\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Severe autoimmune lymphoproliferative syndrome phenotype in a pediatric
patient with a germline FAS gene variant}



\author[1]{Alexandra Dreyzin}%
\author[2]{Jinjun Cheng}%
\author[3]{David Leitenberg}%
\author[1]{Yaser Diab}%
\affil[1]{Children's National Hospital Center for Cancer and Blood Disorders}%
\affil[2]{Children's National Hospital}%
\affil[3]{The George Washington University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup








\sloppy


\textbf{Severe autoimmune lymphoproliferative syndrome phenotype in a
pediatric patient with a germline FAS gene variant}

\textbf{Authors:} Alexandra Dreyzin MD MS\textsuperscript{1}; Jinjun
Cheng MD PhD\textsuperscript{2}, David Leitenberg MD
PhD\textsuperscript{3}, and Yaser Diab MBBS\textsuperscript{1}

\begin{enumerate}
\tightlist
\item
  Center for Cancer and Blood Disorders, Children's National Hospital,
  Washington, DC
\item
  Division of Pathology and Laboratory Medicine, Children's National
  Hospital, Washington, DC
\item
  Department of Microbiology, Immunology and Tropical Medicine, The
  George Washington University School of Medicine and Health Sciences,
  Washington, District of Columbia, USA.
\end{enumerate}

\textbf{Corresponding Author:}

Alexandra Dreyzin, MD MS

Pediatric Hematology/Oncology Fellow

Center for Cancer and Blood Disorders

Children's National Hospital

The George Washington University School of Medicine and Health Sciences

111 Michigan Avenue NW

Washington, DC 20010

Phone: 202-476-2800

Fax: 202-476-3605

\textbf{Word Count:} 849

\textbf{Tables:} 1

\textbf{Figures:} 1

\textbf{Running title:} New FAS mutation in a patient with ALPS

\textbf{Key words:} ALPS, cytopenia, anemia, neutropenia,
thrombocytopenia, FAS, lymphoproliferative

\textbf{List of abbreviations:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Abbreviation & Full term\tabularnewline
\midrule
\endhead
ALPS & Autoimmune lymphoproliferative syndrome\tabularnewline
ANC & Absolute neutrophil count\tabularnewline
DNT & Double-negative T-cell\tabularnewline
G-CSF & G-Colony stimulating factor\tabularnewline
MMF & Mycophenolate mofetil\tabularnewline
\bottomrule
\end{longtable}

\textbf{Severe autoimmune lymphoproliferative syndrome phenotype in a
pediatric patient with a germline FAS gene variant}

\emph{Introduction:}

Autoimmune lymphoproliferative syndrome (ALPS) is caused by a disruption
of the apoptotic pathways that down-regulate lymphocyte
activity.\textsuperscript{1-2} Patients present with lymphadenopathy,
splenomegaly, autoimmunity, and characteristic laboratory markers
including CD3 positive, but CD4 and CD8 negative T-cells, known as
double-negative T-cells (DNTs).\textsuperscript{3} Additional biomarkers
include elevated levels of B12, IgG, IL-10, IL-18, and soluble FAS
ligand.\textsuperscript{4} The severity of disease is widely variable,
with symptoms often presenting in childhood and following a
relapsing/remitting pattern.\textsuperscript{5} While the lymphocyte
proliferation is typically polyclonal and benign, patients with ALPS are
also at increased risk of developing lymphoma, with incidence of Hodgkin
lymphoma over 100 times greater than in the general
population.\textsuperscript{6}

Many genetic mutations have been identified in patients with ALPS, most
commonly affecting the FAS receptor, its ligand, or caspases 8 and 10,
which are downstream in the apoptosis pathway.\textsuperscript{7-8} Of
note, many healthy family members of patients with ALPS have been found
to have similar mutations, suggesting variable penetrance or the need
for a ``two-hit'' process to induce clinical
symptoms.\textsuperscript{6} We present a patient with clinical features
of ALPS and evidence of FAS dysfunction who was found to have a genetic
variant of unknown significance in the FAS gene. Given our patient's
phenotype and laboratory findings, we posit that this mutation is likely
pathogenic and causative of ALPS.

\emph{Case Presentation:}

The patient presented at age 2 with left inguinal lymphadenopathy. The
lymph node biopsy showed peri-cortical expansion with increase in S-100+
histiocytes, although classical emperipolesis was not identified in
either hematoxylin and eosin (H and E) or immunostained sections (Figure
1).

He then developed pancytopenia, with hemoglobin 7.9 g/dl, platelet count
2 k/mcl, and absolute neutrophil count (ANC) 80 k/mcl. He required
intravenous immune globulins as well as steroids for persistent
thrombocytopenia. At age 4, he developed severe hemolytic anemia with
hemoglobin of 2.1 g/dl. He had altered mental status, encephalopathy
with electroencephalogram changes, and white matter ischemia on MRI,
attributed to hypoxia secondary to severe anemia. He was found to have
elevated IL-18 and DNTs, leading to a diagnosis of probable ALPS. After
a prolonged course of steroids, he started mycophenolate mofetil (MMF)
and his blood counts remained stable for several months.

The patient was lost to follow-up for about 6 months and discontinued
MMF during this time. When he returned to care, he was found to have
hepatosplenomegaly, increased DNTs (7\%), and elevated soluble FAS
ligand. He subsequently developed thrombocytopenia with mucosal
bleeding, requiring intravenous methylprednisolone. After restarting
MMF, he developed neutropenic gingivitis, requiring granulocyte-colony
stimulating factor therapy. He was then switched from MMF to sirolimus.
Blood counts normalized and laboratory testing after 6 months showed
normalization of DNTs, B12, and IL-18 levels.

Genetic testing in unsorted blood cells was performed, and he was found
to carry a heterozygous variant in FAS, Exon 9, c.857G\textgreater{}A
(p.Gly286Glu). This sequence change replaces glycine, which is neutral
and non-polar, with glutamic acid, which is acidic and polar, at codon
286 of the FAS protein (p.Gly286Glu).This variant is not present in
population databases. Algorithms developed to predict the effect of this
missense change on protein structure and function were inconclusive
(SIFT: ''Not Available''; PolyPhen-2: ''Possibly Damaging''; Align-GVGD:
''Not Available''). A FAS-mediated apoptosis assay revealed
significantly reduced apoptosis cell loss at 8\% (normal:57-100\%).

\emph{Discussion:}

We present a patient who meets clinical diagnostic criteria for
ALPS\textsuperscript{9} and has a heterozygous germline FAS variant of
previously unknown significance. His clinical phenotype was severe and
included lymphadenopathy, recurrent autoimmune cytopenias, elevated
levels of DNTs, and defective lymphocyte apoptosis. He also met several
secondary accessory criteria, including elevation of soluble FAS ligand,
IL-18, and B12 levels, and typical immunohistological findings. While he
had severe autoimmune cytopenias, he has never had elevation of IgG.
Lymph node biopsy showed S100+ histiocytosis, characteristic of
ALPS.\textsuperscript{10}

The FAS variant found in our patient is not known to be pathogenic. The
variant is on Exon 9 of the FAS receptor gene, which generally encodes
the death domain of the receptor.\textsuperscript{6,11} This is
consistent with the finding of impaired FAS-mediated apoptosis. There
has been one prior report of a similar phenotype with this genetic
variant,\textsuperscript{12} which combined with our patient's
presentation, provides evidence that this variant is pathogenic and is
associated with severe ALPS-FAS phenotype.

The variation in ALPS phenotypes likely relates to the range of genetic
variants associated with the disease. Our patient and the previously
reported patient with the same mutation share many features: both
presented in toddlerhood with lymphadenopathy followed by recurrent
cytopenias.\textsuperscript{12} The severity of our patient's
cytopenias, with acute, life-threatening drops in hemoglobin and
platelets, is remarkable and is consistent with prior work identifying
severity and frequency of cytopenias as predictors of defects in
FAS-mediated apoptosis. \textsuperscript{13} Unlike the case reported by
Gu et al., our patient did have short-term response to steroids. Both
had good response to sirolimus, with stabilization of blood counts and
normalization of DNTs. It is not clear whether severity of cytopenias is
predictive of lymphoma risk or whether disease control with sirolimus
mitigates this risk. \textsuperscript{14} Continued identification of
genetic variants and associated disease course is needed to help with
future risk stratification.



\textbf{TABLE 1 ALPS diagnostic findings identified in our patient}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Diagnostic Criterion} & \textbf{Finding}\tabularnewline
\midrule
\endhead
\textbf{Required criteria} &\tabularnewline
Evidence of Lymphoproliferation & Inguinal lymphadenopathy without
infectious cause requiring lymph node resection\tabularnewline
Double negative T cells & CD3+ CD4-CD8- T cells elevated to
7\%\tabularnewline
\textbf{Primary accessory criteria} &\tabularnewline
Defective lymphocyte apoptosis & FAS-mediated apoptosis decreased at 8\%
(normal range 57-100\%)\tabularnewline
Pathogenic FAS & *FAS mutation identified, although not known to be
pathogenic\tabularnewline
\textbf{Secondary accessory criteria} &\tabularnewline
Elevated biomarkers & Elevated B12, soluble FAS ligand, IL-2, IL-10, and
IL-18\tabularnewline
Autoimmune cytopenias & Severe anemia (Hemoglobin low of 2.1 g/dL),
neutropenia (Absolute neutrophil count low of 80/mcL), thrombocytopenia
(Platelet count low of 2 k/mcL)\tabularnewline
Immunohistologic findings & Lymph node biopsy showed histiocytosis,
positive for CD68 and S100\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure Captions:}

\textbf{Figure 1. Lymph node pathology and flow cytometry results}

The excision biopsy of inguinal lymph node shows a paracortical
expansion with a mixture of small mature lymphocytes and many
histiocytes (Panel A shows 20x and B shows 200x; H\&E stain), which are
highlighted by S100 immunostaining (Panel C, 200x). Panel D displays
flow cytometry data showing increased (\textasciitilde{}16\%) double
negative T-cells (CD4-, CD8-).

\textbf{References:}

\begin{enumerate}
\tightlist
\item
  Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY,
  Strober W, Lenardo MJ, Puck JM. Dominant interfering Fas gene
  mutations impair apoptosis in a human autoimmune lymphoproliferative
  syndrome. Cell. 1995 Jun 16;81(6):935-46.
\item
  Rieux-Laucat, F., Le Deist, F. \& Fischer, A. Autoimmune
  lymphoproliferative syndromes: genetic defects of apoptosis
  pathways.~\emph{Cell Death Differ} ~\textbf{10} , 124--133 (2003).
\item
  Bride K, Teachey D. Autoimmune lymphoproliferative syndrome: more than
  a FAScinating disease. F1000Res. 2017 Nov 1;6:1928.
\item
  Magerus-Chatinet A, Stolzenberg MC, Loffredo MS, Neven B, Schaffner C,
  Ducrot N, Arkwright PD, Bader-Meunier B, Barbot J, Blanche S, Casanova
  JL, Debr\selectlanguage{ngerman}é M, Ferster A, Fieschi C, Florkin B, Galambrun C, Hermine O,
  Lambotte O, Solary E, Thomas C, Le Deist F, Picard C, Fischer A,
  Rieux-Laucat F. FAS-L, IL-10, and double-negative CD4- CD8- TCR
  alpha/beta+ T cells are reliable markers of autoimmune
  lymphoproliferative syndrome (ALPS) associated with FAS loss of
  function. Blood. 2009 Mar 26;113(13):3027-30.
\item
  Magerus-Chatinet A, Neven B, Stolzenberg MC, Daussy C, Arkwright PD,
  Lanzarotti N, Schaffner C, Cluet-Dennetiere S, Haerynck F, Michel G,
  Bole-Feysot C, Zarhrate M, Radford-Weiss I, Romana SP, Picard C,
  Fischer A, Rieux-Laucat F. Onset of autoimmune lymphoproliferative
  syndrome (ALPS) in humans as a consequence of genetic defect
  accumulation. J Clin Invest. 2011 Jan;121(1):106-12.
\item
  Price S, Shaw PA, Seitz A, et al.:~Natural history of autoimmune
  lymphoproliferative syndrome associated with~\emph{FAS} ~gene
  mutations.~\emph{Blood.} ~2014;123(13):1989--99.
\item
  Bleesing JJH, Nagaraj CB, Zhang K. Autoimmune Lymphoproliferative
  Syndrome. 2006 Sep 14 {[}Updated 2017 Aug 24{]}. In: Adam MP, Everman
  DB, Mirzaa GM, et al., editors. GeneReviews® {[}Internet{]}. Seattle
  (WA): University of Washington, Seattle; 1993-2022.~Available from:
  https://www.ncbi.nlm.nih.gov/books/NBK1108/.
\item
  Singh R, Pradhan V, Patwardhan M, Ghosh K. APO-1/Fas gene: Structural
  and functional characteristics in systemic lupus erythematosus and
  other autoimmune diseases. Indian J Hum Genet. 2009;15(3):98-102.
\item
  Oliveira JB, Bleesing JJ, Dianzani U, Fleisher TA, Jaffe ES, Lenardo
  MJ, Rieux-Laucat F, Siegel RM, Su HC, Teachey DT, Rao VK. Revised
  diagnostic criteria and classification for the autoimmune
  lymphoproliferative syndrome (ALPS): report from the 2009 NIH
  International Workshop. Blood. 2010 Oct 7;116(14):e35-40.
\item
  Maric I, Pittaluga S, Dale JK, et al. Histologic features of sinus
  histiocytosis with massive lymphadenopathy in patients with autoimmune
  lymphoproliferative syndrome.~\emph{Am J Surg Pathol} .
  2005;29(7):903-911.
\item
  Martin DA, Zheng L, Siegel RM, Huang B, Fisher GH, Wang J, Jackson CE,
  Puck JM, Dale J, Straus SE, Peter ME, Krammer PH, Fesik S, Lenardo MJ.
  Defective CD95/APO-1/Fas signal complex formation in the human
  autoimmune lymphoproliferative syndrome, type Ia. Proc Natl Acad Sci U
  S A. 1999 Apr 13;96(8):4552-7.
\item
  Gu H, Chen Z, Ma J, Ma J, Fu L, Zhang R, Wang T, Wu R. Case report:
  Effectiveness of sirolimus in a~\emph{de novo} ~FAS mutation leading
  to autoimmune lymphoproliferative syndrome-FAS and elevated DNT/Treg
  ratio. Front Pediatr. 2022 Jul 28;10:868193.
\item
  Seif AE, Manno CS, Sheen C, Grupp SA, Teachey DT. Identifying
  autoimmune lymphoproliferative syndrome in children with Evans
  syndrome: a multi-institutional study. Blood. 2010 Mar
  18;115(11):2142-5.
\item
  Straus SE, Jaffe ES, Puck JM, et al. The development of lymphomas in
  families with autoimmune lymphoproliferative syndrome with germline
  Fas mutations and defective lymphocyte apoptosis. Blood.
  2001;98(1):194-200. Blood. 2017 Jul 13;130(2):232.
\end{enumerate}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

