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Appendix 1 Type: Journal Article

Authors: Maswabi B. C., Molinsky J., Savvulidi F., Zikmund T., Prukova D., Tuskova D., Klanova M., Vockova P., Lateckova L., Sefc L., Zivny J., Trneny M. and Klener P.

Year: 2017

Title: Hematopoiesis in patients with mature B-cell malignancies is deregulated even in patients with undetectable bone marrow involvement

Journal: Haematologica Volume: 102

Issue: 4

Pages: e152-e155

Date: Apr

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Hematopoiesis in patients with mature B cell malignancies is deregulated even in patients with undetectable bone marrow involvement

by Bokang Calvin Lenyeletse Maswabi, Jan Molinsky, Filipp Savvulidi, Tomas Zikmund, Dana Prukova, Diana Tuskova, Magdalena Klanova, Petra Vockova, Lucie Lateckova, Ludek Sefc, Jan Zivny, Marek Trneny, and Pavel Klener

Haematologica 2016 [Epub ahead of print]

Citation: Maswabi BC, Molinsky J, Savvulidi F, Zikmund T, Prukova D, Tuskova D, Klanova M, Vockova P, Lateckova L, Sefc L, Zivny J, Trneny M, and Klener P. Hematopoiesis in patients with mature B cell malignancies is deregulated even in patients with undetectable bone marrow involvement.

Haematologica. 2016; 101:xxx doi:10.3324/haematol.2016.151571

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Published Ahead of Print on January 5, 2017, as doi:10.3324/haematol.2016.151571.

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ƵƚŚŽƌƐǲŽŶƚƌŝďƵƚŝŽŶ͗>DƉĞƌĨŽƌŵĞĚŵŽƐƚŽĨƚŚĞ ĨůŽǁĐǇƚŽŵĞƚƌǇĂŶĚŐĞŶĞĞǆƉƌĞƐƐŝŽŶĂŶĂůǇƐŝƐ ĂŶĚƚŽŽŬŐƌĞĂƚƉĂƌƚŝŶǁƌŝƚŝŶŐŽĨƚŚĞŵĂŶƵƐĐƌŝƉƚ͕:DĂŶĚ&^ŚĞůƉĞĚǁŝƚŚƚŚĞĨůŽǁĐǇƚŽŵĞƚƌǇƐĞƚƵƉ ĂŶĚ ĞǀĂůƵĂƚŝŽŶ ŽĨ ĞǆƉĞƌŝŵĞŶƚƐ͕ d ƚŽŽŬ ƉĂƌƚ ŝŶ ŐĞŶĞ ĞǆƉƌĞƐƐŝŽŶ ĂŶĂůǇƐŝƐ͕ W͕ d͕ D<͕ Ws ĂŶĚ >>

ŚĞůƉĞĚǁŝƚŚ ƐĂŵƉůĞƉƌŽĐĞƐƐŝŶŐĂŶĚƐƚĂŝŶŝŶŐƉƌŽĐĞĚƵƌĞƐ͕>^͕:ĂŶĚDdĐŽŶƚƌŝďƵƚĞĚǁŝƚŚǁƌŝƚŝŶŐŽĨ ƚŚĞŵĂŶƵƐĐƌŝƉƚ͕W<ĐĂŵĞƵƉǁŝƚŚƚŚĞĚĞƐŝŐŶŽĨƚŚĞƉƌŽũĞĐƚĂŶĚƚŽŽŬƐƵďƐƚĂŶƚŝĂůƉĂƌƚŝŶǁƌŝƚŝŶŐŽĨƚŚĞ ŵĂŶƵƐĐƌŝƉƚ

ŽƌƌĞƐƉŽŶĚŝŶŐƵƚŚŽƌ͗ŽŬĂŶŐDĂƐǁĂďŝ ŵĂŝů͗ŵĂƐǁĂďŝΛŐŵĂŝů͘ĐŽŵ

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DĂƚƵƌĞĐĞůůŵĂůŝŐŶĂŶĐŝĞƐƌĞƉƌĞƐĞŶƚƚŚĞŵŽƐƚĐŽŵŵŽŶƚǇƉĞƐŽĨŚĞŵĂƚŽůŽŐŝĐƚƵŵŽƌƐ͘ĞƐƉŝƚĞƚŚĂƚ ůŝƚƚůĞ ŝŶĨŽƌŵĂƚŝŽŶ ŝƐ ĂǀĂŝůĂďůĞ ŽŶ ƚŚĞ ĐŽŵƉŽƐŝƚŝŽŶ ĂŶĚ ĨƵŶĐƚŝŽŶ ŽĨ ŚĞŵĂƚŽƉŽŝĞƐŝƐ ŝŶ ƉĂƚŝĞŶƚƐ ǁŝƚŚ ƚŚĞƐĞŵĂůŝŐŶĂŶĐŝĞƐ͘,ĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐ;,^WͿŵŝŐŚƚďĞŝŶĨůƵĞŶĐĞĚďǇĂƚůĞĂƐƚ ƚŚƌĞĞŬĞǇĨĂĐƚŽƌƐ͗ϭ͘,^WͲŝŶƚƌŝŶƐŝĐŵƵƚĂƚŝŽŶƐ;ƚŚĂƚŵŝŐŚƚƉƌĞĚŝƐƉŽƐĞ ƚŽƚŚĞĚĞǀĞůŽƉŵĞŶƚŽĨƚŚĞƐĞ ŵĂůŝŐŶĂŶĐŝĞƐͿ͕Ϯ͘ŝƌĞĐƚŽƌŝŶĚŝƌĞĐƚŝŵƉĂĐƚŽĨŵĂůŝŐŶĂŶƚůǇŵƉŚŽĐǇƚĞƐƉƌĞƐĞŶƚŝŶƚŚĞďŽŶĞŵĂƌƌŽǁŽƌ ĞǆƚƌĂ ŵĞĚƵůůĂƌǇ͕ ϯ͘ ŐĞͲƌĞůĂƚĞĚ ĐŚĂŶŐĞƐ͘^ϭͲϱdŚĞ Ăŝŵ ŽĨ ƚŚĞ ƐƚƵĚǇ ǁĂƐ ƚŽ ĂŶĂůǇǌĞ ,^W ĐŽŶƚĞŶƚ ŝŶ ƉĂƚŝĞŶƚƐǁŝƚŚƐŽĨĂƌƵŶƚƌĞĂƚĞĚŵĂƚƵƌĞͲĐĞůůŵĂůŝŐŶĂŶĐŝĞƐ͘

tĞĨŝƌƐƚĐŽŶĨŝƌŵĞĚƚŚĞĂŐĞͲƌĞůĂƚĞĚĐŚĂŶŐĞƐŽďƐĞƌǀĞĚďǇ<ƵƌĂŶĚĂ ĞƚĂů͘ŝŶŽƵƌĐŽŚŽƌƚŽĨϮϮĐŽŶƚƌŽů ƐĂŵƉůĞƐ͘^ϲ tŚŝůĞ ƚŚĞ ĂďƐŽůƵƚĞ ŶƵŵďĞƌƐ ŽĨ ŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵ ĐĞůůƐ ;,^Ϳ ĚŝĚ ŶŽƚ ĐŚĂŶŐĞ͕ ǁĞ ŽďƐĞƌǀĞĚĂŶĞŐĂƚŝǀĞ ĐŽƌƌĞůĂƚŝŽŶŽĨ ƚŚĞŝƌ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐǁŝƚŚŝŶĐƌĞĂƐŝŶŐ ĂŐĞ ;&ŝŐƵƌĞ ϭ͕ Ϳ͘ dŚĞ ĂďƐŽůƵƚĞ͕ďƵƚŶŽƚƌĞůĂƚŝǀĞŶƵŵďĞƌƐŽĨŵƵůƚŝͲƉŽƚĞŶƚƉƌŽŐĞŶŝƚŽƌƐ;DWWͿƉŽƐŝƚŝǀĞůǇĐŽƌƌĞůĂƚĞĚǁŝƚŚĂŐĞ

;&ŝŐƵƌĞϭ͕Ϳ͘dŚĞŵŽƐƚƐƚĂƚŝƐƚŝĐĂůůǇƐŝŐŶŝĨŝĐĂŶƚĐŚĂŶŐĞǁĂƐŽďƐĞƌǀĞĚŝŶƚŚĞĐŽŵƉĂƌƚŵĞŶƚŽĨŵƵůƚŝͲ ůǇŵƉŚŽŝĚƉƌŽŐĞŶŝƚŽƌƐ;D>WͿ͕ǁŚĞƌĞďŽƚŚƚŚĞĂďƐŽůƵƚĞĂŶĚƌĞůĂƚŝǀĞĨƌĞƋƵĞŶĐŝĞƐƉŽƐŝƚŝǀĞůǇĐŽƌƌĞůĂƚĞĚ ǁŝƚŚĂŐĞ;&ŝŐƵƌĞϭ͕Ϳ͘ŽƚŚĂďƐŽůƵƚĞĂŶĚƌĞůĂƚŝǀĞŶƵŵďĞƌƐŽĨƉƌŽĐĞůůƐǁĞƌĞƐŝŐŶŝĨŝĐĂŶƚůǇůŽǁĞƌŝŶ ƚŚĞĞůĚĞƌůǇĐŽŶƚƌŽůƐĂŵƉůĞƐ;&ŝŐƵƌĞϭ͕Ϳ͘/ŶĂĚĚŝƚŝŽŶƚŽƚŚĞĂŐĞͲƌĞůĂƚĞĚĐŚĂŶŐĞƐ͕ǁĞŚĂǀĞƌĞĐĞŶƚůǇ ĚĞŵŽŶƐƚƌĂƚĞĚƚŚĂƚŚĞĂůƚŚǇĂƵĐĂƐŝĂŶƐŚĂǀĞƐŝŐŶŝĨŝĐĂŶƚůǇŝŶĐƌĞĂƐĞĚƉƌŽƉŽƌƚŝŽŶƐŽĨDͲĚĞƌŝǀĞĚƉƌŽ ĐĞůůƐĐŽŵƉĂƌĞĚƚŽƐŝĂŶƐ͘^ϳdŽĂǀŽŝĚĂŶǇƉŽƚĞŶƚŝĂůĂŐĞͲŽƌƌĂĐĞͲƌĞůĂƚĞĚďŝĂƐĞƐŝŶ,^WĨƌĞƋƵĞŶĐŝĞƐ͕ƚŚĞ ĐŽŶƚƌŽů ĐŽŚŽƌƚ ƵƐĞĚ ŝŶ ƚŚŝƐ ƐƚƵĚǇ ĐŽŵƉƌŝƐĞĚ D ƐĂŵƉůĞƐ ŽďƚĂŝŶĞĚ ĨƌŽŵ ĂŐĞͲŵĂƚĐŚĞĚ ŚĞĂůƚŚǇ ĂƵĐĂƐŝĂŶƐ;ĂůůƉĂƚŝĞŶƚƐǁĞƌĞĂƵĐĂƐŝĂŶƐĂƐǁĞůůͿ͘dŚĞĨůŽǁĐǇƚŽŵĞƚƌǇŐĂƚŝŶŐƐƚƌĂƚĞŐǇŝƐŐŝǀĞŶŝŶĚĞƚĂŝů ŝŶDĂƚĞƌŝĂůƐĂŶĚDĞƚŚŽĚƐƐĞĐƚŝŽŶŽĨƚŚĞŵĂŶƵƐĐƌŝƉƚ;ƐĞĞ^ƵƉƉůĞŵĞŶƚĂƌǇĚĂƚĂͿ͘

ůů ƉĂƚŝĞŶƚ ƐĂŵƉůĞƐ ǁĞƌĞ ŽďƚĂŝŶĞĚ ĂĨƚĞƌ ǁƌŝƚƚĞŶ ŝŶĨŽƌŵĞĚ ĐŽŶƐĞŶƚ ĂĐĐŽƌĚŝŶŐ ƚŽ ƚŚĞ ,ĞůƐŝŶŬŝ ĞĐůĂƌĂƚŝŽŶ ŽĨ ϭϵϳϱ ;ƌĞǀŝƐĞĚ ŝŶ ϭϵϴϱͿ͘ dŚĞ ƐƚƵĚǇ ǁĂƐ ĂƉƉƌŽǀĞĚ ďǇ ƚŚĞ ƚŚŝĐƐ ŽŵŵŝƚƚĞĞ ŽĨ ƚŚĞ ŚĂƌůĞƐ hŶŝǀĞƌƐŝƚǇ 'ĞŶĞƌĂů ,ŽƐƉŝƚĂů ŝŶ WƌĂŐƵĞ͘ dŚĞ ƉĂƚŝĞŶƚ ĐŽŚŽƌƚ ;ŶсϭϮϱ͕ ŵĞĚŝĂŶ ĂŐĞ ϲϱ ǇĞĂƌƐͿ ŝŶĐůƵĚĞĚƐĂŵƉůĞƐŽĨĐŚƌŽŶŝĐůǇŵƉŚŽĐǇƚŝĐůĞƵŬĞŵŝĂ;>>͕ŶсϮϭͿ͕ĚŝĨĨƵƐĞůĂƌŐĞͲĐĞůůůǇŵƉŚŽŵĂ;>>͕

ŶсϯϱͿ͕ ĨŽůůŝĐƵůĂƌ ůǇŵƉŚŽŵĂ ;&>͕ ŶсϮϰͿ͕ ŵĂŶƚůĞ ĐĞůů ůǇŵƉŚŽŵĂ ;D>͕ ŶсϮϳͿ͕ ĂŶĚ ŵƵůƚŝƉůĞ ŵǇĞůŽŵĂ

;DD͕ŶсϭϴͿ͘^ƵƌƉƌŝƐŝŶŐůǇ͕ŵŽƐƚŽĨƚŚĞĂŐĞͲƌĞůĂƚĞĚĐŚĂŶŐĞƐŽďƐĞƌǀĞĚĂŵŽŶŐdZ>ƐĂŵƉůĞƐǁĞƌĞŶŽƚ ĚŝƐĐĞƌŶŝďůĞŝŶƉĂƚŝĞŶƚƐĂŵƉůĞƐ;&ŝŐƵƌĞϭ͕Ϳ͘dŚĞĞǆĐĞƉƚŝŽŶƐƚŽƚŚĞƌƵůĞǁĞƌĞĂďƐŽůƵƚĞĂŶĚƌĞůĂƚŝǀĞ ĨƌĞƋƵĞŶĐŝĞƐŽĨƉƌŽĐĞůůƐ͕ǁŚŝĐŚŶĞŐĂƚŝǀĞůǇĐŽƌƌĞůĂƚĞĚǁŝƚŚŝŶĐƌĞĂƐŝŶŐĂŐĞ;&ŝŐƵƌĞϭ͕Ϳ͘/ŶĐŽŶƚƌĂƐƚ͕

ƚŚĞ ŵŽƐƚ ƐƚĂƚŝƐƚŝĐĂůůǇ ƐŝŐŶŝĨŝĐĂŶƚ ĂŐĞͲƌĞůĂƚĞĚ ĐŚĂŶŐĞ ŽďƐĞƌǀĞĚ ŝŶ dZ>͕ ƚŚĞ ŝŶĐƌĞĂƐĞĚ ĂďƐŽůƵƚĞ ĂŶĚ ƌĞůĂƚŝǀĞŶƵŵďĞƌƐŽĨD>W͕ǁĂƐůŽƐƚŝŶƉĂƚŝĞŶƚƐĂŵƉůĞƐ;&ŝŐƵƌĞϭ͕͕ĂŶĚ&ŝŐƵƌĞϯ͕Ϳ͘dŚĞƉƌĞĐŝƐĞ ŵŽůĞĐƵůĂƌ ŵĞĐŚĂŶŝƐŵ ƵŶĚĞƌůǇŝŶŐ ƚŚĞ ŽďƐĞƌǀĞĚ ůŽƐƐ ŽĨ ĚŝĨĨĞƌĞŶĐĞ ŝŶ ƚŚĞ ĂďƐŽůƵƚĞ ĂŶĚ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐŽĨ,^͕DWWĂŶĚĞƐƉĞĐŝĂůůǇD>WƉŽƉƵůĂƚŝŽŶƐďĞƚǁĞĞŶƚŚĞǇŽƵŶŐĂŶĚƚŚĞĞůĚĞƌůǇƉĂƚŝĞŶƚƐ ƌĞŵĂŝŶƐĞůƵƐŝǀĞ͘dŚĞĚĂƚĂ͕ŚŽǁĞǀĞƌ͕ƐƚƌŽŶŐůǇƐƵŐŐĞƐƚƐƚŚĂƚŚĞŵĂƚŽƉŽŝĞƐŝƐŽĨƚŚĞĞůĚĞƌůǇ͕ďƵƚŶŽƚŽĨ ƚŚĞ ǇŽƵŶŐ ŝƐ ŝŵƉĂĐƚĞĚ ďǇ ƚŚĞƉƌĞƐĞŶĐĞŽĨ ŵĂůŝŐŶĂŶƚ ĐĞůůƐ͕ ĞŝƚŚĞƌŝŶ ƚŚĞ D͕ Žƌ ĞǆƚƌĂͲŵĞĚƵůůĂƌǇ ůŽĐĂƚĞĚ ;&ŝŐƵƌĞƐ Ϯ ͕ ĂŶĚ ϯ ͕ Ϳ͘ Ɛ Ă ĐŽŶƐĞƋƵĞŶĐĞ ƚŚĞ ĚŝƐĞĂƐĞͲƌĞůĂƚĞĚ ĂůƚĞƌĂƚŝŽŶƐ ŽĨ ƚŚĞ

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ŚĞŵĂƚŽƉŽŝĞƐŝƐ ŝŶ ƚŚĞ ĞůĚĞƌůǇ ŵŝŐŚƚ ŽǀĞƌƌŝĚĞ ƚŚĞ ĂŐĞͲƌĞůĂƚĞĚ ĐŚĂŶŐĞƐ ŶŽƌŵĂůůǇ ŽďƐĞƌǀĞĚ ŝŶ ƚŚĞ ŚĞĂůƚŚǇŝŶĚŝǀŝĚƵĂůƐ͘

ĨƚĞƌƚŚĞĂŐĞͲƌĞůĂƚĞĚĂŶĂůǇƐŝƐǁĞĂŶĂůǇǌĞĚ,^WĐŚĂŶŐĞƐŝŶƚŚĞǁŚŽůĞƉĂƚŝĞŶƚĐŽŚŽƌƚĐŽŵƉĂƌĞĚƚŽ ĂŐĞͲŵĂƚĐŚĞĚ ŚĞĂůƚŚǇ ĐŽŶƚƌŽůƐ͘ tŚŝůĞ ĂďƐŽůƵƚĞ ŶƵŵďĞƌƐ ŽĨ ,^ ;ĨƌŽŵ ƵŶƐĞůĞĐƚĞĚ ďŽŶĞ ŵĂƌƌŽǁ ŵŽŶŽŶƵĐůĞĂƌĐĞůůƐ͕DDͿǁĞƌĞŶŽƚĐŚĂŶŐĞĚ͕ƚŚĞŝƌƌĞůĂƚŝǀĞŶƵŵďĞƌƐ;ĨƌŽŵ>ŝŶ^Ͳϯϰ^нϯϴ^ͲDDͿ ǁĞƌĞƐŝŐŶŝĨŝĐĂŶƚůǇŝŶĐƌĞĂƐĞĚ;ϰϰ͘ϰйцϭϲ͘ϴǀĞƌƐƵƐϯϯ͘ϱйцϭϰ͘ϵ͕ƉсϬ͘ϬϮͿ;&ŝŐƵƌĞϮ͕Ϳ͘tŚŝůĞĂďƐŽůƵƚĞ ŶƵŵďĞƌƐŽĨDWWǁĞƌĞƐŝŐŶŝĨŝĐĂŶƚůǇĚĞĐƌĞĂƐĞĚ;Ϭ͘ϭϴϵϲйцϬ͘ϮϬϲϭǀĞƌƐƵƐϬ͘ϯϰϵϱйцϬ͘ϬϴϬϴ͕ƉсϬ͘Ϭϭ͕

&ŝŐƵƌĞ ϮͿ͕ ƚŚĞŝƌ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐ ǁĞƌĞ ŶŽƚ ĐŚĂŶŐĞĚ ;&ŝŐƵƌĞ ϮͿ͘ ŽƚŚ ƚŚĞ ĂďƐŽůƵƚĞ ĂŶĚ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐŽĨD>WǁĞƌĞƐŝŐŶŝĨŝĐĂŶƚůǇĚĞĐƌĞĂƐĞĚŝŶƉĂƚŝĞŶƚƐĐŽŵƉĂƌĞĚƚŽĐŽŶƚƌŽůƐ͗Ϭ͘ϬϰϯϭϳйцϬ͘ϬϱϱϮϰ ǀĞƌƐƵƐϬ͘ϭϰϮϭйцϬ͘ϭϯϳϬ;ƉфϬ͘ϬϬϬϭͿ͕ĂŶĚϳ͘ϳϴϵцϲ͘ϱϴϰǀĞƌƐƵƐϭϴ͘ϯϮцϭϭ͘ϯϴ;ƉфϬ͘ϬϬϬϭͿ͕ƌĞƐƉĞĐƚŝǀĞůǇ

;&ŝŐƵƌĞϮ͕Ϳ͘dŚĞD>WĚŽǁŶͲƌĞŐƵůĂƚŝŽŶǁĂƐĂůƐŽŽďƐĞƌǀĞĚŝŶƚŚĞĂŶĂůǇƐĞƐŽĨŝŶĚŝǀŝĚƵĂůĚŝĂŐŶŽƐĞƐ͕

ǁŚĞƌĞ Ăůů ƐƵďƚǇƉĞƐ ŽĨ ŵĂƚƵƌĞ ͲĐĞůů ŵĂůŝŐŶĂŶĐĞƐ ƐŚŽǁĞĚ ƌĞĚƵĐĞĚ ĂďƐŽůƵƚĞ ĂŶĚ ƌĞůĂƚŝǀĞ D>W ŶƵŵďĞƌƐ ;&ŝŐƵƌĞ Ϯ ͕ Ϳ͘ ^ŝŵŝůĂƌůǇ͕ ďŽƚŚ ĂďƐŽůƵƚĞ ;ĨƌŽŵ ƵŶƐĞůĞĐƚĞĚ DDͿ ĂŶĚ ƌĞůĂƚŝǀĞ ;ĨƌŽŵ ϯϰ^нϯϴ^нDDͿŶƵŵďĞƌƐŽĨƉƌŽĐĞůůƐǁĞƌĞŶŽƚƐƚĂƚŝƐƚŝĐĂůůǇĚŝĨĨĞƌĞŶƚŝŶƉĂƚŝĞŶƚƐĐŽŵƉĂƌĞĚƚŽ ĐŽŶƚƌŽůƐ;&ŝŐƵƌĞϮ͕Ϳ͘dŚĞĨĂĐƚƚŚĂƚ,^WĚĞƌĞŐƵůĂƚŝŽŶƐǁĞƌĞŽďƐĞƌǀĞĚĂůƐŽŝŶƉĂƚŝĞŶƚƐĂŵƉůĞƐǁŝƚŚ ƵŶĚĞƚĞĐƚĂďůĞD ŝŶĨŝůƚƌĂƚŝŽŶ ƐƚƌŽŶŐůǇ ƐƵŐŐĞƐƚƐ ƚŚĂƚ ĞǀĞŶ ĞǆƚƌĂͲŵĞĚƵůůĂƌǇůŽĐĂƚĞĚ ůǇŵƉŚŽŵĂ ŵŝŐŚƚ ŝŵƉĂĐƚ ŶŽƌŵĂů ŚĞŵĂƚŽƉŽŝĞƐŝƐ ͞ŽŶ ĚŝƐƚĂŶĐĞ͘͟ WƌĞĐŝƐĞ ŵŽůĞĐƵůĂƌ ŵĞĚŝĂƚŽƌƐ ƚŚĂƚ ĐŽƵůĚ ƌĞŵŽƚĞůǇ ĚĞƌĞŐƵůĂƚĞ ŚĞŵĂƚŽƉŽŝĞƚŝĐ ƉƌŽĐĞƐƐ ŵŝŐŚƚ ŝŶĐůƵĚĞ ĐǇƚŽŬŝŶĞƐ͕ ŐƌŽǁƚŚ ĨĂĐƚŽƌƐ͕ Žƌ ŽƚŚĞƌ ƚǇƉĞƐ ŽĨ ŵĞƐƐĞŶŐĞƌƐ ;Ğ͘Ő͘ ŵŝĐƌŽZEͿ ƉƌŽĚƵĐĞĚ ďǇ ŵĂůŝŐŶĂŶƚ ůǇŵƉŚŽĐǇƚĞƐ ĂŶĚ ƐĞĐƌĞƚĞĚ ŝŶƚŽ ƚŚĞ ƉůĂƐŵĂƚŝĐ ƉŽŽů͘

tĞ ĨƵƌƚŚĞƌ ĂŶĂůǇǌĞĚ͕ ǁŚĞƚŚĞƌ ƚŚĞ ĚĞƚĞĐƚĞĚ ĐŚĂŶŐĞƐŝŶ ĂďƐŽůƵƚĞ ĂŶĚͬŽƌ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐ ŽĨ ,^W ŵŝŐŚƚ ďĞ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƚŚĞ ĞǆƚĞŶƚ ŽĨ D ŝŶĨŝůƚƌĂƚŝŽŶ ďǇ ŶĞŽƉůĂƐƚŝĐ ĐĞůůƐ͘ ϳϳ ŽƵƚ ŽĨ ϭϮϱ ƉĂƚŝĞŶƚƐ

;ϲϭ͘ϲйͿ ŚĂĚ ĚĞƚĞĐƚĂďůĞ D ŝŶĨŝůƚƌĂƚŝŽŶ ďǇ ŝŵŵƵŶŽŚŝƐƚŽĐŚĞŵŝĐĂů ;/,Ϳ ĂŶĂůǇƐŝƐ ŽĨ ƚƌĞƉŚŝŶĞ ďŝŽƉƐǇ ƐƉĞĐŝŵĞŶƐǁŝƚŚĂƌĂŶŐĞďĞƚǁĞĞŶϮ͘ϱйĂŶĚŶĞĂƌůǇϭϬϬйĂŶĚĂŵĞĚŝĂŶŽĨϰϬй;&ŝŐƵƌĞϯͿ͘ďƐŽůƵƚĞ ŶƵŵďĞƌƐ ŽĨ Ăůů ĂŶĂůǇǌĞĚƉŽƉƵůĂƚŝŽŶƐŽĨ ,^W ǁŝƚŚ ƚŚĞ ĞǆĐĞƉƚŝŽŶ ŽĨ ,^;ŝ͘Ğ͘DWW͕D>WĂŶĚ ƉƌŽͿ ŶĞŐĂƚŝǀĞůǇĐŽƌƌĞůĂƚĞĚǁŝƚŚƚŚĞĞǆƚĞŶƚŽĨDŝŶǀŽůǀĞŵĞŶƚ;&ŝŐƵƌĞϯͿ͘WůĂƵƐŝďůǇ͕ƚŚĞĚĂƚĂƌĞĨůĞĐƚƐƚŚĞ ƐŝƚƵĂƚŝŽŶ ǁŚĞŶ ŵĂůŝŐŶĂŶƚ ůǇŵƉŚŽĐǇƚĞƐ ƉƌĞƐĞŶƚ ŝŶ ƚŚĞ D ŽƉƉƌĞƐƐ ŶŽƌŵĂů ŚĞŵĂƚŽƉŽŝĞƐŝƐ͘ EŽƚ ƐƵƌƉƌŝƐŝŶŐůǇ͕ ƚŚĞ ŵŽƐƚ ƐŝŐŶŝĨŝĐĂŶƚƐƵƉƉƌĞƐƐŝŽŶ ŽĨ ƚŚĞ ĂďƐŽůƵƚĞ,^W ŶƵŵďĞƌƐ ǁĂƐ ŽďƐĞƌǀĞĚŝŶ>>͕

ǁŚŝĐŚŚĂƐƚŚĞŚŝŐŚĞƐƚĞǆƚĞŶƚŽĨDŝŶǀŽůǀĞŵĞŶƚĨƌŽŵĂůůŽĨƚŚĞĂŶĂůǇǌĞĚĐĞůůŵĂůŝŐŶĂŶĐŝĞƐ;&ŝŐƵƌĞϮ

͕ Ϳ͘ tŚŝůĞ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐ ŽĨ ,^ ĂŶĚ D>W ĚĞŵŽŶƐƚƌĂƚĞĚ ŶĞŐĂƚŝǀĞ ĐŽƌƌĞůĂƚŝŽŶ ǁŝƚŚ D ŝŶĨŝůƚƌĂƚŝŽŶ͕ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐ ŽĨ DWW ƐŚŽǁĞĚ ƉŽƐŝƚŝǀĞ ĐŽƌƌĞůĂƚŝŽŶ ǁŝƚŚ ƚŚĞ ĞǆƚĞŶƚ ŽĨ ŝŶĨŝůƚƌĂƚŝŽŶ

;&ŝŐƵƌĞϯͿ͘dŚĞƐĞĚĂƚĂƉŽŝŶƚƚŽĂŵŽƌĞĐŽŵƉůĞǆĚĞƌĞŐƵůĂƚŝŽŶ ŽĨŚĞŵĂƚŽƉŽŝĞƐŝƐƚŚĂŶŵĞƌĞ ƐƉĂƚŝĂů ŽƉƉƌĞƐƐŝŽŶ ŽĨ ŚĞŵĂƚŽƉŽŝĞƐŝƐ ďǇ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ŵĂůŝŐŶĂŶƚ ͲĐĞůůƐ͘ dŚĞƌĞ ǁĂƐ ŶŽ ƐŝŐŶŝĨŝĐĂŶƚ ĐŽƌƌĞůĂƚŝŽŶďĞƚǁĞĞŶƌĞůĂƚŝǀĞŶƵŵďĞƌƐŽĨƉƌŽĂŶĚDŝŶĨŝůƚƌĂƚŝŽŶ;&ŝŐƵƌĞϯͿ͘

(6)

,ĞŵĂƚŽƉŽŝĞƐŝƐŝƐĂĐŽŵƉůĞǆƉƌŽĐĞƐƐŽƌĐŚĞƐƚƌĂƚĞĚďǇĚŽǌĞŶƐŽĨŚĞŵĂƚŽƉŽŝĞƚŝĐƚƌĂŶƐĐƌŝƉƚŝŽŶĨĂĐƚŽƌƐ ĂŶĚŽƚŚĞƌŝŵƉŽƌƚĂŶƚŐĞŶĞƐ͘tĞĂƐŬĞĚǁŚĞƚŚĞƌƚŚĞŽďƐĞƌǀĞĚĚŝĨĨĞƌĞŶĐĞƐŝŶƚŚĞĐŽŵƉŽƐŝƚŝŽŶŽĨ,^W ďĞƚǁĞĞŶ ƉĂƚŝĞŶƚƐ ĂŶĚ ŚĞĂůƚŚǇ ĐŽŶƚƌŽůƐ ǁŽƵůĚ ďĞ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƚƌĂŶƐĐƌŝƉƚŝŽŶĂů ĚĞƌĞŐƵůĂƚŝŽŶ ŽĨ ƚŚĞƐĞŝŵƉŽƌƚĂŶƚŵŽůĞĐƵůĞƐ͘'ĞŶĞĞǆƉƌĞƐƐŝŽŶĂŶĂůǇƐĞƐŽĨϮϳƐĞůĞĐƚĞĚŐĞŶĞƐŝŶĐůƵĚŝŶŐŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵĐĞůůͬĐĞůůͬdĐĞůůƚƌĂŶƐĐƌŝƉƚŝŽŶĨĂĐƚŽƌƐ͕ĂƉŽƉƚŽƐŝƐƌĞůĂƚĞĚŐĞŶĞƐ͕ĐĞůůĐǇĐůĞƌĞŐƵůĂƚŽƌƐ͕ƐƚĞŵĐĞůů ŵĂƌŬĞƌƐĂŶĚĂĚŚĞƐŝŽŶŵŽůĞĐƵůĞƐ͕ǁŝƚŚ'W,ĂƐĂŶŝŶƚĞƌŶĂůĐŽŶƚƌŽůǁĞƌĞĐĂƌƌŝĞĚŽƵƚŽŶƐŽƌƚĞĚ,^

ƐĂŵƉůĞƐŽďƚĂŝŶĞĚĨƌŽŵϲϯƉĂƚŝĞŶƚƐ;D>сϭϲ͖&>сϭϯ͕>>сϭϱ͕>>сϳĂŶĚDDсϭϮͿĂŶĚϭϯŚĞĂůƚŚǇ ǀŽůƵŶƚĞĞƌƐ͘tĞĐŽŶƐŝĚĞƌĞĚĂƉĂƌƚŝĐƵůĂƌŐĞŶĞƚŽďĞĞǆƉƌĞƐƐĞĚŽŶůǇŝĨŝƚƐƚƌĂŶƐĐƌŝƉƚŝŽŶǁĂƐĚĞƚĞĐƚĂďůĞ ďĞĨŽƌĞ ϰϬ ĐǇĐůĞƐ ŽĨ ƋWZ ŝŶ ŵŽƌĞ ƚŚĂŶ Ϯϱй ŽĨ ƚŚĞ ƉĂƌƚŝĐƵůĂƌ ƐĂŵƉůĞƐ͘ hƐŝŶŐ ƚŚŝƐ ĐƌŝƚĞƌŝŽŶ ǁĞ ŽďƐĞƌǀĞĚ ϯ ŐƌŽƵƉƐ ŽĨ ŐĞŶĞ ĞǆƉƌĞƐƐŝŽŶ ƉĂƚƚĞƌŶƐ ŝŶ ,^ ƐĂŵƉůĞƐ͘ 'ƌŽƵƉ ϭ ĐŽŶƐŝƐƚĞĚ ŽĨ ŐĞŶĞƐ ĞǆƉƌĞƐƐĞĚŝŶхϮϱйĐĂƐĞƐ;ďŽƚŚƉĂƚŝĞŶƚƐĂŶĚĐŽŶƚƌŽůƐͿ;^ƵƉƉůĞŵĞŶƚĂƌǇƚĂďůĞϭͿ͘'ƌŽƵƉϭĂĐŽŵƉƌŝƐĞĚ ŐĞŶĞƐǁŝƚŚƐŝŐŶŝĨŝĐĂŶƚůǇŝŶĐƌĞĂƐĞĚƌĞůĂƚŝǀĞŵZEĞǆƉƌĞƐƐŝŽŶŝŶƚŚĞƉĂƚŝĞŶƚƐĐŽŵƉĂƌĞƚŽĐŽŶƚƌŽůƐ͕ĂŶĚ ŝŶĐůƵĚĞĚƚƌĂŶƐĐƌŝƉƚŝŽŶĨĂĐƚŽƌƐZhEyϭ͕/<&ϭ;/<ZK^Ϳ͕>ϭϭ͕'dϮ͕&KyWϭ͕^W/ϭ͕Dz͕ĂƉŽƉƚŽƐŝƐ ƌĞůĂƚĞĚŐĞŶĞƐD>ϭ͕>Ϯ>ϭ͕ƐƚĞŵĐĞůůŵĂƌŬĞƌƐĂŶĚĂĚŚĞƐŝŽŶŵŽůĞĐƵůĞƐϰϰ͕WZKDϭ;ϭϯϯͿ͕ĂŶĚ ĂŶ ĞĨĨůƵǆ ƉƵŵƉ ϭ͘ 'ƌŽƵƉ ϭď ŝŶĐůƵĚĞĚ ŐĞŶĞƐ ǁŝƚŚ ƐŝŵŝůĂƌ ĞǆƉƌĞƐƐŝŽŶ ĨƌĞƋƵĞŶĐǇ ďĞƚǁĞĞŶ ƚŚĞ ƉĂƚŝĞŶƚ ĂŶĚ ĐŽŶƚƌŽů ƐĂŵƉůĞƐ͕ ĂŶĚ ŝŶĐůƵĚĞĚ ƚƌĂŶƐĐƌŝƉƚŝŽŶ ĨĂĐƚŽƌƐ /Z&ϴ͕ D/ϭ͕ &KyKϭ͕ ^Kyϭϭ͕ ĂŶĚ Ă ƐƚĞŵ ĐĞůů ŵĂƌŬĞƌ ϯϰ͘ 'ƌŽƵƉ Ϯ ĐŽŶƐŝƐƚĞĚ ŽĨ ŐĞŶĞƐ ƚŚĂƚ ǁĞƌĞ ĞǆƉƌĞƐƐĞĚ ŝŶ ƉĂƚŝĞŶƚƐ͕ ďƵƚ ŶŽƚ ŝŶ ĐŽŶƚƌŽůƐ͕ĂŶĚŝŶĐůƵĚĞĚĐĞůůƉƌŽůŝĨĞƌĂƚŝŽŶ͕ĚŝĨĨĞƌĞŶƚŝĂƚŝŽŶĂŶĚĂƉŽƉƚŽƐŝƐƌĞůĂƚĞĚŐĞŶĞƐEϭ͕EKd,ϭ͕

ĂŶĚWϳϬ͘'ƌŽƵƉϯĐŽŵƉƌŝƐĞĚŐĞŶĞƐǁŝƚŚƵŶĚĞƚĞĐƚĂďůĞĞǆƉƌĞƐƐŝŽŶŝŶďŽƚŚƉĂƚŝĞŶƚƐĂŶĚĐŽŶƚƌŽůƐĂŶĚ ŝŶĐůƵĚĞĚƚƌĂŶƐĐƌŝƉƚŝŽŶĨĂĐƚŽƌƐ &ϭ͕/Z&ϰ͕WZDϭ͕>&ϭ͕>ϲ͕ĂŶĂŶƚŝͲĂƉŽƉƚŽƚŝĐŐĞŶĞ>Ϯ͕ĂŶĚĂŶ E&ŬĂƉƉĂƌĞŐƵůĂƚŽƌD>dϭ͘dŚĞŐĞŶĞĞǆƉƌĞƐƐŝŽŶĚĂƚĂƐŚŽǁĞĚƐŝŐŶŝĨŝĐĂŶƚůǇŚŝŐŚĞƌĞǆƉƌĞƐƐŝŽŶŽĨŵŽƐƚ ŽĨ ƚŚĞ ƚĞƐƚĞĚ ƚƌĂŶƐĐƌŝƉƚŝŽŶ ĨĂĐƚŽƌƐ ŝŶ ƉĂƚŝĞŶƚƐ ĐŽŵƉĂƌĞĚ ƚŽ ĐŽŶƚƌŽůƐ ;^ƵƉƉůĞŵĞŶƚĂƌǇ ƚĂďůĞ ϭͿ͘ dŚŝƐ ĨŝŶĚŝŶŐ ƐƵŐŐĞƐƚƐ ƚŚĂƚ ,^Ɛ ŽďƚĂŝŶĞĚ ĨƌŽŵ ƉĂƚŝĞŶƚƐ ǁŝƚŚ ŵĂƚƵƌĞ ĐĞůů ŵĂůŝŐŶĂŶĐŝĞƐ ĂƌĞ ŵŽƌĞ ƚƌĂŶƐĐƌŝƉƚŝŽŶĂůůǇĂĐƚŝǀĞĐŽŵƉĂƌĞĚƚŽ,^ƐŽďƚĂŝŶĞĚĨƌŽŵĂŐĞͲŵĂƚĐŚĞĚŚĞĂůƚŚǇǀŽůƵŶƚĞĞƌƐ͘<ŝŬƵƐŚŝŐĞ ĞƚĂů͘ĚĞƐĐƌŝďĞĚƵƉƌĞŐƵůĂƚŝŽŶŽĨ/<&ϭ͕ďƵƚŶŽƚZhEyϭŽƌ^W/ϭŝŶ>>ͲĚĞƌŝǀĞĚ,^ĐŽŵƉĂƌĞĚƚŽŶŽƌŵĂů ,^͘^ϭ tŚĞƚŚĞƌ ƚŚĞƵƉƌĞŐƵůĂƚĞĚ ƚƌĂŶƐĐƌŝƉƚŝŽŶŵŝŐŚƚĐŽƌƌĞůĂƚĞ ǁŝƚŚ ƚŚĞŽďƐĞƌǀĞĚ ŝŶĐƌĞĂƐĞĚ ƌĞůĂƚŝǀĞ ŶƵŵďĞƌƐ ŽĨ ,^Ɛ ŝŶ Wd ĐŽŵƉĂƌĞĚ ƚŽ dZ>ƌĞŵĂŝŶƐ ƚŽ ďĞĞůƵĐŝĚĂƚĞĚ͘ dŚĞ ŝŶĐƌĞĂƐĞĚ ƚƌĂŶƐĐƌŝƉƚŝŽŶĂů ĂĐƚŝǀŝƚǇ ĂŶĚ ŝŶĐƌĞĂƐĞĚ ĞǆƉƌĞƐƐŝŽŶ ŽĨ ƚƌĂŶƐĐƌŝƉƚŝŽŶ ĨĂĐƚŽƌƐ͕ ŝŶĐůƵĚŝŶŐ ĞĂƌůǇ ůǇŵƉŚŽŝĚ ĚŝĨĨĞƌĞŶƚŝĂƚŝŽŶ ĂƐƐŽĐŝĂƚĞĚƚƌĂŶƐĐƌŝƉƚŝŽŶĨĂĐƚŽƌƐ;Ğ͘Ő͘/<&ϭ͕^W/ϭ͕>ϭϭͿ͕ŝŶƐŽƌƚĞĚ,^ĚŽĞƐŶŽƚĐŽƌƌĞůĂƚĞǁŝƚŚƚŚĞ ŽďƐĞƌǀĞĚƐƵƉƉƌĞƐƐŝŽŶŽĨƚŚĞĞĂƌůŝĞƐƚůǇŵƉŚŽŝĚƉƌŽŐĞŶŝƚŽƌƐ͘dŚŝƐŵŝŐŚƚďĞĞǆƉůĂŝŶĞĚďǇƚŚĞďǇƐƚĂŶĚĞƌ ĞĨĨĞĐƚ ŽĨ ƚŚĞ ŽŶŐŽŝŶŐ ŵĂůŝŐŶĂŶƚ ƉƌŽĐĞƐƐ ĂƐƐƵŵŝŶŐ ƚŚĞ ƉƌŽĚƵĐƚŝŽŶ ŽĨ ĞǆƚĞƌŶĂů ĨĂĐƚŽƌƐ ƚŚĂƚ ŵŝŐŚƚ ŵŽĚŝĨǇ ƚŚĞ ĨƵŶĐƚŝŽŶ ĂŶĚ ĚŝĨĨĞƌĞŶƚŝĂƚŝŽŶ ŽĨ ŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵ ĐĞůůƐ ƚŚƌŽƵŐŚ ŝŶŝƚŝĂƚŝŽŶ ŽĨ ǀĂƌŝŽƵƐ ĞƉŝŐĞŶĞƚŝĐĐŚĂŶŐĞƐ͘

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<EKt>'DEd

&ŝŶĂŶĐŝĂů ƐƵƉƉŽƌƚ͗ DŝŶŝƐƚƌǇ ŽĨ ,ĞĂůƚŚ ŽĨ ƚŚĞ ǌĞĐŚ ZĞƉƵďůŝĐ ŐƌĂŶƚ s ϭϱͲϮϳϳϱϳ ;ůů ƌŝŐŚƚƐ ƌĞƐĞƌǀĞĚͿ͕ 'ƌĂŶƚŐĞŶĐǇŽĨƚŚĞǌĞĐŚZĞƉƵďůŝĐŐƌĂŶƚ'ZϭϰͲϭϵϱϵϬ^͕ŚĂƌůĞƐ hŶŝǀĞƌƐŝƚǇĞŶƚĞƌŽĨ ǆĐĞůůĞŶĐĞ ŐƌĂŶƚ hE ϮϬϰϬϮϭ͕ DŝŶŝƐƚƌǇ ŽĨ ĚƵĐĂƚŝŽŶ͕ zŽƵƚŚ ĂŶĚ ^ƉŽƌƚƐ /ŶƐƚŝƚƵƚŝŽŶĂů ^ƵƉƉŽƌƚ ĨŽƌ

>ŽŶŐƚĞƌŵĞǀĞůŽƉŵĞŶƚŽĨZĞƐĞĂƌĐŚKƌŐĂŶŝǌĂƚŝŽŶƐWZsKh<WϮϰͬ>&ϭͬϯĂŶĚWZsKh<ͲϮϳͬ>&ϭͬϭ͘

^ƉĞĐŝĂůƚŚĂŶŬƐƚŽ/ƌĞŶĂ,ƌĚůŝĐŬŽǀĂ͘

(8)

Z&ZE^

ϭ͘ <ŝŬƵƐŚŝŐĞ z͕ /ƐŚŝŬĂǁĂ &͕ DŝǇĂŵŽƚŽ d͕ Ğƚ Ăů͘ ^ĞůĨͲƌĞŶĞǁŝŶŐ ŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵ ĐĞůů ŝƐ ƚŚĞ ƉƌŝŵĂƌǇƚĂƌŐĞƚŝŶƉĂƚŚŽŐĞŶĞƐŝƐŽĨŚƵŵĂŶĐŚƌŽŶŝĐůǇŵƉŚŽĐǇƚŝĐůĞƵŬĞŵŝĂ͘ĂŶĐĞƌĞůů͘ϮϬϭϭ͖ϮϬ;ϮͿ͗ϮϰϲͲ Ϯϱϵ͘Ϯ͘ DĂũĞƚŝZ͘ůŽŶĂůĞǀŽůƵƚŝŽŶŽĨƉƌĞͲůĞƵŬĞŵŝĐŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĐĞůůƐƉƌĞĐĞĚĞƐŚƵŵĂŶĂĐƵƚĞ ŵǇĞůŽŝĚůĞƵŬĞŵŝĂ͘ĞƐƚWƌĂĐƚZĞƐůŝŶ,ĂĞŵĂƚŽů͘ϮϬϭϰ͖Ϯϳ;ϯͲϰͿ͗ϮϮϵͲϮϯϰ͘

ϯ͘ ,Ƶy͕^ŚĞŶ,͕dŝĂŶ͕ĞƚĂů͘<ŝŶĞƚŝĐƐŽĨŶŽƌŵĂůŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐŝŶĂ EŽƚĐŚϭͲŝŶĚƵĐĞĚůĞƵŬĞŵŝĂŵŽĚĞů͘ůŽŽĚ͘ϮϬϬϵ͖ϭϭϰ;ϭϴͿ͗ϯϳϴϯͲϯϳϵϮ͘

ϰ͘ ƌƵŶƐ /͕ ĂĚĞĚĚƵ ZW͕ ƌƵĞĐŬŵĂŶŶ /͕ Ğƚ Ăů͘DƵůƚŝƉůĞ ŵǇĞůŽŵĂͲƌĞůĂƚĞĚ ĚĞƌĞŐƵůĂƚŝŽŶŽĨ ďŽŶĞ ŵĂƌƌŽǁͲĚĞƌŝǀĞĚϯϰ;нͿŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐ͘ůŽŽĚ͘ϮϬϭϮ͖ϭϮϬ;ϭϯͿ͗ϮϲϮϬͲϮϲϯϬ͘

ϱ͘ <ŝŬƵƐŚŝŐĞz͕DŝǇĂŵŽƚŽd͘,ĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĐĞůůĂŐŝŶŐĂŶĚĐŚƌŽŶŝĐůǇŵƉŚŽĐǇƚŝĐůĞƵŬĞŵŝĂ ƉĂƚŚŽŐĞŶĞƐŝƐ͘/Ŷƚ:,ĞŵĂƚŽů͘ϮϬϭϰ͖ϭϬϬ;ϰͿ͗ϯϯϱͲϯϰϬ͘

ϲ͘ <ƵƌĂŶĚĂ<͕sĂƌŐĂĨƚŝŐ:͕ĚĞůĂZŽĐŚĞƌĞW͕ĞƚĂů͘ŐĞͲƌĞůĂƚĞĚĐŚĂŶŐĞƐŝŶŚƵŵĂŶŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵͬƉƌŽŐĞŶŝƚŽƌĐĞůůƐ͘ŐŝŶŐĞůů͘ϮϬϭϭ͖ϭϬ;ϯͿ͗ϱϰϮͲϱϰϲ͘

ϳ͘ DŽůŝŶƐŬǇ:͕DĂƐǁĂďŝ͕WƌƵŬŽǀĂ͕ĞƚĂů͘^ŝŐŶŝĨŝĐĂŶƚůǇŚŝŐŚĞƌŶƵŵďĞƌƐŽĨƉƌŽĐĞůůƐŝŶŚĞĂůƚŚǇ ĂƵĐĂƐŝĂŶƐ ĐŽŵƉĂƌĞĚ ƚŽ ƐŝĂŶƐ͗ /Ɛ ƚŚĞƌĞ ĂƐƐŽĐŝĂƚŝŽŶ ǁŝƚŚ ŝŶĐŝĚĞŶĐĞ ŽĨ >>͍͘ ůŽŽĚ ĞůůƐ DŽů ŝƐ͘

ϮϬϭϲ͖ϱϳ͗ϭϭϴͲϭϭϵ͘

(9)

Legend to Figures

&ŝŐƵƌĞϭ͘ŽƌƌĞůĂƚŝŽŶŽĨŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐǁŝƚŚĂŐĞ͘ŐĞͲƌĞůĂƚĞĚĐŚĂŶŐĞƐŽĨ ĂďƐŽůƵƚĞ ;͕ Ϳ ĂŶĚ ƌĞůĂƚŝǀĞ ;͕ Ϳ ŶƵŵďĞƌƐ ŽĨ ŚĞŵĂƚŽƉŽŝĞƚŝĐ ƐƚĞŵ ĂŶĚ ƉƌŽŐĞŶŝƚŽƌ ĐĞůůƐ ;,^WͿ ƉŽƉƵůĂƚŝŽŶƐ ŝŶ ƚŚĞ DͲĚĞƌŝǀĞĚ ƐĂŵƉůĞƐ ŽďƚĂŝŶĞĚ ĨƌŽŵ ŚĞĂůƚŚǇ ĐŽŶƚƌŽůƐ ;dZ>Ϳ ;ŶсϮϮͿ ;͕ Ϳ͕ ĂŶĚ ƉĂƚŝĞŶƚƐ;WdͿǁŝƚŚŵĂƚƵƌĞĐĞůůŵĂůŝŐŶĂŶĐŝĞƐ;ŶсϭϮϱͿ;͕Ϳ͘>ŝŶĞĂƌƌĞŐƌĞƐƐŝŽŶĂŶĂůǇƐŝƐĚĞŵŽŶƐƚƌĂƚĞƐ ĐŽƌƌĞůĂƚŝŽŶ ŽĨ ,^W ĨƌĞƋƵĞŶĐŝĞƐ ǁŝƚŚ ĂŐĞ͘ WĞĂƌƐŽŶǲƐ ĐŽƌƌĞůĂƚŝŽŶ ĐŽĞĨĨŝĐŝĞŶƚƐ ;ƌͿ͕ ĂŶĚ ƉͲǀĂůƵĞƐ ĂƌĞ ƐŚŽǁŶ͘

&ŝŐƵƌĞϮ͘ŶĂůǇƐŝƐŽĨŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌƐĐĞůůƐĨƌĞƋƵĞŶĐŝĞƐŝŶǀĂƌŝŽƵƐĚŝĂŐŶŽƐŝƐĂŶĚ ŝŶŝŶĨŝůƚƌĂƚĞĚǀĞƌƐƵƐƵŶͲŝŶĨŝůƚƌĂƚĞĚƉĂƚŝĞŶƚƐ͘ ďƐŽůƵƚĞ;͕ͿĂŶĚƌĞůĂƚŝǀĞ;͕ͿŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵ ĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐĨƌĞƋƵĞŶĐŝĞƐŝŶĂůůƉĂƚŝĞŶƚƐĂŵƉůĞƐ;͕ͿĂŶĚƉĂƌƚŝĐƵůĂƌĐĞůůŵĂůŝŐŶĂŶĐŝĞƐ;͕Ϳ ĐŽŵƉĂƌĞĚƚŽĂŐĞͲŵĂƚĐŚĞĚĐŽŶƚƌŽůƐ͘^ƵďͲĂŶĂůǇƐĞƐŽĨƐĂŵƉůĞƐǁŝƚŚĚĞƚĞĐƚĂďůĞĂŶĚƵŶĚĞƚĞĐƚĂďůĞD ŝŶĨŝůƚƌĂƚŝŽŶĂƌĞĚĞŵŽŶƐƚƌĂƚĞĚ͘ WͲǀĂůƵĞƐ ŽĨ ƵŶƉĂŝƌĞĚ ^ƚƵĚĞŶƚ͛Ɛ ƚͲƚĞƐƚƐ ĂƌĞ ƐŚŽǁŶ ĂƚƚŚĞ ƚŽƉ ŽĨĞĂĐŚ ĐŽůƵŵŶ͘͞ŶƐ͟ƐƚĂŶĚƐĨŽƌŶŽƚƐŝŐŶŝĨŝĐĂŶƚ͘DĞĂŶƐĂŶĚƐƚĂŶĚĂƌĚĚĞǀŝĂƚŝŽŶƐĂƌĞŐƌĂƉŚŝĐĂůůǇĞǆƉƌĞƐƐĞĚ͘

&ŝŐƵƌĞ ϯ͘ŽƌƌĞůĂƚŝŽŶ ŽĨ ďŽƚŚ ŝŶĨŝůƚƌĂƚŝŽŶ ĞǆƚĞŶƚ ĂŶĚ ǇŽƵŶŐĞƌ ǀĞƌƐƵƐ ŽůĚĞƌ ƉĂƚŝĞŶƚƐ ǁŝƚŚ ŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚƉƌŽŐĞŶŝƚŽƌĐĞůůƐĨƌĞƋƵĞŶĐŝĞƐ͘ KǀĞƌǀŝĞǁŽĨ ƚŚĞĞǆƚĞŶƚ ŽĨDŝŶǀŽůǀĞŵĞŶƚ ƉĞƌĚŝĂŐŶŽƐŝƐ;ͿĂŶĚĐŽƌƌĞůĂƚŝŽŶŽĨĂďƐŽůƵƚĞ;ͿĂŶĚƌĞůĂƚŝǀĞ;Ϳ,^WĨƌĞƋƵĞŶĐŝĞƐǁŝƚŚƚŚĞĞǆƚĞŶƚŽĨ DŝŶĨŝůƚƌĂƚŝŽŶŝŶƉĂƚŝĞŶƚƐĂŵƉůĞƐǁŝƚŚĚĞƚĞĐƚĂďůĞDŝŶĨŝůƚƌĂƚŝŽŶ͘WĞĂƌƐŽŶĐŽƌƌĞůĂƚŝŽŶĐŽĞĨĨŝĐŝĞŶƚƐ;ƌͿ͕

ĂŶĚƉͲǀĂůƵĞƐĂƌĞƐŚŽǁŶ͘WĂŶĞůƐĂŶĚƐŚŽǁĂďƐŽůƵƚĞ;ͿĂŶĚƌĞůĂƚŝǀĞ;ͿŚĞŵĂƚŽƉŽŝĞƚŝĐƐƚĞŵĂŶĚ ƉƌŽŐĞŶŝƚŽƌĐĞůůƐ;,^WͿĨƌĞƋƵĞŶĐŝĞƐŝŶDͲĚĞƌŝǀĞĚƉĂƚŝĞŶƚ;WdͿĂŶĚĐŽŶƚƌŽů;dZ>ͿƐĂŵƉůĞƐŽďƚĂŝŶĞĚ ĨƌŽŵ ƚŚĞ ǇŽƵŶŐ ;ч ϰϱͿ ĂŶĚ ƚŚĞ ĞůĚĞƌůǇ ;хϰϱ ǇĞĂƌƐͿ͘ DĞĂŶƐ ĂŶĚ ƐƚĂŶĚĂƌĚ ĚĞǀŝĂƚŝŽŶƐ ƚŽŐĞƚŚĞƌ ǁŝƚŚ

^ƚƵĚĞŶƚ͛ƐƚͲƚĞƐƚƉͲǀĂůƵĞƐĂƌĞƐŚŽǁŶĂƚƚŚĞƚŽƉŽĨƚŚĞƉĂŝƌĞĚĐŽůƵŵŶƐ͘͞ŶƐ͟ƐƚĂŶĚƐĨŽƌŶŽŶͲƐŝŐŶŝĨŝĐĂŶƚ͘

(10)

Age (Years)

202530354045505560657075800

10

20

30

40

50

60

70

80 Age (Years)2025303540455055606570758001020304050607080 Age (Years)

202530354045505560657075800

510

15

20

25

30

35

40 Age (Years)20253035404550556065707580010203040506070

Age (Years)

202530354045505560657075800.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 Age (Years)

0.0

0.1

0.2

0.3

0.4 2530354045505560657075800.00.10.20.30.4

0.5 Age (Years)

253035404550556065707580-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00 r= -0.57 p= 0.005

r= 0.74 p< 0.0001r= 0.54 p= 0.01r= -0.007 p= 0.97 r= 0.74 p< 0.0001

r= 0.14 p= 0.54

r= -0.42 p= 0.04 r= -0.43 p= 0.04

Age (Years) 253035404550556065707580850.00.10.20.30.40.50.60.70.8 253035404550556065707580850.00.10.20.30.40.50.60.70.8 25303540455055606570758085

0.00

0.05

0.10

0.15

0.20

0.25

0.30 253035404550556065707580850.00.20.40.60.8

1.0 253035404550556065707580850102030405060708090100 253035404550556065707580850102030405060708090100 25303540455055606570758085

0

510

15

20

25

30

35

40 25303540455055606570758085010203040506070

80r= -0.005 p= 0.95r= -0.13 p= 0.13

r= 0.12 p= 0.15r= -0.23 p= 0.01

r= -0.30 p= 0.0006r= -0.09 p= 0.27r= -0.07 p= 0.38r= -0.12 p= 0.17 Age (Years)Age (Years)Age (Years)Age (Years) Age (Years)Age (Years)Age (Years)Age (Years)

A. D.C.B.

Relative number

+ CD90 CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA

-/CD34

CD38 +

(%) -

Relative number

CD90

-CD45RA

/CD34 +

CD38 +

(%) -

Relative number CD19 CD10 +

/CD34 +

CD38 +

(%) +

Absolute numbers of

CD34 CD38 +

-CD90

CD45RA + -

from BMMC (%) Absolute numbers of

+ CD34

- CD38

- CD90

CD45RA

-

CD34 CD38 +

-CD90

-CD45RA

+

CD34 CD38 +

-CD10

CD19 + +

from BMMC (%)

+ CD34

- CD38

+ CD90 CD45RA

-

from BMMC (%)

253035404550556065707580

CTRL HSCCTRL ProBCTRL MLPCTRL MPP PT HSCPT ProBPT MLPPT MPP

CTRL HSCCTRL ProBCTRL MLPCTRL MPP

PT HSCPT ProBPT MLPPT MPP

+ CD34

- CD38

- CD90

CD45RA

-

CD34 CD38 +

-CD90

-CD45RA

+

CD34 CD38 +

-CD10

CD19 + +

from BMMC (%)

Relative number

+ CD90 CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA

-/CD34

CD38 +

(%) -

Relative number

CD90

-CD45RA

/CD34 +

CD38 +

(%) -

/CD34 +

CD38 +

(%) +

(11)

CTRLPT UninfiltratedInfiltrated

0.00

0.25

0.50

0.75

1.00

1.25

1.50nsnsns 0.000.250.500.751.001.25ns0.01< 0.0001 00.10.20.30.4< 0.00010.008< 0.0001 0.000.250.500.751.001.251.501.752.00nsnsns 0

10

20

30

40

50

60

70

80

901000.020.020.03 0102030405060708090

100 05101520253035

< 0.0001< 0.0001< 0.0001 010203040506070ns

PT UninfiltratedInfiltratedPT UninfiltratedInfiltratedPT UninfiltratedInfiltrated nsns

CTRLCTRLCTRL CTRLPT UninfiltratedInfiltratedCTRLPT UninfiltratedInfiltratedCTRLPT UninfiltratedInfiltratedCTRLPT UninfiltratedInfiltrated

nsnsns

A. FLDLBCLMCLCLLMM020406080 FLDLBCLMCLCLLMM020406080100 FLDLBCLMCLCLLMM

010

20

30

40 CTRLFLDLBCLMCLCLLMM020406080

CTRLFLDLBCLMCLCLLMM-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 CTRLFLDLBCLMCLCLLMM

0.0

0.2

0.4

0.6

0.8

1.0 FLDLBCLMCLCLLMM

0.0

0.1

0.2

0.3

0.4 FLDLBCLMCLCLLMM

0.0

0.5

1.0

1.5

2.0nsnsnsnsns0.04ns0.030.02<0.0001 CTRL

0.020.010.00030.007<0.0001 CTRL

nsnsnsns0.002 0.0060.050.02nsns CTRLCTRL

0.05nsnsns0.050.0030.00050.00020.02<0.0001 CTRL

ns0.0070.005nsns

B. D.C.

HSCProBMLPMPP HSCProBMLPMPP HSCProBMLPMPP HSCProBMLPMPP

CD34 CD38 +

-CD90

CD45RA + -

+ CD34

- CD38

- CD90

CD45RA

-

CD34 CD38 +

-CD90

-CD45RA

+

CD34 CD38 +

-CD10

CD19 + +

from BMMC (%)

CD34 CD38 +

-CD90

CD45RA + -

+ CD34

- CD38

- CD90

CD45RA

-

CD34 CD38 +

-CD90

-CD45RA

+

CD34 CD38 +

-CD10

CD19 + +

from BMMC (%)

Relative number

+ CD90 CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA /CD34 +

CD38 +

(%) -

/CD34 +

CD38 +

(%) +

Relative number

+ CD90 CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA

-/CD34

CD38 +

(%) -

Relative number

- CD90

CD45RA /CD34 +

CD38 +

(%) -

/CD34 +

CD38 +

(%) +

(12)

E.

Infiltrated and Uninfiltrated samples (%)

Uninfiltrated Infiltrated Bone Marrow Infiltrated vs Uninfiltrated

Infiltration of B.M by IHC (%)

Bone Marrow Infiltration Extent

DLBCL FL MCL MM CLL

r= -0.30 p = 0.007

r= -0.40 p= 0.0004

r= -0.39 p= 0.0004

r= 0.40

p= 0.0003 r= -0.41

p= 0.0002 r= 0.05

p= 0.64

Bone Marrow Infiltration Extent by IHC (%) r= -0.15

p= 0.18

0 10 20 30 40 50 60 70 80 90100 0.0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 10 20 30 40 50 60 70 80 90100 0.0

0.1 0.2 0.3 0.4 0.5 0.6

0 10 20 30 40 50 60 70 80 90100 0.000

0.025 0.050 0.075 0.100 0.125 0.150

0 10 20 30 40 50 60 70 80 90100 0

10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 70 80 90100 0

10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 70 80 90100 0

5 10 15 20 25 30 35 40

0 10 20 30 40 50 60 70 80 90 100 0

10 20 30 40 50 60 70 80

0 10 20 30 40 50 60 70 80 90 100 0.0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

r= -0.25 p= 0.02

D.

A.

Absolute numbers of CD34+CD38-CD90+CD45RA- from BMMC (%) Absolute numbers of CD34+CD38-CD90-CD45RA- from BMMC (%) Absolute numbers of CD34+CD38-CD90-CD45RA+ from BMMC (%) Absolute numbers of CD34+CD38-CD10+CD19+ from BMMC (%)

Relative number CD90+CD45RA-/CD34+CD38- (%) Relative number CD90-CD45RA-/CD34+CD38- (%) Relative number CD90-CD45RA+/CD34+CD38- (%) Relative number CD19+CD10+/CD34+CD38+ (%)

HSC MPP MLP ProB

B.

C.

p <0.0001

p <0.0001 p <0.01 p <0.005

HSC MPP MLP ProB

0.00 0.25 0.50 0.75 1.00 1.25 1.50

0.00 0.25 0.50 0.75 1.00 1.25

0.0 0.1 0.2 0.3 0.4

-0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

0 10 20 30 40 50 60 70 80 90 100

0 5 10 15 20 25 30 35 40

0 10 20 30 40 50 60 70 Absolute numbers of CD34+CD38-CD90+CD45RA- from BMMC (%)

CTRLS ≤45 Years

PTs ≤45 Years

CTRLs >45 Years

PTs >45 Years

Absolute numbers of CD34+CD38-CD90-CD45RA- from BMMC (%)

PTs ≤45 Years

PTs >45 Years

Absolute numbers of CD34+CD38-CD90-CD45RA+ from BMMC (%)

PTs ≤45 Years

PTs >45 Years

Absolute numbers of CD34+CD38-CD10+CD19+ from BMMC (%)

PTs ≤45 Years

PTs >45 Years

Relative number CD90+CD45RA-/CD34+CD38- (%) Relative number CD90-CD45RA-/CD34+CD38- (%) Relative number CD90-CD45RA+/CD34+CD38- (%) Relative number CD19+CD10+/CD34+CD38+ (%)

PTs ≤45 Years

PTs >45 Years CTRLS ≤45

Years PTs ≤45 Years

PTs >45 Years CTRLS ≤45

Years PTs ≤45 Years

PTs >45 Years

PTs ≤45 Years

PTs >45 Years

(13)

SUPPLEMENTAL MATERIAL

MATERIALS AND METHODS Clinical samples

Between February 28, 2012 and April 24, 2014 we analyzed 125 bone marrow (BM) samples from newly diagnosed patients with mature B cell malignancies, including CLL (n=21, median 66 , range 40-79), MCL (n=27, median 65, range 48-79), DLBCL (n=35, median 65, range 29-81), FL (n=24, median 63, range 40- 82), and MM (n=18, median 65, range 30-73). 22 BM samples were obtained from healthy volunteers. Of these, 13 age-matched BM samples (median age 63, range 29-78) were used for comparison to the whole PT cohort (n=125), 15 (younger) age-matched BM samples were used for comparison to the younger patients (i.e. <45 years, median age 27, range 22-45), and 7 (older) age-matched BM samples were used for compĂƌŝƐŽŶƚŽƚŚĞĞůĚĞƌůǇƉĂƚŝĞŶƚƐ;шϰϱǇĞĂƌƐ, median age 70, range 63-78). All samples were obtained after written informed consent according to the Helsinki Declaration of 1975 (revised in 1985). The study was approved by Ethics Committee of the Charles University General Hospital in Prague. Bone marrow mononuclear cells (BMMC) were obtained by a standard Ficoll-Hypaque gradient centrifugation. Cells were then twice washed in PBS and incubated with 1% human immunoglobulin at room temp for 10 minutes and then stained ready for fluorescence activated cell sorting (FACS) analysis.

Antibodies, Cell Staining, sorting and storage

Human HSPCs, namely HSC

(

Lin^-CD34⁺CD38^-CD90⁺CD45RA^-), multipotent progenitors (MPP, Lin^- CD34⁺CD38^-CD90^-CD45RA^-), multi-lymphoid progenitors (MLP, Lin^-CD34⁺CD38^-CD90^-CD45RA⁺), and proB cells (CD34⁺CD38⁺CD10⁺CD19⁺) were stained and subsequently sorted using a FACS Aria IIu (BD Biosciences, San Jose, CA, USA) and evaluated using BD FACS Diva 6.2 software.^1-4

To exclude lineage-positive cells BMMCs were stained with a biotinylated lineage antibody cocktail (Miltenyi Biotech,Bergisch-gladbach, Germany) followed by APC-Cy7 streptavidin (Biolegend, Fell, Germany). Lineage-negative cells were then stained with PE-anti-CD34 (8G12), Pe-Cy7-anti-CD38 (HIT2), FITC-anti-CD90 (eBio 5E10) and APC-anti-CD45 (HI100), and sorted to obtain HSC, MPP and MLP populations. For the analysis of the proB population, BMMCs were stained with PE-anti-CD34 (8G12), Pe- Cy7-anti-CD38 (HIT2), FITC-anti-CD19 (HIB19), and APC-anti-CD10 (ebioOCB). Relevant isotype matched controls and fluorescence minus one (FMO) controls were used to determine the level of background staining and as gating controls, respectively. Exclusion of non-viable cells was done using propidium iodide (PI) staining excited by UV laser and detected at Hoechst detector after filtering by Long pass filter 600LP