B cells, meaning anti-CD19 CAR-T targets B cell tumors but also normal B cells. Despite considerable differences in CAR-T constructs used in different studies (1, 2, 3, 4), specifically

  • different short-chain variable fragments, scFv (the anti-CD19 antibody variable domain) that would be expressed on the CAR-T cell surface to bind to the CD19 expressed by the B cell tumor, and
  • different stimulating elements (CD28 or 4-1BB +/- CD3 ) that would get inserted into its cell membrane to signal within,

so far anti-CD19 CAR-Ts have yielded impressive anti-tumor responses in relapsed or refractory populations. This is interpreted as a considerable advance since these were patients who’d failed standard therapies.

Reversible Neurotoxicities as also Fatalities have occurred in various Anti-CD19 CAR-T Immunotherapy Trials

Since such studies began, different groups, including Juno, Novartis and Kite, testing CAR-T immunotherapy for B cell cancers have reported severe side effects including CRS or Cytokine release syndrome – Wikipedia and neurotoxicity as well as fatalities.

  • CRS ranged from low-grade to high-grade fevers, muscle pain (myalgia) and severe hypotension (5).
  • CNS (Central nervous system – Wikipedia) involvement is striking and unexpected since it occurred even in patients without disease in CNS. Neurotoxicity symptoms ranged from Ataxia – Wikipedia (lack of coordination), confusion and delirium to speech loss (aphasia), hallucinations, headaches, seizures (3, 4, 6, 7, 8, 9).
  • While different studies reported anti-CD19 CAR-T associated-neurotoxicities in 13 to 53% of patients (10), they were reversible with speedy admission into critical care and aggressive management.

Juno’s ROCKET trial: Too Many Fatalities, All from Cerebral Edema. Why: Patient selection criteria and/or CAR-T construct and/or dose?

Juno’s ROCKET trial was different because it had far more fatalities and all from the same cause, cerebral edema, something previously unreported. Juno Therapeutics first reported 3 fatalities among enrolled patients with resistant/refractory B-ALL (11).

Some additional background is necessary at this point. Early in tests of CAR-Ts, scientists observed that depleting patients of lymphocytes (aka lymphodepletion or conditioning) seemed to improve clinical response. Speculation is reducing patient’s own circulating lymphocytes prior to infusing them with CAR-Ts reduces competition for cytokines such as IL-7 and IL-15 that CAR-Ts need to survive and expand. Accordingly, patients in Juno’s ROCKET study also received conditioning consisting of Fludarabine – Wikipedia / Cyclophosphamide – Wikipedia, a nucleoside analog and an alkylating agent, respectively.

FDA put this trial on hold after 3 patients died but then lifted the hold in a record short time, allowing it to resume after removing Fludarabine alone, implying it was the source of increased neurotoxicity. However, two more deaths occurred even after this modification in the conditioning regimen, which led to the FDA stopping this trial.

It’s difficult to decipher what led to these fatalities since all the relevant factors aren’t (yet) in the public domain. However, comparing variables between different trials helps narrow down options (see below from 12) to patient selection and CAR-T construct and dose.

Specifically, was there something different about their patient selection criteria and CAR-T construct, and did they use a different perhaps larger dose? This is because

  • Conditioning regimen of Fludarabine/Cyclophosphamide is commonly used in such trials with no reports of similar problems so that’s out as a probable cause.
  • Juno used autologous CAR-T cells (derived from patients themselves) as did Novartis and Kite.
  • Juno used a similar CAR-T construct to the one currently being used by Kite in its anti-CD19 CAR-T trial (13). However, Juno tested it for ALL while Kite’s ongoing trial is against non-Hodgkin’s lymphoma (NHL). Safe in NHL (at least as of Aug 2017) but not in ALL patients would be an obvious speculation, and determining what the difference entails would be an obvious question to explore.
  • Novartis (14) and Fred Hutchinson Cancer Research Center (15) are using a different CAR-T construct against ALL.
    • Key difference is the signaling domain in their CAR-T is derived from 4-1BB while Juno and Novartis use one derived from CD28 (see below from 12).
    • Studies suggest the CD28 signaling domain enhances anti-tumor activity (16) while the 4-1BB signaling domain confers longer persistence (16, 17).
    • Though the Novartis (14) and FHCRC (15) trials are still ongoing, they already report high percentages of 82 (24 of 29) (18) and 93% (27 of 29) (19), respectively, complete response, which is disappearance of all clinical evidence of disease. No reports yet (as of Aug 2017) of fatalities due to cerebral edema.
  • Unfortunately, patient selection criteria and CAR-T dose Juno used in their ROCKET trial aren’t yet available in the public domain (as of Aug 2017) so conclusions about their role can only be speculative.
    • Dose is an especially interesting consideration since one FHCRC study (19) done in collaboration with and funded by Juno showed a correlation between CAR-T cell dose and time of peak CAR-T expansion.
    • What was additionally interesting about this study is that even though they infused CD4 and CD8 CAR-T cells at a 1:1 ratio, they found higher absolute numbers of CD8s at the peak of expansion.
    • Since activated CD8 Cytotoxic T cell – Wikipedia are presumably the effector cells that actually kill the tumor cells, whether dose-dependent expansion of this type of 2nd generation CD8 CAR-Ts could contribute to cerebral edema in relapsing/refractory B-ALL at the dose Juno used in its ROCKET trial is a possibility to consider.

Bibliography

1. Grupp, Stephan A., et al. “Chimeric antigen receptor–modified T cells for acute lymphoid leukemia.” New England Journal of Medicine 368.16 (2013): 1509-1518.; http://www.nejm.org/doi/pdf/10.1…

2. Kochenderfer, James N., et al. “Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation.” Blood 122.25 (2013): 4129-4139. https://pdfs.semanticscholar.org…

3. Davila, Marco L., et al. “Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.” Science translational medicine 6.224 (2014): 224ra25-224ra25. https://www.researchgate.net/pro…

4. Lee, Daniel W., et al. “T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.” The Lancet 385.9967 (2015): 517-528. https://www.researchgate.net/pro…

5. Lee, Daniel W., et al. “Current concepts in the diagnosis and management of cytokine release syndrome.” Blood 124.2 (2014): 188-195. http://www.bloodjournal.org/cont…

6. Brentjens, Renier J., et al. “CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.” Science translational medicine 5.177 (2013): 177ra38-177ra38. https://www.researchgate.net/pro…

7. Maude, Shannon L., et al. “Chimeric antigen receptor T cells for sustained remissions in leukemia.” New England Journal of Medicine 371.16 (2014): 1507-1517. http://www.nejm.org/doi/pdf/10.1…

8. Park, Jae H., et al. “Implications of minimal residual disease negative complete remission (MRD-CR) and allogeneic stem cell transplant on safety and clinical outcome of CD19-targeted 19-28z CAR modified T cells in adult patients with relapsed, refractory B-cell ALL.” (2015): 682-682.

9. Schuster, Stephen J., et al. “Sustained remissions following chimeric antigen receptor modified T cells directed against CD19 (CTL019) in patients with relapsed or refractory CD19+ lymphomas.” (2015): 183-183.

10. Wang, Zhenguang, Yelei Guo, and Weidong Han. “Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment.” Protein & Cell (2017): 1-30. https://link.springer.com/conten…

11. DeFrancesco, Laura. “Juno’s wild ride.” (2016): 793-793. https://www.nature.com/nbt/journ…

12. Hartmann, Jessica, et al. “Clinical development of CAR T cells—challenges and opportunities in translating innovative treatment concepts.” EMBO Molecular Medicine (2017): e201607485. http://embomolmed.embopress.org/…

13. A Phase 1-2 Multi-Center Study Evaluating KTE-C19 in Subjects With Refractory Aggressive Non-Hodgkin Lymphoma (ZUMA-1)

14. Determine Efficacy and Safety of CTL019 in Pediatric Patients With Relapsed and Refractory B-cell ALL

15. Laboratory Treated T Cells in Treating Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma, or Acute Lymphoblastic Leukemia – Full Text View – ClinicalTrials.gov

16. Zhao, Zeguo, et al. “Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells.” Cancer cell 28.4 (2015): 415-428. https://www.researchgate.net/pro…

17. Porter, David L., et al. “Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.” Science translational medicine 7.303 (2015): 303ra139-303ra139. http://proaa9ff3.pic19.websiteon…

18. Grupp, Stephan A., et al. “Analysis of a global registration trial of the efficacy and safety of CTL019 in pediatric and young adults with relapsed/refractory acute lymphoblastic leukemia (ALL).” (2016): 221-221.

19. Turtle, Cameron J., et al. “CD19 CAR–T cells of defined CD4+: CD8+ composition in adult B cell ALL patients.” The Journal of clinical investigation 126.6 (2016): 2123. https://www.ncbi.nlm.nih.gov/pmc…

https://www.quora.com/Why-didnt-the-anti-CD19-CAR-T-immunotherapies-from-Novartis-or-Kite-suffer-from-neurotoxicity-adverse-events-as-the-ones-from-Juno-did/answer/Tirumalai-Kamala

What are the limits of flow cytometry analysis of cell viability using propidium iodide staining?

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Many dyes can assess cell viability when using Flow cytometry – Wikipedia. Classic dyes such as such as Propidium iodide – Wikipedia (PI) and 7-Aminoactinomycin D – Wikipedia (7AAD) are

  • Cell membrane-permeable, intercalating with double-stranded nucleic acids, DNA in particular, of cells whose membranes have become more permeable, a sign that such cells are in cell membrane damaging types of distress, dying or dead.
  • Cheap.
  • Added at the end of the staining protocol, thus requiring little by way of extra time or steps.

An inexpensive, positively charged dye that cannot cross an intact plasma membrane, PI has fairly discreet excitation and emission spectra, excites at 488nm (maximum 535nm) and emits within the 570 to 630nm range (red fluorescence emission).

Full disclosure: For reasons explained below, I preferred 7AAD to PI for flow cytometry cell viability assessment and switched away from both to amine dyes as soon as they arrived on the scene.

PI Disadvantages

  • PI’s emission spectrum overlaps a little with that of FITC (Fluorescein isothiocyanate – Wikipedia) and a lot with that of PE (Phycoerythrin – Wikipedia). Compensating for PI’s spectral overlap with FITC and PE is much more challenging (1) compared to 7AAD. Having been successfully conjugated to thousands upon thousands of antibodies, these are two of the most versatile, proven workhorses in flow cytometry. PI’s overlap with FITC and PE thus severely limits the scope of a multi-staining flow cytometry antibody panel.
  • Need to use dead cell compensation control for PI and 7AAD, usually by heat killing 70oC for 30 minutes an aliquot of the cells being stained, which adds an additional variable to the experiment.
  • PI could be genotoxic/mutagenic to cells (2).
  • PI can intercalate with RNA as well (3), a quality long vastly under-appreciated by regular flow cytometry, since morphological assessment isn’t its strength. OTOH, the newer Imaging Flow Cytometry, which combines flow cytometry with fluorescence microscopy, shows increased cytoplasmic PI staining can lead to higher false positives when using standard flow cytometry protocols (4, 5).
  • RNA binding of PI is the reason RNase is used with PI in microscopy (6). However, treating samples with RNase prior to running them on flow cytometry isn’t a widespread practice (5) since RNase doesn’t penetrate live cells. It also requires fixation, which does not work for PI.
  • This brings us to cell fixation and the fact that PI only works when using live, not fixed, cells, a major drawback since running fixed cells is a major advantage of high throughput flow cytometry. Cells are typically fixed after surface staining in order to permeabilize their membranes to additionally stain intracellular proteins. Since PI binds DNA non-covalently, when cells are fixed after staining, dye bound to dead cells’ DNA could dissociate and even stain live cell DNA. After all, fixation destroys cell membrane integrity.

Being able to use them on both live and fixed cells is a major reason for the shift away over the past 10 years from PI and 7AAD towards amine dyes. Amine dyes such as Molecular Probes (Invitrogen-Thermo Fisher Scientific) Live/Dead® dye combinations

  • Are also cell membrane permeable, meaning they work on the same principle, only entering cells with compromised plasma membranes.
  • However, rather than binding DNA, they covalently bind amine groups of cellular proteins. Such dyes would thus only bind the few amines present on the cell surface of live cells but many, many more on intracellular proteins within cells with compromised cell membranes, resulting in a marked increase in fluorescence in distressed/dying/dead cells.
  • Covalent binding renders amine dyes impervious to cell fixation, meaning they remain bound only to amines of intracellular proteins within cells that were already dead to start with and won’t leak out to enter previously live, now fixed cells. This is how they can be used to assess viability even on fixed cells.
  • Are available in a wide range of excitation and emission profiles.
  • Many vendors offer amine-reactive beads to use as dead cell marker compensation control.
  • While greatly outweighed by their benefits, disadvantages of amine-reactive dyes are the extra time and step when staining for fixed cells:
    • need to stain with them first before permeabilizing and fixing cells for main staining protocol.
    • amine-dye staining step needs to be preceded by a saline wash-out step to remove free proteins to minimize non-specific staining of the solution used to suspend the cells, which would waste the reagent and make far less of it available for binding amines of intracellular proteins within cell membrane-compromised cells.
  • Since amine-reactive dyes are washed out before the staining steps are done, they track cells that died during the experiment but not the ones that die during the sorting process, when using flow cytometry to sort out subsets of live cells. For this reason and due to the extra steps, classic DNA-binding dyes such as PI and 7AAD are still preferred when using flow cytometry sorting.

Bibliography

1. Telford, William, Karen Tamul, and Jolene Bradford. “Measurement and Characterization of Apoptosis by Flow Cytometry.” Current Protocols in Cytometry (2016): 9-49.

2. The Molecular Probes Handbook

3. Deitch, ARLINE D., H. O. R. A. T. I. O. Law, and R. deVere White. “A stable propidium iodide staining procedure for flow cytometry.” Journal of Histochemistry & Cytochemistry 30.9 (1982): 967-972. http://journals.sagepub.com/doi/…

4. Rieger, Aja M., et al. “Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.” Journal of immunological methods 358.1 (2010): 81-92.

5. Rieger, Aja M., and Daniel R. Barreda. “Accurate assessment of cell death by imaging flow cytometry.” Imaging Flow Cytometry: Methods and Protocols (2016): 209-220.

6. Fried, Jerrold, Amaury G. Perez, and Bayard D. Clarkson. “Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.” The Journal of cell biology 71.1 (1976): 172-181. http://jcb.rupress.org/content/j…

https://www.quora.com/What-are-the-limits-of-flow-cytometry-analysis-of-cell-viability-using-propidium-iodide-staining/answer/Tirumalai-Kamala